BAM 10 (1&2)

BAM 10 (1&2), 2000

Articles                 5       Dissimilar nuclear protein binding at human b -myosin heavy chain proximal and distal
                                     MCAT elements in response to increased skeletal muscle activity
                                 DRVyas, JJ McCarthy, GL Tsika, and RW Tsika     [Full text pdf ]

17 Troponin T isoform regulation and structure-function relationships
J-P Jin, Q-Q Huang, O Ogut, A Chen and J Wang [Full text pdf ]

27 Disuse induced-atrophy and contractile impairment of human skeletal muscle fibres
G D’Antona, MA Pellegrino, R Rossi, C Naccari Carlizzi, C Reggiani and R Bottinelli [Full text pdf ]

33 Time-course of exercise and apoptosis in dystrophin-deficient muscle of mice
K Rossini, A Donà, M Sandri, C Destro, M Donà and U Carraro [Full text pdf ]

39 Novel functions (work-rest and day-night regimens) in a new cardiomyostimulator for cardiac bioassist
VS Chekanov, JC Chachques, U Carraro, RCJ Chiu, and LW Stephenson [Full text pdf ]

Myology News 49 Program and abstracts of Basics&Applications of Muscle Plasticity Abano Terme, Padova (Italy), June 11-13, 2000.

Dissimilar Nuclear Protein Binding at Human b-Myosin Heavy Chain Proximal and Distal MCAT Elements in Response to Increased Skeletal Muscle Activity

Dharmesh R.Vyas⁽¹⁾, John J. McCarthy⁽¹⁾, Gretchen L. Tsika⁽¹⁾, and Richard W. Tsika^{(1, 2,
3)}

(1) Department of Biomedical Sciences, College of Veterinary Medicine, (2) Department of Biochemistry, College of Medicine, and (3) Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, Missouri

Abstract

MCAT regulatory elements mediate muscle-specific transcription and can act as inducible promoter elements in response to hormonal and mechanical stimuli. Herein we investigate whether two distinct MCAT elements [distal MCAT (-290 to -284), proximal MCAT (-210 to –203: b e3 region)] within a region shown by us to confer mechanical overload (MOV) responsiveness onto a 293-base pair human b MyHC transgene (b 293) function comparably as MOV-inducible elements. As compared to the distal MCAT element, electrophoretic mobility shift assay (EMSA) analysis revealed low levels of binding at the proximal MCAT element when using skeletal muscle [control plantaris (CP) and MOV-P] nuclear extract and in vitro translated transcription enhancer factor 1 (TEF-1) proteins. Direct and competition EMSA analysis showed that only the low mobility binding complex (LMC) formed at the distal MCAT element was highly enriched when using MOV-P nuclear extract, and was only partially competed for by the chicken cardiac troponin T element (cTnT); an element shown to form a high-order LMC involved in muscle-specific expression. Methylation interference (MI) assay identified strong DNA-protein interactions throughout the distal MCAT core element and weaker interactions at the adjacent nucleotides. Our data tentatively indicate that the nuclear protein(s) forming the high-order low mobility complexes at the distal MCAT and cTnT MCAT elements differ, or alternatively, are the same protein(s) binding with different affinity. Also, the proximal and distal MCAT elements display different binding affinities for TEF-1 isoproteins; a property that may determine the magnitude of their involvement in the MOV-induction of transgene b 293 in skeletal muscle of adult transgenic mice.

Key words: b MyHC, gene transcription, hypertrophy, MCAT element, mechanical overload, neuromuscular activity, skeletal muscle, TEF-1.

Basic Appl Myol 10 (1&2): 5-16, 2000

Address correspondence to:

Richard Tsika, Ph.D., University of Missouri-Columbia, Department of Biochemistry and Department of Biomedical Sciences, 1600 E. Rollins Ave, W112 Vet. Medicine Bldg., Columbia, Missouri, 65211, tel. 001 (573) 884 4547, fax 001 (573) 884 6890, Email tsikar@missouri.edu.

Troponin T Isoform Regulation and Structure-Function Relationships

Jian-Ping Jin, Qi-Quan Huang, Ozgur Ogut, Aihua Chen and Jennifer Wang

Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, USA

Abstract

Three fiber type-specific genes have evolved in vertebrates to encode cardiac, slow and fast skeletal muscle troponin Ts (TnT). From the transcript of each gene, alternative RNA splicing produces multiple TnT isoforms with structural differences in three variable regions of the polypeptide chain. The expression of TnT isoform is regulated during heart and muscle development and adaptation. Muscles containing different TnT isoforms show differences in their sensitivity to Ca²⁺-activation and cooperativity of contraction. The NH₂-terminal region of TnT is hypervariable among isoforms and may play a role in modulating the contractility corresponding to the cellular environment and functional state of the muscle. Molecular evolution of the NH₂-terminal variable region of TnT indicates its tolerance to structural variation and role in the functional divergence of striated muscle. Physical property of the NH₂-terminal domain of TnT modulates conformation and function of other regions of TnT, explaining the functional significance of TnT isoform regulation. Species-specific timing of cardiac TnT isoform switching is synchronized in the heart and skeletal muscle, suggesting control by a genetic program. Therefore, the regulation of TnT isoform expression is not only a response to functional demands but may contribute to both physiological adaptation and pathogenesis.

Key words: alternative splicing, development, gene regulation, muscle, protein conformation.

Basic Appl Myol 10 (1&2): 17-26, 2000

Address correspondence to:

Dr. J.-P. Jin, Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970, USA, phone (216) 368 5525, fax (216) 368 3952, Email jxj12@po.cwru.edu.

Disuse Induced-Atrophy and Contractile Impairment of Human Skeletal Muscle Fibres

Giuseppe D’Antona, Maria A. Pellegrino, Rosetta Rossi, Carmine Naccari Carlizzi, Carlo Reggiani and Roberto Bottinelli

Institute of Human Physiology, University of Pavia, Pavia, Hospital "Casa di Cura Columbus", Milano and Department of Anatomy and Physiology, University of Padova, Italy

Abstract

In this study we described the effect of four months of disuse and immobilization on Myosin Heavy Chain (MHC) isoform composition and contractile properties of single fibres in human muscles. We compared muscle samples collected from the vastus lateralis of three groups of two subjects: 1) subjects (60-70 y old) who underwent to the replacement of a total knee prosthesis after four months with the leg immobilized in extended position, 2) control adult subjects (30-40 y old) sampled by needle biopsy and 3) control aged subjects (60-70 y old) who underwent to knee surgery for various reasons. We found in immobilized muscles an increase in MHC-2X , a decrease of slow MHC and the expression of a developmental MHC isoform, indication of fibre regeneration. The proportion of hybrid fibres, i.e. fibres containing two or more MHC isoforms, was increased. Slow fibres showed significant reductions in cross sectional area and in specific tension (force/cross sectional area) developed during maximal isometric contractions. Unloaded shortening velocity was, however, not modified. No alteration was detectable in fast 2A fibres when compared with corresponding fibres of adult and old controls. Slow fibres seemed, therefore, selectively affected by the combination of immobilization and disuse examined in this study.

Key words: disuse, immobilization, muscle atrophy, myosin isoforms.

Basic Appl Myol 10 (1&2): 27-32, 2000

Address correspondence to:

Carlo Reggiani, Dept. of Anatomy and Physiology, University of Padova, via Marzolo 3, 35131 Padova, Italy, phone +39 049 8275 313, fax +39 049 8275 301, Email reggiani@unipv.it.

Time-Course of Exercise and Apoptosis in Dystrophin-Deficient Muscle of Mice

Katia Rossini⁽¹⁾, Andrea Donà⁽¹⁾, Marco Sandri^{(1, 2)}, Chiara Destro⁽¹⁾, Massimo Donà⁽¹⁾ and Ugo Carraro⁽¹⁾

(1) C.N.R. Unit for Muscle Biology and Physiopathology, Department of Biomedical Sciences, University of Padova, Padova, Italy and (2) Institute of Experimental and Laboratory Medicine, University of Padova, Padova, Italy

Abstract

Apoptosis is a process of cell death occurring in many tissues. That apoptosis precedes necrosis in death of dystrophin-deficient muscle fibers of mdx is now accepted. Furthermore, we were the first to describe an increase of apoptotic myonuclei in mdx mice two days after spontaneous running exercise. To investigate the role of apoptosis in muscular dystrophy and to determine minimal time/characteristics of physical exercise able to induce a bout of muscle apoptosis, in the present work we studied contribution of apoptosis to exercise-induced death of muscle fiber by a time-course analysis in mdx mice. The runners were housed in a cage with an exercise wheel and allowed to run spontaneously for two hours or for an entire night (about 12 hours) and, the morning after, Tibialis Anterior of both hindlimbs removed. We checked the activity of mice by monitoring the covered distance and the time when the wheel was moving.

Apoptosis was assessed by the terminal deoxynucleotidyl transferase assay and expressed as number of apoptotic nuclei for mm³ of muscle tissue, and by electron microscopy for morfological features.

In 12-hours running groups mdx mice present a significant minor activity (18±0.7) in comparison with 2-hours mdx runners (36±3.5 p < 0.001). Consequently, it seems that mdx mice have an activity peak after 2-hours exercise and then it decreases maybe because mdx muscular fibers are frailer, more fatiguable and susceptible at exercise-induced damage.

Control non-runner mdx mice present 40±13 apoptotic myonuclei/mm³, 2-hours runner mice 84±13 (p = 0.04 against control) and 12-hours mice 158±32 (p = 0.04 against control; p = 0.23 against 2-hours), while in 12-hours mdx runners interstitial nuclei/mm³(188±46) significantly decrease in comparison with 2-hours group (348±50). Electron microscopy confirm that apoptotic myonuclei increase after 2-hours running in comparison with sedentary and some more after 12-hours running. Besides it shows that apoptotic process involves satellite cells.

Besides confirming that apoptosis present in mdx mice at rest dramatically increases after exercise, results suggest that inflammation and interstitial apoptosis increase during a short-time exercise while the apoptotic process in myofibers (doubled in 2-hours runners in comparison with sedentary group) becomes more manifested after a long-term exercise, even if mdx mice activity decreases.

Key words: apoptosis, Duchenne muscular dystrophy, muscle, programmed cell death.

Basic Appl Myol 10 (1&2): 33-38, 2000

Abbreviations

TUNEL, Terminal dUTP Nick End Labelling; DMD, Duchenne Muscular Dystrophy; PBS, Phosphate Buffered Solution; BSA, Bovine Serum Albumin.

Novel Functions (Work-Rest and Day-Night Regimens) in a New Cardiomyostimulator for Cardiac Bioassist

Valeri S. Chekanov, Juan C. Chachques⁽¹⁾, Ugo Carraro⁽²⁾, Ray C.J. Chiu⁽³⁾, and Larry W. Stephenson⁽⁴⁾

Milwaukee Heart Institute, Heart Care Associates of Sinai Samaritan Medical Center, Milwaukee, Wisconsin, USA, (1) Hospital Broussais, Paris, France, (2) University of Padova, Padova, Italy, (3) McGill University, Montreal General Hospital, Montreal, Quebec, Canada, and (4) Wayne State University School of Medicine, Detroit, Michigan, USA

Abstract

The LD-PACE II, a new cardiomyostimulator was manufactured by CCC del Uruguay with the support of the Illini Group, Chicago, Illinois. The LD-PACE II was designed for use in cardiomyoplasty, aortomyoplasty, and skeletal muscle ventricle. All parameters specified as programmable can be changed in a noninvasive manner (using a programming interface wand connected to a computer using the Windows 95/98 environment). Most of the functions of the new stimulator are similar to functions of previously used devices (Transform, Medtronic; EKS 445, Russia).

In the case of bradycardia or atrio-ventricular blockage, the LD-PACE II will act as a pacemaker with a basic pacing rate between 36 and 120 BPM. In order to prevent the stimulator from inappropriately sensing events, there is a ventricular refractory period (195-480 ms). Hysteresis (0-20%) allows the patient's heart rate to temporarily fall below the lower rate without inducing immediate pacing. Synchronization delay (2-350 ms) obtains the optimal time of muscle contraction. Adaptive delay allows the automatic change in the delay time with a change of heart rate. The cardiosynchronization ratio is programmed from 1:1-1:16. Muscle output is inhibited if the heart rate is higher than the synchronization upper rate (120-226 BPM). The adaptive ratio allows for the automatic change of the cardiosynchronization ratio with an increase in heart rate. Delivery of a muscle pulse train is triggered by paced or sensed ventricular events. Characteristics of the pulse train are changes in pulse amplitude (0.44-0.75 V), pulse width (0.061-0.076 ms), pulses per burst (1-8), and pulse interval (15.6-132.8 ms). The adaptive pulse train duration will automatically decrease the train duration with an increase in heart rate and inhibit muscle contraction during the diastolic phase. The LD-PACE II does however have two new functions which prove to be extremely important for clinical use based on experimental research.

1. Work-rest regimen. In the conventional stimulation regimen, the latissimus dorsi muscle (LDM) works 24 hours daily with no rest except for short periods between contractions. The LD-PACE II is able to deliver alternating periods of muscle contractions and rest. Work and rest periods may be programmed independently between 1 and 120 minutes in increments of one minute. The work-rest regimen may be useful clinically if muscle contractions are needed for cardiac assist postoperatively. Morphological (light microscopy, transmission electron microscopy) and electrophysiological data show that a short period of work followed by a long period of rest does not damage the ischemic muscle.

2. Day-night regimen. This feature is also brand new. It allows for a change in the ratio of muscle contractions according to a patient's activity level. During the day the cardiosynchronization ratio may be set from 1:1 to 1:4 and during the night it may be set for 1:8 to 1:16. This allows the LDM to have a long rest period, prevents overuse, and prolongs battery life. Data confirmed that there is no depression in hemodynamics when the ratio is slowed or turned off during the night.

The first experimental tests of the LD-PACE II (in Milwaukee, Wisconsin, Detroit, Michigan and Paris, France) showed good functional ability of a high caliber. The first clinical cardiomyoplasty with the LD-PACE II was performed on April 2, 2000 at the Bahamas Heart Center.

Key words: LD-PACE II, dynamic cardiomyoplasty, aortomyoplasty, skeletal muscle ventricle, heart failure.

Basic Appl Myol 10 (1&2): 39-48, 2000

Address correspondence to:

VS. Chekanov, Milwaukee Heart Institute, Heart Care Associates of Sinai Samaritan Medical Center, 945 North Twelfth Street, Box 342, Milwaukee, WI 53402-0342, USA, tel. 001 414 219-7899, fax 001 414 219-6266, E-mail: vchekanov@aol.com.

Myology News Sponsored by

University of Padova, Department of Biomedical Sciences - C.N.R. Unit for Muscle Biology and Physiopathology

National Project of The Ministry of University and Scientific&Technological Research: Italian Trial of Demand Dynamic Cardiomyoplasty

The Sixth Abano Terme Meeting on Rehabilitation

BAM’2000

Basics&Applications of Muscle Plasticity

Foundations of Muscle Plasticity - Dynamic Cardiomyoplasty & Cardiac-Bio-Assists – Cell & Gene Muscle Therapy

Abano Terme, Padova (Italy), June 11-13, 2000

HOTEL SAVOIA, via P. d’Abano 49, ABANO TERME (Padova), Italy - Tel +39 049 8231111; Fax +39 049 667777; Internet: http://www.savoiaterme.it/ ; E-mail: hotelsavoia@intercity.shiny.it

International Scientific Committee

U Carraro, Padova, Italy; H Blau, Stanford, CA, USA; A Carpentier, Paris, France; D Casarotto, Padova, Italy; JC Chachques, Paris, France; J Chamberlain, Ann Arbor, MI, USA; V Chekanov, Milwaukee, WI, USA; R C-J Chiu, Montreal, Quebec, Canada; M Grounds, Perth, Australia; E Hoffman, Washington, DC, USA; G Itoh, Aichi, Japan; L Larsson, University Park, PA, USA; A Margreth, Padova, Italy; R Sabbadini, San Diego CA, USA; S Salmons, Liverpool, UK; WP Santamore, Philadelphia, PA, USA; S Schiaffino, Padova, Italy; A Wernig, Bonn, Germany; RS Williams, Dallas, TX, USA

Aim

Reactions of normal muscles to unusual requests and of diseased muscles to normal demands

BAM’2000 Abstracts and Manuscripts Publication

Authors may submit a Manuscript in BAM COMMUNICATION style by July 18, 2000. Please find Information for Authors at: http://www.bio.unipd.it/bam/bam.html

Conference Site

HOTEL SAVOIA, via P. d’Abano 49, ABANO TERME (Padova), Italy

Tel +39 049 8231111; Fax +39 049 667777; Internet: http://www.savoiaterme.it/; E-mail: hotelsavoia@intercity.shiny.it

PROGRAM

June 10, 2000 (Saturday)

Morning: Tour to Venice. Late Afternoon: Opening Ceremony and BAM’2000 Gala Dinner

Locanda Cipriani in the beautiful historical island of Torcello, Venice Laguna.

June 11, 2000 (Sunday)

9.00 - 13.30 Foundations of Muscle Plasticity (I) (II)

9.00 – 11.00 Foundations of Muscle Plasticity: (I) Signalling Pathways

Chairpersons: H Blau, S Schiaffino

9.00 – R Sanders Williams, University of Texas South-western Medical Center, Dallas, TX, USA: Calcineurin-dependent signalling events in muscle plasticity

9.40 – S Hughes, The Randall Institute, King’s College, London: Hedgehog and the control of muscle cell diversification

10.10 – S Schiaffino, Department of Biomedical Sciences and C.N.R. Unit for Muscle Biology and Physiopathology, University of Padova, Italy: Nerve Activity-dependent Muscle Gene Regulation: A New Role for RAS

10.40 - RW Tsika, University of Missouri-Columbia: Muscle-specific transcription of beta-myosin heavy chain transgene requires A/T-rich and NF-AT elements

11.00 - Coffee Break

11.30 - 13.00 Foundations of Muscle Plasticity: (II) The Muscle Transcriptome

Chairpersons: S Williams, S Schiaffino

11.30 - TA Prolla, University of Winsconsin, Madison, WI, Usa: The Transcriptional Profile of the Aging Process in Skeletal and Cardiac Muscle

12.10 - G Lanfranchi, Department of Biology, University of Padova, Italy: Gene expression profiling of human skeletal muscle during ageing and differentiation using an arrayed collection of 3’-end muscle cDNAs

12.30 - G Valle, Department of Biology, University of Padova, Italy: A data base of transcripts expressed in human skeletal muscle

13.00 - 14.30 Lunch and Posters

14.30 - 18.00 Apoptosis of Sarcomeric Muscles (Myocardium and Skeletal Muscle)

14.30 - 16.00 Apoptosis of Sarcomeric Muscles (I)

Chairpersons: RA Gottlieb and U Carraro

14.30 - RA Gottlieb, Division of Hematology, Department of Molecular & Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA: Mitochondria:The Ignition Chamber for Apoptosis

15.00 - R Sabbadini, Heart Institute and Department of Biology, San Diego State University, San Diego California, USA: G Protein-coupled receptors, calcium deregulation and apoptosis in the heart

15.30 - P Bernardi, Department of Biomedical Sciences, University of Padova, Italy: Mitochondria and muscle cell death

16.00 - G Vescovo, Division of Internal Medicine, Adria General Hospital, Ro, Italy: Skeletal muscle apoptosis: a determinant of muscle atrophy in CHF?

16.30 - Coffee Break

17.00 - 18.00 Apoptosis of Sarcomeric Muscles (II)

Chairpersons: R Sabbadini and G Vescovo

17.00 - M Sandri, Department of Biomedical Sciences and C.N.R. Unit for Muscle Biology and Physiopathology, University of Padova, Italy: Apoptosis and muscular dystrophies

17.15 - R Matsuda, Department of Life Sciences, University of Tokyo, Japan: Dystrophic processes can be separated into two distinct stages in mdx skeletal muscle

17.30 - A Jakubiec-Puka: Sarcolemma damage in extended-stimulated muscles

17.40 - K Rossini, Department of Biomedical Sciences and C.N.R. Unit for Muscle Biology and Physiopathology, University of Padova, Italy: Activity-induced apoptosis in mdx mice

17.50 - C Destro, Department of Biomedical Sciences and C.N.R. Unit for Muscle Biology and Physiopathology, University of Padova, Italy: Fas/FasL system regulates apoptosis of macrophages and myoblasts during muscle regeneration

18.00 – TELETHON Lecture: Helen Blau, Department of Molecular Pharmacology, Stanford, CA, USA

BAM’2000 Keynote:Gene Expression and Signal Transduction

19.00 - Concert

June 12, 2000 (Monday)

9.00 - 13.00 - Dynamic Cardiomyoplasty

9.00 - 11.00 Dynamic Cardiomyoplasty (I)

Chairpersons: WP Santamore and V Chekanov

9.00 - A Carpentier & J Chachques, Broussais Hospital, Paris, France: Present and future of Dynamic Cardiomyoplasty

9.30 - JK Kirklin, Division of Cardiothoracic Surgery, University of Alabama, Birmingham, USA: Cardiomyoplasty-skeletal muscle assist randomized trial (C-SMART): 6 month results

10.00 - C Werling, Department of Cardiac Surgery, Herzzentrum Ludwigshafen, Germany: Dynamic Cardiomyoplasty : Clinical experience after seven years

10.15 - J Trainini, Hospital Peron, Buenos Aires, Argentina: Chronic aortic counterpulsation with latissimus dorsi: clinical follow-up. Cardiomyoplasty comparison

10.30 - R Lorusso, Cardiac Surgery, Spedali di Brescia, Italy: Cardiomyoplasty and implantable defibrillator in heart failure patients: positive impact on patient survival

10.40 - R Scelsi, Department of Human Pathology, University of Pavia, Italy: Pathological findings in LD wrap after short- and long-term dynamic cardiomyoplasty

10.50 - V Chekanov, Milwaukee Heart Institute, Heart Care Associates, Milwaukee, Wisconsin, USA: A new alternative for the use of electrical stimulation – atherosclerosis prevention

11.00 Coffee break

11.30 - 13.00 Dynamic Cardiomyoplasty (II)

Chairpersons: JK Kirklin, A Carpentier

11.30 - WP Santamore, Philadelphia, PA, USA: Vascular delay and intermittent stimulation: Keys to success in Cardiomyoplasty

11.50 – D Casarotto & C Muneretto, Cardiac Surgery, Universities of Padova and Brescia, Italy: Three-year experience of Demand Dynamic Cardiomyoplasty

12-00 R Riccardi, Fondazione Maugeri, IRCCS, Centro Medico Montescano, Pavia, Italy: Demand stimulation after long-term Dynamic Cardiomyoplasty

12.10 - GL Rigatelli & M Barbiero, Legnago General Hospital, Legnago (Vr), Italy: Systolic assistance by Demand Dynamic Cardiomyoplasty? The answer from aortic peak flow determined by intravascular Doppler flow wire

12.20 - M Trivellato, on behalf of the Registry partners, Ospedale Geriatrico, Padova, Italy: The Registry of Demand Dynamic Cardiomyoplasty

12.30 - VS Chekanov, Milwaukee Heart Institute, Heart Care Associates of Sinai Samaritan Medical Center, Milwaukee, Wisconsin, USA: Novel functions (work-rest and day-night regimens) in a new cardiomyostimulator for cardiac bioassist

12.40 - F Monese & F Di Gregorio, MEDICO spa, & U Carraro, Padova, Italy: Demand Dynamic Cardiomyoplasty and LD wrap mechanography: new characteristics of a myostimulator (demand LD-cardio pacer) to physiologically activate LD wrap and clinically determine its dynamic characteristics

12.50 – U Carraro, Department of Biomedical Sciences and C.N.R. Unit for Muscle Biology and Physiopathology, University of Padova, Italy: Dynamic Cardiomyoplasty:It is time to wrap!

13.00 Lunch and posters

14.15 – 17.30 – Cell and Gene Therapy in Sarcomeric Muscles: Basics and Applications

Chairpersons: F Rossi and GS Butler-Browne

14.15 - GS Butler-Browne, UFR Biomedicale des St Pères, Paris, France: Satellite cells: life-span and telomeres

14.35 - L Gorza Department of Biomedical Sciences and C.N.R. Unit for Muscle Biology and Physiopathology, University of Padova, Italy: Cell-surface localization of the glucose-regulated protein GRP94 in skeletal myoblasts is involved in myotube formation

14.50 - M Shiozuka, Department of Human Sciences, Waseda University, Japan: A possible role of SPARC (secreted protein-acidic and rich in cysteine) in myogenesis

15.00 - J-P Jin, Department of Physiology & Biophysics, Case Western Reserve, Cleveland, OH, USA: Troponin T gene regulation during muscle development and adaptation, and functional significance

15.10 - Y Atomi, Department of Life Science, University of Tokyo, Tokyo Japan: Role of stress protein aB-crystallin for stabilization of tubulin/microtubule in vitro and in myoblast cells

15.20 - T Obinata, Department of Biology, Chiba University, Japan: Actin filament organization is regulated by cofilin, a small G-actin-binding protein, during myofibril assembly

15.30 - I Dell’Aica, Department of Biomedical Sciences and C.N.R. Unit for Muscle Biology and Physiopathology, University of Padova, Italy: Telomerase activity in skeletal muscles of the mdx mice

15.40 Coffee Break

16.00 - F Rossi, Department of Molecular Pharmacology, Stanford, CA, USA : Myoblast-mediated delivery of tightly regulated therapeutic genes

16.20 - J Huard, Growth and Development Lab, Children’s Hospital of Pittsburg, PA, USA: The use of muscle derived cells expressing stem cell antigens to improve the efficiency of cell transplantation to dystrophic muscle

16.40 - A Wernig, Dept. Physiology Univ. Bonn and Klinikum Karlsbad-Langensteinbach, Germany: Human muscle tissue grown in mice

17.00 - L Vitiello, Department of Biology, University of Padova, Italy: Gene transfer into skeletal muscle by means of lipopolyplexes

17.10 - M Cantini CRIBI Center and Department of Biomedical Sciences, University of Padova, Italy: J774 macrophage cell line secretes a specific myogenic factor capable to inducing myoblast proliferation and to delaying their differentiation

17.20 - Shuttle bus to Padova University

Palazzo del Bò, Università di Padova

18.10 - Visit to historical Anatomical Theatre

Myology Lectures

18.40 - G Marechal, Department of Physiology and Pharmacology, UCL, Brussels, Belgium: The effects of nitric oxide on the contraction of skeletal muscle

19.00 - A Margreth, CNR Unit for Muscle Biology and Physiopathology c/o Department of Biomedical Sciences, University of Padova, Italy: Species-specific biochemical properties and adaptive changes of the sarcoplasmic reticulum of human skeletal muscle under pathological conditions

20.00: BAM Friends, Get Together

June 13, 2000 (Tuesday)

9.00 - 10.00 - Cellular Mechanisms and Progression of Muscular Dystrophies

Chairpersons: L Larsson and C Angelini

9.00 - C Angelini, Neuromuscular Center, Department of Neurology, University of Padova, Italy: Clinico molecular correlations in dystrophinopathies and carriers

9.20 - A Stracher, Stony Brooks, New York, USA: Ca++/calpain hypothesis for neuromuscular degeneration

9.30 - R Betto, CNR Unit for Muscle Biology and Physiopathology, University of Padova, Italy: Role of purinergic receptors in the pathogenesis of sarcoglycanopathies

9.40 - G Siciliano, Metabolic and muscle adaptation to aerobic training in mitochondrial myopathies

9.50 - M Bonifati, Neuromuscular Center, Department of Neurology, University of Padova, Italy: A multicenter double-blind randomized trial of deflazacort versus prednisone in Duchenne muscular dystrophy: analysis after 2 years

Coffee Break

10.30 - 13.30 - Mechanisms of Cellular Adaptation to Workload and Exercise-induced Muscle Damage

Chairpersons: Y Mounier and C Reggiani

10.30 - L Larsson, Noll Physiological Research Center & Department of Cellular and Molecular Physiology, School of Medicine, Hershey Medical Center, The Pennsylvania State University, USA: Effects of aging on the regulation of muscle contraction at the motor unit, muscle cell, and molecular levels

11.00 - C Reggiani, Department of Human Anatomy and Physiology, University of Padova, Italy: Disuse induced-atrophy and contractile impairment of human skeletal muscle fibres

11.15 - Y Mounier, Laboratoire de Plasticité Neuromusculaire, UST Lille, France: Troponine C plasticity in unloading conditions

11.30 - H Kern, University of Vienna: Denervated muscles in human: First results of training with electrical stimulation

11.45 - M Falempin, Laboratoire de Plasticité Neuromusculaire, UST Lille, France: Sole plantar stimulation: a countermeasure for rat soleus atrophy observed during unloading

12.00 - A Wernig, Dept. Physiology Univ. Bonn and Klinikum Karlsbad-Langensteinbach, Germany: Locomotor (Laufband) therapy in SCI persons

12.15 - W Mayr, Dept. of Biomedical Engineering and Physics, and Dept. of Plastic and Reconstructive Surgery, University of Vienna Medical School, Austria: Functional electrostimulation as a countermeasure against muscular atrophy in long-term space flights

12.30 - A Martinuzzi, Walking energy cost in children with neurogenic motor impairment

12.40 - C Chisari: Impaired muscle oxidative metabolism in polymyositis and dermatomyositis evaluated in vivo through lactic acidaemia assay

12.50 - O Rossetto, Department of Biomedical Sciences, University of Padova: Recovery of human neuromuscular junction after botulinum neurotoxin therapy

13.00 - A Megighian, Human Physiology, University of Padova: Effects of tenotomy on rat slow muscle regeneration

Lunch and posters

14.30 - 18.00 - Cardiac-Bio-Assists

Chairpersons: J Jarvis and NW Guldner

14.30 - J Jarvis, Department of Anatomy and Cell Biology, University of Liverpool, UK: Skeletal muscle ventricles in the pig: left ventricular off-loading demonstrated by observation of pressure-volume loops

15.00 - NW Guldner, Clinic of Cardiac Surgery, Medical University of Lubeck, Germany: Biomechanical Hearts Performed in a One-Step Operation and Trained Dynamically under Support of Clenbuterol

15.30 - B Voss, Department of Cardiac Surgery, Deutsches Herzzentrum München, Germany: Dynamic Cardiomyoplasty in a growing organism

15.45 - R Lorusso, Cardiac Surgery, Spedali di Brescia, Italy: Muscle power after vascular delay in a sheep model of muscle transposition.

16.00 - V Chekanov, Milwaukee Heart Institute, Heart Care Associates, Milwaukee, Wisconsin, USA: Pharmacological support of angiogenesis using deferoxamine in biological glue for cardiomyoplasty

16.15 - A Shafy/JC Chachques, Department of Cardiac Surgery, Broussais Hospital, Paris, France: Association of cellular cardiomyoplasty with multisite cardiac pacing

16.30 - E Monnet, Department of Clinical Sciences, Colorado State University, Fort Collins CO, USA: Is injury to the toracodorsal nerve present after long-term dynamic cardiomyoplasty in goats?

16.45 - P Klapproth, Clinic of Cardiac Surgery, Medical University of Lubeck, Germany: Methods to evaluate dynamic training of skeletal muscle ventricles and biomechanical hearts

17.00 - E Giardini, Institute of Plastic Surgery, University of Padova, Italy: Vascular delay of a LD in an experimental rat model for dynamic cardiomyoplasty

17.15 - U Carraro, Department of Biomedical Sciences, University of Padova, Italy: Myoblast cell therapy to augment muscle mass in Cardiac-Bio-Assists

Coffee Break and First Meeting of THE CARDIAC-BIO-ASSIST ASSOCIATION
BAM’2000

Basics&Applications
of Muscle Plasticity

Foundations of Muscle Plasticity – Cell&Gene Muscle Therapy Dynamic Cardiomyoplasty&Cardiac-Bio-Assists

Abano Terme, Padova (Italy), June 11-13, 2000

HOTEL SAVOIA, via P. d’Abano 49,
ABANO TERME (Padova), Italy
Tel +39 049 8231111; Fax +39 049 667777;
Internet: http://savoiaterme.it/ ;
E-mail: hotelsavoia@intercity.shiny.it

POSTERS

1. MYOTUBES OVER-EXPRESSING Fas L PROTEIN UNDER MCK PROMOTER ARE RESISTANT TO Fas-Fas L-INDUCED PAPOOSES, A Bon, M Sandri, C Radu, S Tiozzo, E Sartori, L Vitiello, M Cantini, CRIBI Center, and Departments of Biomedical Sciences and Biology, University of Padova, Italy

2. PROPOSAL OF VALUTATIVE AND REHABILITATIVE PROTOCOL IN FACIOSCAPULOHUMERAL DYSTROPHY, Chisari C, Giannini E, Mussini F, Simonella C, Bresci M, Rossi B, Neurorehabilitation Unit, Dept. of Neuroscience, University of Pisa

3. SURFACE EMG TO STUDY MUSCLE FUNCTION IN ASYMPTOMATIC DIABETIC NEUROPATHY, Chisari C, Piaggesi A, Baccetti F, Rizzo L, Giannini E, Simonella C, Rossi B, Neurorehabilitation, Dept. of Neuroscience, and Chair of Metabolic Diseases, Dept. of Endocrinology and Metabolism, University of Pisa

4. Impaired muscle oxidative metabolism in polymyositis and dermatomyositis evaluated in vivo through lactic acidaemia assay, Chisari C, Stampacchia G, Giannini E, Neri R, Mosca M, Rossi B, Neurorehabilitation Unit, Dept. of Neuroscience, and Reumatology Unit, Dept of Medicine, University of Pisa

5. Correlative ³¹P-MRS - M wave analysis of high and low frequency electrically-induced fatigue in rabbit tibialis anterior muscle, J-L Darques, D Bendahan , M Roussel , Y Le Fur , S Confort-Gouny , F Tagliarini , PJ Cozzone , Y Jammes, Laboratoire de Physiopathologie Respiratoire (UPRES EA 2201), Faculté de Médecine and Centre de Résonance magnétique biologique et médicale, UMR CNRS 6612, Univ. Méditerranée, Marseille, France.

6. SOLE PLANTAR STIMULATION: A COUNTERMEASURE FOR RAT SOLEUS ATROPHY OBSERVED DURING UNLOADING, L De Doncker, F Picquet, M Falempin, Laboratoire de Plasticité Neuromusculaire - SN4 - UST Lille 1, Villeneuve d’Ascq, Lille, France

7. TELOMERASE ACTIVITY IN MUSCLE OF NORMAL AND MDX MICE AND IN MUSCLE-REGENERATION, I Dell’Aica, M Sandri, K Rossini, C Destro, U Carraro. C.N.R. Unit for Muscle Biology and Physiopathology, Department of Biomedical Sciences,

University of Padova, Italy.

8. Fas/FasL SYSTEM REGULATES APOPTOSIS OF MACROPHAGES AND MYOBLASTS DURING MUSCLE REGENERATION, C Destro, M Sandri, K Rossini, C Sandri, I Dell’Aica, U Carraro, C.N.R. Unit for Muscle Biology and Physiopathology, Department of Biomedical Sciences, University of Padova, Italy.

9. VASCULAR DELAY OF LD IN AN EXPERIMENTAL RAT MODEL FOR DYNAMIC CARDIOMYOPLASTY, E Giardini, F Mazzoleni, K Rossini (1), A El Messlemani (1), A Donà (1), U Carraro (1), Institute of Plastic Surgery, and (1) C.N.R. Unit for Muscle Biology and Physiopathology, Department of Biomedical Sciences, University of Padova, Italy

10. METHODS TO EVALUATE DYNAMIC TRAINING OF SKELETAL MUSCLE VENTRICLES AND BIOMECHANICAL HEARTS, P Klapproth, NW Guldner, M Grssherr (1), HH Sievers, Clinic of Cardiac Surgery, and (1) Clinic of Anaesthesiology, Medical University of Lubeck, Germany

11. SORTING AND DYNAMICS OF MYOSIN ALKALI LIGHT CHAIN EXPRESSED IN CULTURED CARDIOMYOCYTES AND CARDIAC FIBROBLASTS, MM Khan, M Komiyama, Y Shimada, Department of Anatomy and Cell Biology, School of Medicine, Chiba University, Japan

12. IDENTIFICATION OF MUSCLE DERIVED STEM CELLS CAPABLE OF DIFFERENTIATING INTO OSTEOGENIC LINEAGES AND IMPROVING BONE HEALING, JY Lee, Z Qu-Petersen, B Cao, S Kimura, R Jankowski, D Musgrave, P Bosch, C Gates, P Robbins, A Wernig, J Huard, Growth and Development Laboratory, Department of Orthopaedic Surgery, University of Pittsburg and Children’s Hospital of Pittsburgh, PA, Department of Molecular Genetics and Biochemistry, University of Pittsburgh, PA, USA and Department of Physiology, Neurophysiology, University of Bonn, Germany

13. WALKING ENERGY COST IN CHILDREN WITH NEUROGENIC MOTOR IMPAIRMENTS, A Martinuzzi, E Trevisi, P Zamparo (1), PE Di Prampero (1), IRCCS "E. Medea" Polo Regionale di Conegliano, (1) Dip. Scienze e Tecnologie Biomediche, Università di Udine.

14. EFFECTS OF TENOTOMY ON RAT SLOW MUSCLE REGENERATION, A Megighian, D Danieli Betto, E Germinario, M Midrio, Department of Human Anatomy and Physiology, University of Padova, Italy

15. DEMAND DYNAMIC CARDIOMYOPLASTY AND LD WRAP MECHANOGRAPHY: NEW CHARACTERISTICS OF A MYOSTIMULATOR (DEMAND LD-CARDIO PACER) TO PHYSIOLOGICALLY ACTIVATE LD WRAP AND CLINICALLY DETERMINE ITS DYNAMIC CHARACTERISTICS, F Monese, F Di Gregorio, U Carraro (1), MEDICO s.p.a., Rubano ( Padova), (1) Department of Biomedical Sciences, University of Padova, Italy

16. GENE TRANSFER INTO SKELETAL MUSCLE BY MEANS OF LIPOPOLYPLEXES, F Pampinella, M Pozzobon, E Zanetti, P Gamba, GL Lukacs, M Cantini, L Vitiello, CRIBI Center and Departments of Biomedical Sciences, Biology and Pediatrics, University of Padova, Italy, Cell and Lung Biology, Research Institute, Hospital for Sick Children, Toronto, Canada

17. THE TRANSDUCTION OF SKELETAL MUSCLE WITH ADENO-ASSOCIATED VIRUS IS FIBER DEPENDENT, R Pruchnic, BH Cao, Z Qu, X Xiao, J Li, RJ Samulski, M Epperly, J Huard, Department of Orthopaedic Surgery, Department of Molecular Genetics and Biochemistry, University of Pittsburgh, PA, University of North Carolina, Chapel Hill, NC, USA

18. EXERCISE LACTATE ANAEROBIC THRESHOLD IN HEREDITARY SPASTIC PARAPLEGIA, L Pasquali, ML Manca, E Pastorini, FM Santorelli, G Siciliano, Department of Neurosciences, Neurological Clinics, University of Pisa, Italy

19. J774 MACHROPHAGE CELL LINE SECRETES A SPECIFIC MYOGENIC FACTOR CAPABLE TO INDUCING MYOBLAST PROLIFERATION AND TO DELAYING THEIR DIFFERENTIATION, C Radu, S Tiozzo, A Bon, G Zaniolo, E Sartori, M Cantini, CRIBI Center and Departments of Biomedical Sciences and Biology, University of Padova, Italy

20. TIME-COURSE OF EXERCISE AND APOPTOSIS IN THE MDX MICE, K Rossini, A Donà, M Sandri, U Carraro, C.N.R. Unit for Muscle Biology and Physiopathology, Department of Biomedical Sciences, University of Padova, Italy.

21. IS A HIGH INTRACELLULAR LIPID CONTENT AN ADAPTATIONAL RESPONSE TO A LOW CAPILLARY SUPPLY IN SKELETAL MUSCLE?, AM Saenger, Institute of Zoology, University of Salzburg, Austria

22. ENDURANCE TRAINING AFFECTS WHITE AXIAL MUSCLE IN THE CYPRINID SPECIES CHALCALBURNUS CHALCOIDES MENTO (AGASSIZ, 1832), CYPRINIDAE, TELEOSTEI, AM Saenger, U Putscher, Institute of Zoology, University of Salzburg, Austria

23. ASSOCIATION OF CELLULAR CARDIOMYOPLASTY WITH MULTISITE CARDIAC PACING, A Shafy, JC Chachques, P Argyriadis, L Shen, C Rajnoch, A Berrebi, JN Fabiani, A Carpentier, Department of Cardiac Surgery, Broussais Hospital, Paris, France

24. A POSSIBLE ROLE OF SPARC (SECRETED PROTEIN-ACIDIC AND RICH IN CYSTEINE) IN MYOGENESIS, M Shiozuka, Department of Basic Human Sciences, School of Human Sciences, Waseda University, Saitama, Japan

25. DYNAMIC CARDIOMYOPLASTY IN A GROWING ORGANISM , B Voss, M Thielmann, M El-Mehsen, PA Schnabel (1), S Hagl (1), R Lange, Department of Cardiac Surgery, Deutsches Herzzentrum München, and (1) Departments of Cardiac Surgery and Pathology, University Hospital Heidelberg, Germany

26. DEVELOPMENTAL RELATIONSHIP OF MYOSIN BINDING PROTEINS (CONNECTIN, MYOMESIN and C-PROTEIN) TO MYOSIN IN CHICKEN SOMITES AS STUDIED BY CONFOCAL MICROSCOPY, Y Yang, T Obinata, Y Shimada, Department of Anatomy and Cell Biology, School of Medicine, Chiba University, Japan

BAM’2000

Basics&Applications
of Muscle Plasticity

Foundations of Muscle Plasticity – Cell&Gene Muscle Therapy Dynamic Cardiomyoplasty&Cardiac-Bio-Assists

Abano Terme, Padova (Italy), June 11-13, 2000

HOTEL SAVOIA, via P. d’Abano 49,
ABANO TERME (Padova), Italy

ABSTRACTS

CLINICO MOLECULAR CORRELATIONS IN DYSTROPHINOPATHIES AND CARRIERS

C Angelini, E Pegoraro, P Melacini, M Fanin

Department of Neurology, University of Padova, Via Giustiniani 5, 35128 Padova, Italy

Natural history. We monitored 50 severe dystrophinopathy (DMD) patients for age of loss of Gowers’ sign (8 years) and of loss of independent ambulation that occurred at a mean age of 10.4±1.75 years. In deflazacort treated DMD patients the age of loss of independent ambulation may be extended beyond 13 years of age. 125 mild dystrophinopathy Becker patients were classified according to clinical status as subclinical, benign, moderate or severe. Significant decrease of dystrophin abundance was observed with increased clinical severity (p<0.05).We observed a significant lower level of dystrophin in patient with rapid course. In patients with large deletions or duplications in the proximal rod region prognosis was better showing a stable course. These patients often suffered from cramp/myalgia syndrome or episodic myoglobinuria.

Cardiac involvement was detected in 65% of cases. A right ventricle volume increase was seen in all mild dystrophinopathy groups. A left ventricular dilatation was observed in 25% of cases and decreased ejection fraction in 29% of our cases. The ejection fraction reduction and increase left ventricular value was age related. Since sudden death may occur as a consequence severe left ventricular dysfunction in mild dystrophinopathy should be monitored. This cardiological picture in mild dystrophinopathy patient does not correlate with skeletal muscle involvement.

Cardiac transplantation was performed in two Becker patients and in a Duchenne Muscular Dystrophy carrier. We studied their explanted heart specimen and endomyocardial biopsies by immunohistochemistry and Western blot for both dystrophin and utrophin. Utrophin was over-expressed in these specimen. Our results suggest that in upregulation of utrophin was unable to compensate for the development of life-threatening evolution. The adverse cardiac course may also be explained by the absence of regeneration in myocardium. Heart transplant improves functional outcome and quality of life of dystrophinopathy patients.

RIGHT VENTRICULAR DYSFUNCTION CAN BE TREATED BY CARDIOMYOPLASTY

P Argyriadis, JC Chachques, G Fontaine, JL Hébert, A Shafy, JP Couetil, F Fontaliran, A Carpentier

Broussais and Jean Rostand Hospital, France

The role of the right ventricle (RV) was underestimated for many years. Current knowledge demonstrate that the RV is more than a conduit, it is a pump having systolic and diastolic functions. This pump may fail due to dysplasia, myocarditis, idiopathic or ischemic cardiomyopathies and congenital diseases (Ebsteins’s anomaly). In patients suffering for RV failure associated with tricuspid valve insufficiency the results of single tricuspid valve repair or replacement are disappointing. Our approach consist of performing the cardiomyoplasty (CMP) surgical procedure to improve RV function associated with tricuspid valve repair.

Methods: Among 183 patients who underwent CMP by our team (112 cases operated at Broussais Hospital and 71 patients by our team abroad) we selected six specific patients who underwent CMP for RV failure at Broussais Hospital. Our series included 4 males and 2 females whit a mean age of 38 years old (range 15 to 63 years). Ethiology of RV failure was dilated cardiomyopathy in 4 and ischemic in 2 cases. All the patients with dilated cardiomyopathy and one with ischemic had tricuspid valve regurgitation. Five patients were in NYHA functional class III and one in class IV. The mean radioisotopic EF of RV was 29% (12-31) and EF of LV was 37% (26-56). The mean cardiothoracic ratio (CTR) was 0,61.

Four patients underwent CMP combined with tricuspid valve repair and one patient CMP with tricuspid valve replacement. Only the patient had a single CMP. The procedures were performed under cardiopulmonary bypass and moderate systemic hypothermia. After completion of tricuspid repair or replacement, the left latissimus dorsi muscle (LDM) was inserted over the RV and then the free edge of the muscle was fixed to the diaphragm (anterior wrapping).

Results: A ten-years follow-up showed very encouraging results. Functional class and right ventricular function improved in all cases. Five patients have a remarkable quality of life. Four of them are in NYHA functional class I and one in class II. The mean postoperative EF of RV is 32% (16-51) and EF is 51% the mean CTR is 0,54. One patient died 7 years later the operation due to stroke (atrial fibrillation without anticoagulant treatment because of hepatic cirrhosis) but he was in functional class I-II.

In conclusion, the results of our study, showing no perioperative mortality and no longer-term RV dysfunction related deaths, are very encouraging. Functional class and right ventricular function improved in all cases. We believe that surgical indications for RV failure and eventually also for Ebstein’s anomaly should be liberalized, associating tricuspid valve right ventricular cardiomyoplasty.

ROLE OF STRESS PROTEIN aB-CRYSTALLINE FOR STABILIZATION OF TUBULIN/MICROTUBULE
IN VITRO AND IN MYOBLAST CELLS

Y Atomi, Y Jujita, M Tanaka, E Ohto, K Toro

Department of Life Science, University of Tokyo,
Tokyo Japan

aB-Crystalline, one of the small heat shock proteins, is constitutively expressed in nonlenticular tissues as well as in lens. It can function as a molecular chaperone for other lens crystallines and some to stability and maintenance of cytoskeletal network dynamics. Previously we have reported that the protein that specifically decreased in rat atrophied soleus muscle was aB-crystalline. aB-Crystalline localized at Z and I bands in skeletal muscle expresses fiber-type specific manner. Z and I bands are composed of cytoskeletons, such as actin, intermediate filament desmin. In the present study, we reexamined precisely the cellular localization of aB-crystallin in L6 myoblast cells in relation to three cytoskeletal networks. We observed relatively distinct distribution of aB-crystalline with F-actin, and more concomitant distribution with intermediate filaments (vimentin). Surprisingly, the localization of aB-crystallin was strikingly coincident with microtubule lattice. Microtubule assembly using L6 cell lysate with taxol, a microtubule polymerizing drug, resulted in coprecipitation of aB-crystalline in the microtubule fraction. In addition, purified aB-crystalline bound to microtubules reconstituted from phosphocellulose-purified tubulin at a rate of 1 to 1 aB-crystalline monomer/tubulin dimer ratio. Electron micrograph showed aB-crystalline complex regularly bound on MT. Sense modification of glial and myoblast aB-crystalline production resulted in resistance to nocodazole-induced MT disassembly more stable tubulin turnover, while antisense modification showed reverse direction of MT stability. Direct resistant effect of aB-crystalline to nocodazole was also observed in vitro.

These results indicate that one of the functions of aB-crystallin in living myoblast and probably myotube cells is the increased stability of microtubule network and its component, tubulin subunits. It seemed that aB-crystallin might work as stabilizing buffer for cytoskeleton on the way of remodeling relating muscle plasticity.

MITOCHONDRIA IN MUSCLE CELL DEATH

P Bernardi¹, W Irwin¹, E Fontaine²

¹Dipartimento di Scienze Biomediche Sperimentali, Università di Padova, Viale G. Colombo 3, I-35121 Padova and ²Laboratoire de Bioénergétique Fondamentale et Appliquée, Université Joseph Fourier - BP 53X, F-38041 Grenoble

Mitochondria, the main sources of energy for eukaryotic cells through oxidative phosphorylation, also play a key role in the pathways to cell death. The mode of cell death may be influenced by the availability of ATP, and its very occurrence may critically depend on release of mitochondrial proteins like cytochrome c, apoptosis-inducing factor and caspases 3 and 9. Ca^2±dependent onset of the permeability transition, caused by opening of a cyclosporin A-sensitive pore modulated by cyclophilin D, may play a major role in cell death through ATP depletion, disruption of Ca²⁺ homeostasis, and possibly through release of the above mentioned proteins. Dysregulation of Ca²⁺ homeostasis, proteolysis and a decreased ability to cope with oxidative stress are clearly involved in the pathogenesis of Duchenne’s muscular dystrophy (DMD) downstream of the genetic lesion. Mitochondria appear as likely targets that may amplify the initial insult, resulting in the irreversible events leading to cell demise. We have identified novel inhibitors of the permeability transition pore that are effective in skeletal muscle mitochondria, and validated bupivacaine in a short-term model of muscle cell death involving mitochondrial depolarization and pore opening as early events. We will discuss the role of mitochondria in muscle cell death, with particular emphasis on the role of the permeability transition pore in Ca²⁺ homeostasis and in the release of cytochrome c. Of particular relevance appear to be the differences between fibers of the striated portion of the esophagus (which in DMD is spared from the disease despite the genetic lesion) and of skeletal muscle (which undergoes Ca²⁺ dysregulation and cell death). Our results show that esophagus striated muscle fibers are resistant to bupivacaine toxicity after 24 hrs of culture, a condition that leads to Ca²⁺ deregulation and hypercontracture of skeletal muscle cells and results in cell death. The basis for this striking difference, which correlates with the resistance to degeneration of this type of muscle in DMD, is under active investigation.

ROLE OF PURINERGIC RECEPTORS IN THE PATHOGENESIS OF SARCOGLYCANOPATHIES

R Betto¹, D Biral¹, D Sandonà², E Tarricone²

¹CNR Muscle Biology and Physiopathology Unit, and ²Department of Biomedical Sciences, University of Padova, Viale G. Colombo 3, I-35121 Padova

?-Sarcoglycan is a member of a transmembrane complex of proteins (the sarcoglycans) associated to dystrophin at sarcolemma of skeletal and cardiac muscles. Mutations of sarcoglycans genes are responsible for severe forms of muscular dystrophy. Moreover, the lack of dystrophin in Duchenne muscular dystrophy causes the absence or reduction of many of the associated proteins, included ?-sarcoglycan. The function of the sarcoglycan complex is still elusive. We demonstrated that ?-sarcoglycan is an ecto-ATPase (Betto et al., J. Biol. Chem. 1999), a protein that control the concentration and, thus, the action of extracellular ATP. Extracellular nucleotides are important signaling molecules modulating various physiological responses in different tissues by acting through P2 purinergic receptors distributed on cell surfaces. P2 receptors are distinguished into P2X, ionotropic ligand-gated channels, and P2Y, G-protein-coupled metabotropic receptors. We have used cultured muscle fibers to analyze the possible action of extracellular nucleotides in modulating the activity of purinergic receptors. We demonstrate that P2X4 and P2X7 purinergic receptors, ligand gated cationic channels, are expressed is skeletal muscles. BzATP, a specific agonist of these receptors, caused intracellular calcium transients, which amplitude decreased with the age of the animal, being barely evident in fibers from 50-day old normal mice. Diversely, in muscle fibers from mdx mouse stimulation of P2X receptors evoked large calcium transients even in fibers from adult mice. Consistent with the different sensitivity to nucleotides, P2X4 and P2X7 receptor expression was almost absent in adult muscle fibers from normal mice and still evident in adult mdx fibers. We propose that the higher sensitivity to P2X agonists of the dystrophic muscle fibers, associated to the lack of ?-sarcoglycan, contribute to muscle fiber calcium overload and cell death.

Funded by CNR institutional funds and by TELETHON Italy (grant #629).

GENE EXPRESSION AND SIGNAL TRANSDUCTION

HM Blau

Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA, USA

Plasticity of cell fate is becoming increasingly apparent. Myoblasts can be engineered to express a range of genes typical of totally distinct cell types and perform tissue-specific post-translational modifications characteristic of cells such as liver. Among the gene products expressed by myoblasts at physiological levels are human growth hormone, clotting Factor IX, erythropoietin, macrophage colony-stimulating factor (M-CSF), and vascular endothelial growth factor (VEGF). Studies of VEGF, in particular, have highlighted the importance of regulated expression in order to avoid toxic effects and modulate levels over time. The development of inducible gene expression systems is clearly of utmost importance to the future of gene therapy. To date, myoblasts provide longterm high-level expression of foreign genes and are therefore perhaps the most efficient delivery method currently available for delivering secreted proteins. The plasticity of myoblasts has provoked interest in other cell types and their potential to adapt to distinct settings and perform diverse functions. However, in most tissues, only a small subpopulation of cells is capable of responding to environmental clues and change their fate accordingly. The study of such cells is limited by their rarity, which prompted us to develop assays and model systems apt to investigating biological events at the single cells level, in living cells. In particular, protein-protein interactions are involved in most physiological decisions directing cell fate. In most cases however, the techniques available to study these events are limited to biochemical methods which require large amount of homogeneous material and do not provide information on the behavior of single cells. We have developed a novel assay that allows to monitor protein-protein interactions dynamically in intact mammalian cells. In this approach, chimeric proteins composed of proteins of interest fused to complementing b-galactosidase (b-gal) deletion mutants are generated and expressed within cells. The b-gal activity resulting from the forced interaction of non-functional weakly complementing b-gal peptides (D? and D?) serves as a measure of the extent of interaction of the non-b-gal portions of the chimeras. We have shown in myogenic cells that this technique allows real time monitoring not only of cytoplasmic (Rossi et al., PNAS 94:8405), but also transmembrane proteins. Chimeric proteins containing the extracellular and transmembrane domains of the Epidermal Growth Factor Receptor (EGFR) fused to D? and D? were constructed, inserted in a retroviral vector and expressed in mammalian cells. Enzymatic b-gal activity served to monitor the formation of the EGF-induced receptor dimers in a time-dependent manner, as assessed by fluorescence activated cell sorting assays. Treatment with different EGF-related factors resulted in responses whose intensity was proportional to the reported affinity of such ligands for the wild type receptor, demonstrating that the chimeric receptors retained their ligand specificity. Furthermore this approach led to the identification of a previously unknown level of regulation of the EGFR activity, acting directly on receptor dimerization. This approach is the first to allow the monitoring of complex formation at the membrane of intact, viable mammalian cells. It may prove to be a valuable tool for both basic research, and more application-oriented uses such as drug screening. Finally this technique is unique in that it may allow the identification of novel interactions restricted to the membrane of mammalian cells.

Another important step in regulating cell fate decision is the integration of extracellular signals at the level of transcription. In eukaryotes, chromatin-embedded genes are inactive in their basal state. Nevertheless, many genes involved in controlling cell growth and developmental processes are actively repressed. Their activation is controlled by extracellular signals that trigger the release of transcriptional repressors and the binding of activators, often to the same promoter element. Transcription factor networks that follow this model include MyoD, Myc, E2F, nuclear hormone receptors and many others. We created a tetracycline (tet) regulatable synthetic system that mimics this phenomenon in cultured primary myoblasts and that allows monitoring of the effect of different amounts of bound activator or repressor on transcription from individual promoters. We found that increased binding of a transcriptional activator alone resulted in a dose-dependent increase in transcription from each single promoter. Decreasing binding of a transcriptional repressor had the same effect. Upon co-expression of activator and repressor, the graded response was converted to a threshold response and no intermediate levels of gene expression were observed. This finding fits well with the notion that growth and differentiation are generally all-or-none responses. Our results suggest that a repressor-activator interplay functions to establish a switch that facilitates all-or-none responses to extracellular signals.

A MULTICENTER DOUBLE-BLIND RANDOMIZED TRIAL OF DEFLAZACORT VERSUS PREDNISONE
IN DUCHENNE MUSCULAR DYSTROPHY:
ANALYSIS AFTER 2 YEARS

MD Bonifati, G Ruzza, P Bonometto, A Berardinelli, K Gorni, S Orcesi, G Lanzi, C Angelini

From the Neuromuscular Center (Dr. Angelini, Bonifati, Ruzza and Bonometto), Department of Neurology, University of Padova, Padova, Italy and Department of Child Neurology (Dr. Berardinelli, Dr. Gorni, Dr. Orcesi and Dr. Lanzi), Istituto Mondino, University of Pavia, Italy

Duchenne muscular dystrophy is a progressive muscle disease caused by absence of dystrophin, a sarcolemmal protein. Actually no risolutive therapy is available but steroids have demonstrated to slow down slightly the progression of the disease and delaying loss of independent ambulation. Recently deflazacort has been stated to be as efficacious as prednisone but with less side effects but no comparative trial have not yet been done. Thus we have conducted a double-blind, randomized, multicentric trial in 18 Duchenne muscular dystrophy (DMD) boys whose age ranged from 5.2 to 14.6 years (mean 7.3 yrs) for treatment with either deflazacort (0.9 mg/kg/day) or prednisone (0.75 mg/kg/day). To reduce side effects after one year treatment schedule was shifted on an alternate day regimen. The two groups were randomized and stratified on the basis of age and functional score at the onset of treatment. Thus, at the beginning of treatment the two groups were similar in mean age and functional parameters.

We followed the patients every 3 months in the first year and then every four months, evaluating MRC scale in four limb muscles, two in the right upper limb (deltoid and triceps) and two in the right lower limb (ileopsoas and quadriceps femoris) and performance of four functions (walking for 10 meters, climbing stairs, Gowers’ maneuvre, and rising from a chair). We evaluated the differences in MRC and functional score with respect to baseline. Statistical significance was checked by the Mann-Whitney test. Side effects were monitored by a questionnaire and by routine blood examination (serum creatine kinase, glucose, ionds, hematocrit and complete blood count) and weight and height were recorded at each visit. Change in body was evaluated with the student t-test. After 24 months there were 2 drop out in deflazacort group and 3 in the prednisone group for loss of independent ambulation. The two steroids were equally effective improving motor function and functional performances in the first 6 months and then slowing down the course of the disease. The two groups were quite similar both in MRC score and in functional score.

About side effects at 9 months, the average weight increase respect to baseline value was 5% (2 Kg) in the deflazacort group but 18% in the prednisone group (p< 0.005) and after 24 months it was 19% in the deflazacort group and 41% in the prednisone group. In only 3 patients on deflazacort had the increase in body weight exceeded 20% with respect to baseline, but all the remained patients in the prednisone group had an increase in body weight of over 20%.

Two fractures occurred in the deflazacort group. Bone formation and growth evaluated with X-ray of the left hand for bone age did not appear different in the two groups. Eye examination revealed a slight cataract in 3 patients in the deflazacort group and in 1 in the prednisone group. Other minor and slight side effects such as behavioural changes, increased appetite and cushingoid appearance were observed in both groups.

Different corticosteroids could have different effects on muscle and other tissues since it has been demonstrated that different glucocorticoids differ in their binding to the steroid receptor, in their affinity properties, and in their tissue specific metabolism and interaction with transcription factors.

In conclusion since the two types of steroids that we studied are similar in their clinical efficacy, other considerations are important when deciding which one should be used to treat a DMD boy.

Steroid treatment with deflazacort appears to cause less side effects than prednisone, particularly weight gain, which could be important to maximize motor performances and to avoid long term complications such as spinal deformity and respiratory fatigue. The slight increase in cataract in the deflazacort has to been evaluated in a long term follow up.

DYNAMIC CARDIOMYOPLASTY: PATIENT SELECTION CONSIDERATIONS AS APPLIED IN THE
C-SMART STUDY TO REDUCE HOSPITAL MORTALITY

RC Bourge, JB Young, JK Kirklin, F McGrew, C Lui, A Keogh, M Jessup, D Humen, AD Roettger on behalf of the C-SMART Investigators

The University of Alabama at Birmingham, Birmingham, Alabama, USA

Introduction: Dynamic cardiomyoplasty (DCMP) is a surgical approach to treat patients with heart failure. In this procedure, the latissimus dorsi muscle is wrapped around the failing ventricles. The muscle is then stimulated with a pacemaker therefore the procedure eventually (after muscle training) provides both constraint of cardiac dilatation and systolic augmentation.. DCMP has been extensively studied within the context clinical trials for several decades with some reported multicenter experiences demonstrating relative high mortality, especially in the peri-operative period. An analysis of the early experiences of non-controlled trials led to steps to reduce operative mortality in a subsequent controlled study, C-SMART.

Methods: Three FDA-approved phases of clinical study have been executed by Medtronic, Inc. A single center feasibility study was initiated in 1985 and enrolled 13 patients, 54% NYHA class IV. A multi-center Phase II trial began in 1991 and concluded enrolment in 1993. Sixty-eight patients were enrolled in Phase II with significantly fewer patients in NYHA class IV (3%). Finally, a large, Phase III randomized trial (C-SMART or Cardiomyoplasty-Skeletal Muscle Assist Randomized Trial) comparing cardiomyoplasty with standard medical therapy in NYHA class III patients was undertaken in September 1994. C-SMART was stopped in October 1998 due to slow patient recruitment after enrolling 103 patients.

Results: Hospital mortality, defined as death during initial hospitalization or 30 days post cardiomyoplasty surgery, has been reduced from 31% during Phase I to 3.9% in the Phase III C-SMART study. Several key analyses, utilizing worldwide data, shaped selection criteria for the C-SMART trial and quite possibly may have positively impacted patient outcome.

First: NYHA class IV patients had significantly higher hospital mortality than patients in class III. Three hundred sixty Phase I and II patients were enrolled worldwide by April 1994 with baseline NYHA class was III in 80% and class IV in 14% of patients. Hospital mortality was 6% and 36% of these patients, respectively. Several smaller series have also corroborated these results.

Second: There was a strong correlation between poor exercise performance, depressed LV function, and high pulmonary vascular resistance with the incidence of hospital mortality. A multiple logistic regression analysis performed on 166 patients in April 1994 revealed that patients with poor exercise performance measured by peak VO₂ continued to be the strongest factor associated with hospital mortality. Finally, patients with increasing number of coronary lesions stenosed to > 70% experienced higher hospital mortality. 261 patients enrolled from April 1991 to May 1995 were analyzed and the risk of early death following cardiomyoplasty surgery was >20% when 2-3 coronary beds had a ? 70% stenotic lesion.

Conclusions: Incorporation of patient selection criteria which excludes the end stage heart failure patient has lead to progress in reducing hospital mortality following cardiomyoplasty surgery. Experiences gained from over a decade of clinical research will only support newer technologies emerging in the area of skeletal muscle assistance for the treatment of chronic heart failure.

CARDIOMYOPLASTY:LONG-TERM RESULTS AND RECENT TECHNICAL INNOVATIONS

JC Chachques, E Braunberger, JP Marino, A Berrebi, N D’Attelis, D Bensasson, A Carpentier

Department of Cardiovascular Surgery, Broussais Hospital, Paris, France

The management of patients with end stage heart failure (HF) is a daily challenge in cardiac surgery. Cardiac translaptions and mechanical assist devices do not cover all the needs. Since 1985, cardiomyoplasty has been used in our institution for heart failure patient refractory to medical therapy, 112cases were operated at Broussais Hospital and 71 patients by our team abroad, in the scope of an international cooperative program. We report the longest follow-up of this technique and the improvements introduced in recent years.

Methods: Between 1985 and 2000, 112 patients aged 15 to 72 years (mean 51 years) were operated in Broussais Hospital; 86 were aged in NYHA class ??? and 26 in class ?V. Ejection fraction (EF) everaged 17%, EDLV volume 178± 31 ml/m2. The cause of HF was ischemia in 59 patients, dilated cardiomyopathy in 46 patients and ventricular tumors in 60%. The technique has envolved from "open fixation" (58 patients), to "non-suture wrapping" (41 patients), to "mini-invasive technique" (13 patients). Two-stage operation in high risk patients with mitral valve insufficiency or severe arrhythmia were performed in 6 patients. Associated procedures were necessary in 24 patients (CABG=14, valve=10).

Results: Hospital mortality was 53% between 1985-1987, 13% between 1988-1997, and 8% since the introduction of mini-invasive techniques. Actuarial survival at 10 years was 70% for preop and 1.4 postop. Ten patients required transplantation. Investigation in the survivors showed significant improvement in EF (17±4% to 28±6%) and cardiac index (1.9 to 2.8 L/mm/m2).

In conclusion: Cardiomyoplasty has been associated with better result due to technical improvements, the most significant being mini-invasive techniques, the latest the use of growth factors to enhance vascularisation. Risk factors have been identified resulting in more precise indications, a lower hospital mortality, and a wider use of this operation.

NOVEL FUNCTIONS (WORK-REST AND DAY-NIGHT REGIMENS) IN A NEW CARDIOMYOSTIMULATOR FOR CARDIAC BIOASSIST

VS Chekanov

Milwaukee Heart Institute, Heart Care Associates of Sinai Samaritan Medical Center, Milwaukee, Wisconsin, USA

The LD-PACE II, a new cardiomyostimulator was manufactured by CCC del Uruguay with the support of the Illini Group, Chicago, Illinois.

The LD-PACE II was designed for use in cardiomyoplasty, aortomyoplasty, and skeletal muscle ventricle. All parameters specified as programmable can be changed in a noninvasive manner (using a programming interface wand connected to a computer using the Windows 95/98 environment). Most of the functions of the new stimulator are similar to functions of previously used devices (Transform, Medtronic; EKS 445, Russia).

In the case of bradycardia or atrio-ventricular blockage, the LD-PACE II will act as a pacemaker with a basic pacing rate between 36 and 120 BPM. In order to prevent the stimulator from inappropriately sensing events, there is a ventricular refractory period (195-480 ms). Hysteresis (0-20%) allows the patient’s heart rate to temporarily fall below the lower rate without inducing immediate pacing. Synchronization delay (2-350 ms) obtains the optimal time of muscle contraction. Adaptive delay allows the automatic change in the delay time with a change of heart rate. The cardiosynchronization ratio is programmed from 1:1-1:16. Muscle output is inhibited if the heart rate is higher than the synchronization upper rate (120-226 BPM). The adaptive ratio allows for the automatic change of the cardiosynchronization ratio with an increase in heart rate. Delivery of a muscle pulse train is triggered by paced or sensed ventricular events. Characteristics of the pulse train are changes in pulse amplitude (0.44-0.75 V), pulse width (0.061-0.076 ms), pulses per burst (1-8), and pulse interval (15.6-132.8 ms). The adaptive pulse train duration will automatically decrease the train duration with an increase in heart rate and inhibit muscle contraction during the diastolic phase. The LD-PACE II does however have two new functions which prove to be extremely important for clinical use based on experimental research.

2. Day-night regimen. This feature is also brand new. It allows for a change in the ratio of muscle contractions according to a patient’s activity level. During the day the cardiosynchronization ratio may be set from 1:1 to 1:4 and during the night it may be set for 1:8 to 1:16. This allows the LDM to have a long rest period, prevents overuse, and prolongs battery life. Data confirmed that there is no depression in hemodynamics when the ratio is slowed or turned off during the night.

PHARMACOLOGICAL SUPPORT OF ANGIOGENESIS USING DEFEROXAMINE IN BIOLOGICAL GLUE FOR CARDIOMYOPLASTY

V. Chekanov, M. Maternowski, V. Nikolaychik

Milwaukee Heart Institute, Heart Care Associates, Milwaukee, Wisconsin, USA

Background. When using the latissimus dorsi muscle (LDM) for cardiomyoplasty we would like to have a muscle that is powerful enough to augment cardiac contractions and viable enough to provide indirect myocardial revascularization. Iron, which is released during LDM mobilization, is cytotoxic to the vascular endothelium.

Ischemia also leads to microcirculatory impairment caused, in part, by the generation of reactive free radicals. Strong metal chelators like deferoxamine can counteract the iron-catalyzed formation of these deleterious substances. Deferoxamine has protected human endothelial cells in vitro from reoxygenation injury and may activate an angiogenic response.

Aims. The aims of this study were to investigate the effect of deferoxamine (an iron chelator) on fibroblast, smooth muscle cells and endothelial cells in vitro, and its use in accelerating indirect myocardial revascularization.

Methods. Series 1. In vitro three different concentrations of deferoxamine (DEF; 10 mM, 100 mM, and 1 mM) were added to fibroblasts, smooth muscle cells, and endothelial cells. Expression of bFGF and VEGF was investigated.

Series 2. The latissimus dorsi muscle in adult sheep was mobilized and left in situ. Autologous biological glue (ABG) with added deferoxamine was applied to the muscle. The percent area occupied by capillaries was investigated 56 days later.

Series 3. In adult sheep an ameroid constrictor was placed on a coronary artery in order to create a coronary stenosis. Blood flow was measured (using colored microspheres) prior to placement of the constrictor. Cardiomyoplasty (CMP) was subsequently performed; ABG with added deferoxamine was placed between the LDM and the myocardium. A second blood flow measurement was taken after a two month electrical stimulation protocol.

Results

Table 1. Expression of bFGF (activity in%).

Fibroblasts Smooth Muscle Cells Endothelial Cells

Control 100 100 100

10 µM DEF 386 201 152

100 µM DEF 782 341 182

1 mM DEF 1054 532 213

Table 2. Expression of VEGF (activity in%).

Fibroblasts Smooth Muscle Cells Endothelial Cells

Control 100 100 100

10 µM DEF 185 219 115

100 µM DEF 368 326 105

1 mM DEF 651 589 98

Table 3. Percent area of LDM occupied by capillaries (56 days after mobilization).

Distal (%) Middle (%) Proximal (%)

Control 3.04±0.46 3.36±0.25 4.15±0.15

ABG only 5.17±0.29 5.40±0.81 5.93±0.37

ABG+DEF 7.93±0.80 9.03±0.81 9.76±1.03

Table 4. Myocardial blood flow (ml/min/gm).

Nonischemic myocardium before CMP 0.594±0.067

Nonischemic myocardium after CMP, no DEF 0.651±0.102

Nonischemic myocardium after CMP + DEF 0.814±0.141

Ischemic myocardium before CMP 0.083±0.019

Ischemic myocardium after CMP, no DEF 0.151±0.036

Ischemic myocardium after CMP + DEF 0.369±0.042

In conclusion:

1. Deferoxamine triggered an angiogenic response in the in vitro fibroblasts and smooth muscle cells by releasing at least two growth factors: bFGF and VEGF. These two growth factors when present in vivo will recruit the more quiescent endothelial cells in the LDM to amplify and create new capillaries.

2. A combination of ABG and added iron chelator (DEF) prevents further ischemia of the mobilized LDM, increases LDM angiogenic potential, and increases myocardial revascularization. It may be useful in patients with ischemic cardiomyopathy in order to promote indirect myocardial revascularization.

A NEW ALTERNATIVE FOR THE USE OF ELECTRICAL STIMULATION – ATHEROSCLEROSIS PREVENTION

V. Chekanov, M. Maternowski, G. Tchekanov, M. Mortada, R. Eisenstein

Milwaukee Heart Institute, Heart Care Associates, Milwaukee, Wisconsin, USA

Background. A study of the long-term results of patients with ischemic cardiomyopathy who underwent cardiomyoplasty and patients with atrio-ventricular block who underwent pacemaker implantation led us to find the answers to the following questions. Although intended solely for latissimus dorsi training and contraction, does the cardiomyostimulator also protect cardiomyoplasty patients against aggravation of atherosclerosis in the coronary arteries? Although intended to safeguard patients with AV block against fatal arrhythmias, does the pacemaker also protect them against development of atherosclerosis in the coronary arteries?

Aim. To investigate the influence of low-frequency electrical impulses on prevention and development of atherosclerosis.

Methods. Control study. Eighteen rabbits were placed on a high cholesterol diet (HCD) to induce atherosclerosis. No electrical stimulation was applied to the control animals. The morphology of the coronary arteries, thoracic and abdominal aorta was analyzed in three groups of animals at three, eight and eleven weeks respectively after beginning HCD. Atherosclerosis was assessed histologically using a 0-4 grading scale.

Experimental study. In rabbits, an electrode was sutured to the left psoas major muscle close to the abdominal aorta. A pacemaker was implanted in a muscle pocket on the right side near the right psoas major muscle. Immediately after implantation, stimulation was started at 10 Hz single pulses with varying impulse rates, voltages and at different times corresponding to the beginning of HCD. Atherosclerosis was assessed histologically using a 0-4 grading scale.

Results

Table 1. Influence of HCD on development of atherosclerosis.

	Thoracic Aorta – top	Thoracic Aorta – bottom	Abdominal Aorta – top	Abdominal Aorta - bottom
3 wk HCD	3.0	1.0	0.7	1.0
8 wk HCD	3.2	1.4	1.5	1.2
11 wk HCD	3.8	3.0	3.0	3.3

Table 2. Effect of 8 weeks of electrical stimulation begun after 3 weeks HCD.

Thoracic Aorta – top Thoracic Aorta – bottom Abdominal Aorta – top Abdominal Aorta – bottom

Control
(11 wk HCD) 3.8 3.0 3.0 3.3

30 CPM,
2 V, HCD 3.6 2.4 2.5 2.6

30 CPM,
3 V, HCD 3.4 1.0 0.6 0.5

30 CPM,
4 V, HCD 3.5 1.0 0.6 0.5

60 CPM,
3 V, HCD 3.5 0.7 0.5 0.6

120 CPM, 3 V, HCD 3.1 2.2 2.7 2.1

Table 3. Effect of 8 weeks of electrical stimulation (30 CPM, 3 V, normal diet) begun after 8 weeks HCD (control had 8 weeks HCD followed 8 weeks normal diet).

Thoracic Aorta – top Thoracic Aorta – bottom Abdominal Aorta – top Abdominal Aorta –
bottom

Control 2.7 1.2 1.2 1.0

Experimental 2.7 0.5 0.4 0.3

Table 4. Effect of 11 weeks of electrical stimulation (30 CPM, 3 V, 24 or 8 hours daily) begun together with HCD.

Thoracic Aorta – top Thoracic Aorta – bottom Abdominal Aorta – top Abdominal Aorta – bottom

Control
(11 wk HCD) 3.0 2.7 2.7 1.7

24 hr ES 1.7 1.2 1.2 0.5

8 hr ES 3.0 2.0 2.0 1.5

In conclusion, with or without electrical stimulation, all animals fed HCD were found to have atheroma on the top of the thoracic aorta (2-4 on a scale of 4). Intramyocardial vessels were found to have similar evidence of advanced atherosclerosis. Electrical stimulation applied near the abdominal aorta did not change the atherosclerosis level in the heart or aortic root-both of which were beyond the field of the electrical stimulation. The abdominal aorta subjected to electrical stimulation had levels of atherosclerosis that were intriguingly different from those of the control animals, especially when 30 CPM and 3 or 4 V were used. Data showed that electrical stimulation, when applied near the abdomina aorta that had initial signs of atherosclerosis, might prevent and even decrease fatty deposits despite continuation of HCD. Continuous 24 hour electrical stimulation has a better effect than 8 hour daily stimulation. Our data also showed that in case of moderate levels of atherosclerotic build-up, a normal diet can result in lesion regression and this is significantly enhanced by electrical stimulation.

SURFACE EMG TO STUDY MUSCLE FUNCTION IN ASYMPTOMATIC DIABETIC NEUROPATHY

Chisari C.¹, Piaggesi A.², Baccetti F.², Rizzo L.², Giannini E.¹, Simonella C.¹, Rossi B.¹

(1) Neurorehabilitation, Dept. of Neuroscience, University of Pisa and (2) Chair of Metabolic Diseases, Dept. of Endocrinology and Metabolism, University of Pisa

Objective. To test if the quantitative analysis of myoelectric fatigue, through surface EMG, can reveal modification of muscle characteristic in the earlier stages of diabetic polyneuropathy (PN).

Patients and methods. We evaluated 24 non insulin-dependent diabetic patients (NIDDP), in stable metabolic control and asymptomatic as regard neuropathy. The patients were subdivided in two group: 12 with diabetic neuropathy (ND+), as ascertained by bilateral compromission of nerve conduction velocity of both peroneal and sural nerves, 12 without diabetic neuropathy (ND-). In all subjects we studied functional properties of muscle fibers through an electrically elicited contraction of the tibialis anterior (TA). The motor point stimulation protocol consisted of pulse train (each stimulus 0.1 msec in width) lasting 43 seconds delivered at 35 Hz. We recorded the surface electromyografic (EMG) signal and analysed the median frequency of the power spectrum (MDF), the muscle fibers conduction velocity (CV) and the average rectified value (ARV).

Results. At the beginning of stimulation MDF (71.8±22.4 Hz vs 119.8±27.9 Hz vs 124.6±32.8 Hz; ANOVA p<0.05), CV (4.07±0.51 m/s vs 5.11±0.48 m/s vs 5.39±0.52 m/s; ANOVA <0.05) and ARV (355.1±151.3 mV vs 606.1±230.5 mV vs 634.6±221.7 mV; ANOVA p<0.01) were all significantly decreased in ND+ patients compared both to ND- and controls respectively. After 23" and 43" from the beginning of stimulation a more important percentual decrease in CV was observed in ND+ compared to ND- and Controls (-26.29% and -48.9% in ND+, -9.1% and 27.1% in ND-, -11.88 and –29.68% in C; ANOVA p<0.01). No significant differences emerged for MDF and ARV either after 23" and 43".

Discussion. Data obtained in this study showed significantly lower MDF, CV and ARV basal value in ND+ compared with ND- and Controls. Accordingly with Hakanson (1956) this indicates that the mean size of tibialis anterior fibers is reduced in ND+. Furthermore the percentual decrement of CV, during the sustained contraction, is significantly greater in ND+ respect to ND- and Controls. This decrement, which is related to modifications in muscle membrane excitability and represents the direct effect of the electrical fatigue phenomenon (Merletti and De Luca, 1989), suggests a modified myofibral composition in TA of ND+ patients: an increased percentage of type 2 fibers.

In conclusion we consider remarkable to dispose of a no invasive tool to evaluate the muscle function also in asymptomatic NIDDP. It seems very important to detect an early functional muscle impairment to prevent the severe and compromising complications at lower limbs. In fact, in this way, it is possible to design early specific rehabilitative program.

PROPOSAL OF VALUTATIVE AND REHABILITATIVE PROTOCOL IN FACIOSCAPULOHUMERAL DYSTROPHY

C Chisari C, E Giannini, F Mussini, C Simonella, M Bresci, B Rossi

Neurorehabilitation Unit, Dept. of Neuroscience, University of Pisa

Background and Objective. The role of exercise in the management of dystrophy has been controversial. Surely passive exercise and stretching results very important to reduce joint contractures, to delay scoliosis and to control body weight. But the active exercise is of unknown value.

In this study we evaluated the efficacy of a period of physiotherapic treatment comprehensive of active exercise and respiratory resistive training in 5 subjects affected by Facioscapulohumeral Dystrophy (FSH).

Methods. We followed 7 FSH patients during a period of 6 month. 5 of them were submitted to a rehabilitative programme and 2, untreated, served as controls. All groups performed a series of functional tests at the beginning and at the end of this period:

1) exercising test on a treadmill with assay of CPK at rest and 1, 3, 6, and 24 hours after the exercise (to test muscle damage susceptibility);

2) MRC scale (testing 24 muscles);

3) Respiratory function measurements, maximal inspiratory and espiratory mouth pressures (MIP-MEP), blood gas analysis;

4) Motor Functional Scale (Vignos).

For 3 patients rehabilitative protocol consisted of passive and active physiokinesiterapy and a walking on treadmill on an individual basis; 2 patients performed the same programme associated with a specific training for respiratory muscles. Each exercise session lasted for 1h or 1.5 h, carried out three times weekly. The patients in the control group did not undergo any training and continued their previous way of life.

Results. All patients treated referred they felt better as regard the activity daily living; objectively we registered improvement of muscle performance: walking for more time and at higher speed, with a better cardiovascular compliance. Respiratory function parameters showed improvement in all treated patients: mild in those patients submitted to the only motor training and greater in the 2 patients performing the specific inspiratory muscle training.

In conclusion, this study showed that the rehabilitative treatment, comprehensive of active exercise, walking and respiratory training is useful in the long-term management of subjects affected by FSH. Of course it results necessary to design individual rehabilitative programme on the basis of objective motor function, muscle damage susceptibility and respiratory capacity evaluations.

Impaired muscle oxidative metabolism in polymyositis and dermatomyositis evaluated in vivo
through lactic acidaemia assay

C Chisari¹, G Stampacchia¹, E Giannini¹, R Neri², M Mosca², B Rossi¹

¹Neurorehabilitation Unit, Dept. of Neuroscience, University of Pisa and ²Reumatology Unit, Dept of Medicine, University of Pisa

Objective. The aim of the present study was to investigate the efficiency of muscle oxidative metabolism in vivo in patients affected by polymyositis (PM) and dermatomyositis (DM).

Method. A group of 14 PM/DM patients and 15 healthy subjects, age and gender matched, performed a submaximal incremental exercise on a treadmill. The exercise consisted of: 11 steps, each of 2’ of duration, at a constant speed (3 Km/h) with a grade that was 0° at the beginning and increasing of 2.5% at each step. The heart rate was monitored during the exercise and the subjects were stopped if they reached the 80% of the maximum heart rate theoretically calculated or if they referred excessive fatigue. Lactic acidaemia was assessed through blood samples obtained at rest and after 1’, 5’, 10’, and 30’ from the end of exercise.

Results. Lactic acidaemia doesn’t show significative difference as regard the basal value. It was significantly different for all recovery period.

The mean number of steps performed was 6.1 vs 11 in patients respect to controls respectively.

Lactate	at rest	1’	5’	10’	30’
Patients	2.15	5.18	4.61	4.24	2.74
Controls	1.63	2.13	1.91	1.64	1.31

Discussion. The data obtained in this study show a greater production of lactate after a submaximal exercise with a slower recovery in subjects affected by idiopathic inflammatory myopathy respect to controls. This reveals a defective utilization of energy in skeletal muscle of these patients and confirms previous works showing impaired oxidative metabolism evaluated by means of phosphorus magnetic resonance spectroscopy in PM and DM. Moreover some authors described a reduced muscle mitochondrial function in these diseases. The symptom principally leaked to abnormal muscle oxidative function is abnormal fatiguability. In this way is reasonable to think that the weakness referred by subjects with inflammatory myopathy has a metabolic genesis too.

In conclusion it seems us very important, in the management of PM and DM patients, to dispose of a simple, no expensive and reproducible test to assess in vivo the muscle oxidative efficiency in inflammatory myopathy.

This can be a useful tool in the screening of those patients with excessive fatiguability that could profit by aerobic rehabilitative program.

DISUSE INDUCED-ATROPHY AND CONTRACTILE IMPAIRMENT OF HUMAN SKELETAL
MUSCLE FIBRES

G D’Antona, M Antonietta Pellegrino, R Rossi, C Naccari Carlizzi, C Reggiani, R Bottinelli

Institute of Human Physiology, University of Pavia, Italy, Hospital "Casa di Cura Columbus", Milano, Italy and Department of Anatomy and Physiology, University of Padova, Italy

In this study we described the effect of four months of disuse and immobilisation on Myosin Heavy Chain (MHC) isoform composition and contractile properties of single fibres in human muscles. We compared muscle samples collected from the vastus lateralis of three groups of two subjects: 1) subjects (60-70 y old) who underwent to the replacement of a total knee prosthesis after four months with the leg immobilised in extended position, 2) control adult subjects (30-40 y old) sampled by needle biopsy and 3) control aged subjects (60-70 y old) who underwent to knee surgery for various reasons. We found in immobilised muscles an increase in MHC-2X , a decrease of slow MHC and the expression of a developmental MHC isoform, indication of fibre regeneration. The proportion of hybrid fibres, i.e. fibres containing two or more MHC isoforms, was increased. Slow fibres showed significant reductions in cross sectional area and in specific tension (force/cross sectional area) developed during maximal isometric contractions. Unloaded shortening velocity was, however, not modified. No alteration was detectable in fast 2A fibres when compared with corresponding fibres of adult and old controls. Slow fibres seemed, therefore, selectively affected by the combination of immobilisation and disuse examined in this study.

Correlative ³¹P-MRS - M wave analysis
of high and low frequency electrically-induced fatigue
in rabbit tibialis anterior muscle

J-L Darques, D Bendahan(1), M Roussel, Y Le Fur, S Confort-Gouny, F Tagliarini, PJ Cozzone, Y Jammes

Laboratoire de Physiopathologie Respiratoire (UPRES EA 2201), Faculté de Médecine and (1) Centre de résonance magnétique biologique et médicale, UMR CNRS 6612, Univ. Méditerranée, Marseille.

We have already demonstrated that electrically-induced muscle fatigue at a high frequency (HFF, 100 Hz) produced a higher reduction in force and M wave amplitude than low frequency fatigue (LFF, 10 Hz). On the other hand, LFF elicited a greater activation of group IV muscle afferents than HFF, suggesting a greater release of muscle metabolites during and after LFF. The present study compared the consequences of 5-min LFF and 3-min HFF trials in the tibialis anterior of anesthetized, paralyzed rabbits, on the M wave and two indices of muscle metabolism (?pH, PCr consumption) explored with ³¹P- nuclear magnetic resonance spectroscopy. After LFF, there was a significant PCr consumption (-27%) and intracellular acidosis (-0.32 pH unit), with no significant changes in M wave amplitude. By contrast, after HFF the M wave amplitude was significantly reduced (-54%) whereas there were no variations in PCr and pH. We conclude that the failure of force during HFF is only associated with an impaired propagation of muscle potentials, whereas LFF is mostly associated with metabolic variations like those measured after muscle fatigue. Thus, the LFF model may be an interesting tool to mimic fatigue.

This work is supported by CNRS, AFM (Association Française contre les Myopathies) and PHRC (Programme Hospitalier de Recherche Clinique).

SOLE PLANTAR STIMULATION: A COUNTERMEASURE FOR RAT SOLEUS ATROPHY OBSERVED
DURING UNLOADING.

L De Doncker, F Picquet, M Falempin

Laboratoire de Plasticité Neuromusculaire - SN4 - UST Lille 1, F - 59655 Villeneuve d’Ascq Cedex

The purpose of this study was to investigate in the rat whether the effects of cutaneous mechanoreceptors stimulation of plantar sole could prevent totally or partially the soleus atrophy which occurred during hindlimb unloading (HU). Eighteen male Wistar rats were divided randomly into three groups: control (n = 6), hindlimb unloaded (HU, n = 6) and hindlimb submitted to daily intermittent sole plantar stimulation (HU + PS, n = 6). HU and HU + PS were subjected to a unloading period of 14 days. Tactile stimulation was achieved by a latex balloon filled with air on the two plantar soles of the animals once a day during 0.23% of the 14-day unloading period. Mechanical, histological and electrophoretical properties of the soleus muscle were analysed in the three groups. Results were analysed using a one-way analysis of variance between the three experimental groups. In agreement with other studies, 14 days of unloading induced significant alterations in the soleus muscles.

Theses changes included 1) decrease in muscle wet weight (43%) and fiber atrophy (-40 to -50% of the fibers cross sectional area), 2) decrease in the maximal strength developed during twitch (- 64%) and tetanus (- 80%), 3) decreases in the isometric contraction time parameters (-37% for the time to peak tension; -23% for the half relaxation time), 4) a shift towards a significant higher percentage of fast type IIA (+ 93%) fibers and 5) changes in the myosin heavy chain isoforms when compared with control soleus.

Plantar sole stimulation applied during HU significantly attenuated but did not prevent the muscle wet weight (% atrophy prevented: 53%), the decrease in the twitch tension (% atrophy prevented 31%) and in the maximal tetanus tension (% atrophy prevended: 25%). However changes in the kinetic parameters during the twitch, in the histological and electrophoretical parameters associated with HU were not counteracted.

These results supported the hypothesis that the reflex contraction of the soleus muscle, induced by the stimulation of the sole plantar mechanoreceptors constitute a efficient form of exercise training as a countermeasure which could be associated with others (stretching, tendon vibration) to soleus atrophy observed after a period of unloading.

This work was supported by grants from the Centre National d’Etudes Spatiales, the Nord-Pas de Calais régional council and the Fond Européen de Développement Régional (FEDER, n° F007).

TELOMERASE ACTIVITY IN MUSCLE OF NORMAL AND MDX MICE AND IN MUSCLE REGENERATION

I Dell’Aica, M Sandri, K Rossini, C Destro, U Carraro

C.N.R. Unit for Muscle Biology and Physiopathology, Department of Biomedical Sciences and University of Padova, viale Colombo 3, 35031 Padova, Italy

Duchenne muscular dystrophy (DMD) is a chronic, X-linked recessive disorder which causes progressive muscle wasting and weakness leading to an early death. Three progressive characteristics accompany the atrophy and coincident clinical weakness: active proliferation of endomysial connective tissue (fibrosis), muscle fiber atrophy and failure of myofiber regeneration. A factor that adversely affects new fiber formation is the reduced replicative capacity of satellite cells.

To verify if the reduced telomerase activity and consequent chromosomal telomeric shortening could affect the proliferative capacity of satellite cells in mice, we measured telomerase activity in neonatal and adult muscle of normal and mdx mice. Moreover we analysed telomerase activity in muscles which underwent repeated cycles of injury/regeneration by local infiltration of myotoxic agent (bupivacaine). The treatment was repeated one, two and three times and the muscle were analysed after 3 days from the last injection. In this case we also detected the presence of proliferating myoblasts using the polyclonal antibody against MyoD, a transcription factor expressed in proliferating myoblasts.

We detected telomerase activity by Telomerase PCR ELISA, this method allows highly specific amplification of telomerase-mediated elongation products combined with nonradioactive detection following a ELISA protocol. In the first step telomerase adds telomeric repeats (TTAGGG) to the 3’ end of the biotin-labeled synthetic P1-TS primer. In a second step these elongation products are amplified by PCR using a second primer. The PCR products can be separated by polyacrylamide gel electrophoresis, blotted onto a positively-charged membrane and visualized by chemiluminescence. At the end an aliquot of PCR products is detected by ELISA protocol.

In neonatal normal and mdx muscle the telomerase activity is elevated while in adult normal and mdx muscle the activity is absent, there is no significant difference between normal and mdx group. In three days regenerating muscle telomerase activity is measurable but lower than in neonatal muscle, after 2 and 3 bupivacaine treatments the level of telomerase activity is comparable with that of adult normal skeletal muscle, even in muscle with high levels of proliferating myoblasts (MyoD positive cells). These results suggest that repeated cycles of regeneration could cause decrease of telomerase and consequent chromosomal telomeric shortening in proliferating myoblasts.

Fas/FasL SYSTEM REGULATES APOPTOSIS OF MACROPHAGES AND MYOBLASTS DURING MUSCLE REGENERATION

C Destro, M Sandri, K Rossini, C Sandri, I Dell’Aica, M Cantini, U Carraro.

C.N.R. Unit for Muscle Biology and Physiopathology, Department of Biomedical Sciences, University of Padova, viale Colombo 3, 35121 Padova, Italy

Acute muscle injuries result in inflammation of the damaged site. Muscle recovery requires an effective interaction between inflammatory and muscle cells and its deficiency leads to a persistent immune-response which could be critical for muscle fiber viability. We observed an evident relation between the peak of FasL (APO1L, CD95L) expression in proliferating myoblasts and the peak of apoptosis of macrophages during regeneration following a damage (induced by local infiltration of myotoxic agent). This observation suggests that apoptosis of macrophages during resolution of muscle inflammation which follows muscle damage is a Fas-mediated process induced by FasL expression in proliferating myoblasts. We tested this hypothesis treating injured muscles of normal mice from day 3 to day 8 with a FasL inhibitor, that is the recombinant protein Fas-Ig. Treated muscles were collected at 3, 5 and 10 days from the damage. Immunohistochemical analyses on treated muscles showed that inflammation persists until day 10. The persistence of inflammatory cells highly impaired myofiber regeneration. Apoptosis of macrophages was absent during Fas-Ig treatment but it was observed at day 10, that is two days after the suspension of the treatment with the Fas-Ig inhibitor. These results clearly show direct macrophage/myoblast interaction during muscle regeneration. In macrophages-myoblasts cocultures (which mimic the in vivo situation of muscle regeneration after a damage) we observed a major number of apoptotic phagocytic cells than in cultures of macrophages alone.

Besides playing an important role in regulating macrophage/ myoblast interaction, Fas/FasL system also seems to be implicated in programmed cell death of myoblasts themselves. During myogenesis not all myoblasts fuse to generate adult myofibers: a possible mechanism of selection could be associated with apoptotic Fas/FasL cascade.

RNasi protection analyses demonstrate that FasL gene and other genes correlated to Fas/FasL cascade (such as FADD, TRAIL, RIP) are up-regulated in cultures of myoblasts where apoptosis is induced by growth factors withdrawal.

Moreover, overexpression of FasL in myoblasts, both in vivo and in vitro, causes their rapid apoptotic death. We therefore conclude that macrophage/ myoblast regulative mechanism is very delicate, since FasL induces macrophages death but its altered expression induces myoblasts death.

VASCULAR DELAY OF LD IN AN EXPERIMENTAL RAT MODEL FOR DYNAMIC CARDIOMYOPLASTY

E Giardini, F Mazzoleni, K Rossini (1), A El Messlemani (1), A Donà (1), U Carraro (1)

Institute of Plastic Surgery, and (1) C.N.R. Unit for Muscle Biology and Physiopathology, Department of Biomedical Sciences, University of Padova, Italy

"Vascular delay" is a surgical technique that renders a skin flap sub-lethally ischemic and produces significant alterations in the characteristics of tissue perfusion. The enhanced perfusion is associated with decreased distal flap necrosis and increased viability. The staging of vascular delay involves a surgical reduction of the vascular supply to tissue, usually by undermining the flap and dividing the perforating vessels. Despite the well-documented effectiveness of vascular delay in skin flaps, vascular delay in muscle flaps has not been widely reported.

The aim of our study was to investigate the effectiveness of vascular delay of a LD delay in an experimental rat model of Dynamic Cardiomyoplastic. We hypothized that muscle function would be improved by a vascular delay procedure that increases distal muscle perfusion of the LD muscle.

In the first group of 6 rats the right LD were subjected to a "vascular delay" procedure, the left LD were tenotomized and resutured in the shortened position it spontaneously attained to mimic transposition effects of cardiomyoplasty, which result in LD distal devascularization and decreased resting tension. After 7 days the LD muscle flaps were studied. In a second experimental group, the right LD muscles of 6 rats were subjected to a vascular delay procedure with LD tenotomized and resutured in the shortened position. One week later both right (vascular delay) and left (control) LD muscles were subjected to a tenotomy procedure. Seven days later the LD flaps were studied.

In both group the LD muscles were excised and fixed in liquid nitrogen at resting length. Muscle damage was graded by histological morphometry (H-E) on cryostat sections.

Muscle damage in vascular delay LD was 21%±12 (SE), in tenotomized LD was 79%±10 (SE), and in muscle tenotomized after vascular delay was 42%+7 (SE).

In conclusion we found that "vascular delay" procedure of LD tenotomized and resutured in the shortened position, that mimics the Dinamic Cardiomyoplastic significantly reduces muscle damage and improves LD muscle flap perfusion and function particularly in the distal portion of muscle.

CELL-SURFACE LOCALIZATION OF THE GLUCOSE-REGULATED PROTEIN GRP94 IN SKELETAL MYOBLASTS IS INVOLVED IN MYOTUBE FORMATION

L Gorza and M Vitadello

CNR-Unit for Muscle Biology and Physiopathology and Department of Biomedical Sciences, University of Padova, Italy

A previous work of ours showed that the glucose-regulated protein GRP94, which belongs to a group of endoplasmic reticulum-resident proteins characterized by chaperonin activity and calcium binding ability and upregulated in response to diverse stress types, shows differences in both its constitutive and induced expression in rabbit skeletal muscle myocytes. We found that GRP94 mRNA and protein are abundant in cardiac and skeletal muscle myocytes of the fetal and newborn rabbits, whereas they cannot be observed in adult skeletal muscle myofibers even after exposure to bacterial lipopolysaccharide, at variance with adult cardiomyocytes (Vitadello et al. Biochem J 332,351, 1998).

The lack of responsiveness displayed by the grp94 gene in adult skeletal muscle myofibers suggested us that GRP94 accumulation in developing skeletal muscle may be devoted to specific events occurring during muscle differentiation. Thus, the aim of present work was to enucleate the role played by this endoplasmic reticulum protein in skeletal muscle maturation by using stable transformants of the muscle cell line C2C12 under- and over-expressing the protein. Our results show that a decrease in GRP94 amount greater than 40% is followed by loss of myoblast fusion competence either in vitro and after in vivo cell transplantation. The absence of fusion was not accompanied by lack of myoblast differentiation, since the quantification of BrdU incorporation in differentiative medium and the percentage of cells displaying nuclear p21 translocation among grp94 antisense clones indicated ability to exit from the proliferative cycle and Western blot analysis showed upregulation of muscle-specific gene expression. Conversely, accelerated myotube formation was observed in GRP94 overexpressing clones. Thus, these results indicate that GRP94 expression in developing myocytes is necessary for complete maturation.

The mechanism through which GRP94 is involved in myoblast fusion was then enlighted by another important finding we obtained, namely the evidence of GRP94 localization at the cell surface, in addition to its presence in the endoplasmic reticulum. Such a localization was detected in C2C12 cells and in mouse neonatal skeletal muscle cells, but not in fibroblasts, derived from primary cultures by mean of immunofluorescence studies and Western blot analyses of cell-surface biotinylated proteins. Comparable assays performed on grp94 antisense clones revealed about 40-fold reduced expression of cell surface GRP94, suggesting that dramatic changes in GRP94 amount at the level of sarcolemma may be responsible for the absence of muscle cell fusion. The possibility that GRP94 itself is involved in the fusion process was explored incubating untransfected C2C12 cells in the differentiation medium in the presence of purified monoclonal anti-GRP94 antibody: at variance with parallel cultures incubated with mouse non-immune immunoglobulins, the growth in the presence of anti-GRP94 antibody significantly reduced myotube formation. Thus, a multifunctional endoplasmic-reticulum protein such as GRP94, when exposed on the muscle cell surface, participates directly to myoblast fusion.

Up to now the presence of GRP94 on the cell-surface has been demonstrated only in neoplastic cells, where GRP94-peptide complexes induce specific cytotoxic T cell response after internalization in dendritic cells (Tamura et al. Science 278:117, 1997; Nicchitta, Curr.Opin. Immunol. 10:103, 1998). Although the nature of the interaction which involves cell-surface GRP94 and promotes fusion of skeletal muscle cells remains to be determined, a major role for environmental cues, such as the presence of extracellular matrix components or the effects of proteases like calpain, ?-meltrin and catepsin B (Yagami-Hiromas et al. Nature, 377: 652, 1995; Gogos et al. J Cell Biol 134:837, 1996; Dourdin et al. Exp. Cell Res 235: 385, 1997) is ruled out by the persistence of fusion impairment shown by grp94 antisense clones transplanted in living muscles. Furthermore, the effects due to reduced cell-surface GRP94 expression can not be explained with reduced externalization of N-cadherin, since in vitro knock-out of N-cadherin and chimeric mice with null-?1 integrin muscle cells have proven to not perturb muscle cell fusion (Charlton et al. J Cell Biol 138: 331, 1997; Fassler et al. Genes Dev. 9:1896, 1995).

A previously considered ubiquitous- and constitutively-expressed protein plays novel and unique roles: not only the chaperonin function of GRP94 is not relieved by other chaperonins of the endoplasmic reticulum, but, far more important, its surface localization appears to be required for and directly involved in the cell fusion process.

MITOCHONDRIA: THE IGNITION CHAMBER FOR APOPTOSIS

RA Gottlieb

Department of Molecular & Experimental Medicine, Division of Hematology MEM220, The Scripps Research Institute, La Jolla, CA, USA

Apoptosis occurs in a wide variety of tissues in response to both physiologic triggers and pathologic insults. Despite the wide range of inducing signals, there are some common features of the cell death program which have been the subject of intensive investigation and which are now becoming better understood.

Cellular stresses perturb the cytosolic sequestration of pro-apoptotic Bcl-2 family members such as Bax, Bad, or Bid. Dephosphorylation of Bad (which releases it from 14-3-3 protein) or proteolytic cleavage of Bid by caspase-8 results in their translocation to the mitochondria. Cytochrome c is made available to join a complex now termed the "apoptosome" or "death-inducing signal complex" (DISC) consisting of Apaf-1, caspase 9, and the cytochrome. Upon binding cytochrome c and dATP or millimolar concentrations of ATP, Apaf-1 recruits caspase 9 through homologous caspase activation and recruitment domains (CARD), culminating in the self-processing of caspase 9 (8,9). Once activated, caspase 9 cleaves its downstream target, procaspase 3, which then serves as an effector caspase to accomplish limited proteolysis of a variety of cellular targets.

The mechanism by which Bax causes cytochrome c release has been the subject of intense investigation, but is still incompletely understood. Bcl-2 family members have a tertiary structure which resembles the bacterial pore-forming colicins suggesting that they could insert into membranes and form pores, a property which has since been demonstrated in planar lipid bilayers. Some have suggested these pores could grow large enough to permit proteins (such as cytochrome c) to pass through. Recombinant Bax has been shown to destabilize lipid bilayer, while Bcl-2 and Bcl-x_L did not. Bax has been demonstrated to interact with the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane and the adenine nucleotide transporter, an inner membrane component of the permeability transition pore. Although these findings provide a means for cytochrome c to diffuse from the intermembrane space into the cytosol, they fail to explain how cytochrome c first dissociates from the electron transport complexes of the inner membrane, where it is normally retained by electrostatic interactions. Both the dissociation of cytochrome c and its egress from the mitochondria are stimulated by Bid and opposed by Bcl-2. The process of cytochrome c release comprises at least two steps: 1) cytochrome c diffuses away from the electron transfer complexes, and 2) crosses the outer mitochondrial membrane, which may not break down until later during apoptosis. Indeed, if we measure outer mitochondrial membrane integrity and cytochrome c release into cytosol, then we can eventually detect cytochrome c release, at about 4 hours, a time when Fas-mediated apoptosis is nearly complete in these Jurkat cells. Thus, cytochrome c dissociation occurs early, and can be detected by cessation of oxygen consumption, while its release into the cytosol occurs late, unless the cell disruption method employed also happens to disrupt the mitochondrial outer membrane.

In addition to cytochrome c, the mitochondrial intermembrane space also sequesters several procaspases and Apoptosis Inducing Factor (AIF). Because cytochrome c dissociates from the electron transfer complex long before it is released into the cytosol, we considered the possibility that a caspase activation complex could form within the mitochondrial intermembrane space. In contrast to Fas ligation, when apoptosis is induced by a chemical trigger—which involves the participation of the mitochondria—caspase activation can be detected first in the mitochondria. In contrast, Fas ligation, which can bypass the mitochondria, results in caspase activation in the cytosol before the mitochondria. It appears that the two pathways may result in processing and activation of pools of caspases that are spatially separated. The significance of mitochondrial caspase activation and the role of Apaf-1 in that process remain to be determined.

Mitochondria may play an additional role in regulating caspase activation, even in the setting of Fas-mediated apoptosis which can lead directly to cytosolic caspase activation. This suggestion is based on recent reports that carnitine can suppress apoptosis. We have shown that carnitine is able to directly inhibit caspases. In contrast, the long-chain acyl derivative, palmitoylcarnitine, has the opposite effect to increase caspase activity. Palmitoylcarnitine is able to reverse the inhibition of caspases mediated by carnitine. This modulation of caspase activity by carnitine and palmitoylcarnitine is tied directly to the mitochondria, because the key enzyme that regulates the formation of palmitoylcarnitine from carnitine and palmitoyl-CoA, carnitine palmitoyl transferase I (CPT I), is located in the mitochondrial outer membrane.

Mitochondria play a central role in the regulation of apoptosis. They are the source of cytochrome c, which serves as a critical cofactor for caspase activation. Through regulation of carnitine and palmitoylcarnitine, they may also fine-tune the activity of caspases. The significance of mitochondrial caspases remains to be determined. The mitochondria play additional critical roles that determine cell viability, to provide the majority of ATP in organs such as the heart and brain, to sequester calcium, preventing it from reaching dangerous levels within the cell. As the center of most of the oxidative activity of the cell, mitochondria can also serve as a source of dangerous free radicals, although whether this continues or possibly increases after cytochrome c dissociation is controversial. A variety of signal transduction pathways involved in apoptosis converge upon the mitochondria, suggesting that this organelle may integrate a variety of signals, including protein kinases, cytoskeletal disturbances, and intracellular pH, to reach a life-or-death decision.

This work was supported in part by NIH R01 AG13501 and HL61518.

BIOMECHANICAL HEARTS, PERFORMED IN A ONE-STEP OPERATION AND TRAINED DYNAMICALLY UNDER SUPPORT OF CLENBUTEROL

NW Guldner, P Klapproth, M Grssherr (1), E Rumpel (2), A Bruegge, R Noel (3), HH Sievers

Clinic of Cardiac Surgery, (1) Clinic of Anaesthesiology, (2) Institute of Anatomy and (3) Institute of Animal Care, Medical University of Lubeck, Germany

Background. Skeletal muscle ventricles (SMVs) were connected to circulation by a second operation after several weeks of a vascular delay and electrical conditioning. This study investigates, whether muscular blood pumps are to performed in a one-step operation and to train within circulation. These devices with a stabilizing pumping chamber were defined as Biomechanical Hearts (BMHs). Their hemodynamics and myosin heavy chain composition were to evaluate in a chronic animal model.

Material and Results. In adult Bore goats (n=5) latissimus dorsi muscle (LDM) was wrapped around a polyurethane chamber which was connected to the descending aorta by vascular prostheses. LDM was stimulated electrically and trained dynamically under support of Clenbuterol (5X150 ?g/week) within circulation. Intra-operative stroke volume of BMHs was 53,8±22,4 ml. After one month of training BMH augmented contractions of the left heart ventricle differed from non supported ones significantly in mean diastolic and end diastolic pressure (P mean +2,9% (p<0.003)

Pdiast -2,4% (p<0.02) and after a BMH supported heart contraction, left hearts maximal rate of pressure generation dp/dt max increased to 20,5%. One BMH, using an LDM of 300 g pumped 34,8 ml per beat and 1.4 L/min continuously after 132 days of training. Percentage of myosin heavy chains type I in BMHs muscle ranged between 31 and 100% depending on duration of training.

In conclusion, Biomechanical Hearts performed in a one-step-operation and trained within circulation showed significant hemodynamic effects. They are expected to become a clinical relevant treatment option of end-stage heart failure.

METHODS TO EVALUATE DYNAMIC TRAINING OF SKELETAL MUSCLE VENTRICLES
AND BIOMECHANICAL HEARTS

P Klapproth, NW Guldner, M Grssherr (1), HH Sievers

Clinic of Cardiac Surgery and (1) Clinic of Anaesthesiology, Medical University of Lubeck, Germany

Background: Skeletal muscle ventricles (SMVs) were performed in adult boor goats by wrapping the latissimus dorsi muscle (LDM) either around an intrathoracic implantable elastic training device (ETD) or around a pumping chamber of a Biomechanical Heart (BMH) integrated as a circulatory loop in the aorta descendence. In both applications (ETD+BMH) the mechanical performance of SMVs was to evaluate during their dynamic training.

Material and methods: ETD: During each electrically induced muscle contraction, the ETD was compressed and a volume shift was performed. In a test rig, which simulated SMVs contraction, was to determine, if the volume shift correlates with the pressure increase within the training device. Thus, the stroke volume and other parameters could become calculated. In adult boor goats (n=5) the time course of the mechanical performance was to evaluate during the dynamic training. BMH: The pumping chamber was integrated by two vascular prostheses into circulation. In order to evaluate the hemodynamics during muscle contraction, a conductance catheter was placed at the long axis of the BMH. Firstly, in a test rig it was to verify, if the measured changes in conductivity of the fluid within the pumping chamber correlates with the stroke volume. Secondly, this method was to apply in adult boor goats (n=4).

Results: ETD: The in vitro examination of the method of indirect stroke volume evaluation shows a linear regression with a correlation of R=0.996 and a maximum relative error of 2.65%. In vivo, with 5 boor goats the dynamic training was observed weekly over a period of 6 months with tracing peak/load pressure, stroke volume, stroke work, contraction and half-value of relaxation time, maximum and minimum of pressure rise dp/dt as well as mean flow. BMH:

In vitro, the conductance measured volumes fit with a polynom of 2nd order to the control (R=0.9986) with a maximum relative error of 5.4%. In vivo, pressure ñ volume loops were achieved intraoperatively (n=3) during construction of the BMH and 132 days postoperative (n=1). The conductance measurement in combination with an intracavitary pressure assessment delieved peak/load pressure, stroke volumen, ejection fraction, stroke work, maximum peak ejection /filling rate, mean diastolic pressure increase and end diastolic pressure decay as well as the maximum pressure rise dp/dt of the supported heart beat.

Conclusion: Both methods, the method of indirect stroke volume determination as well as the conductance catheter method are suitable to evaluate the current state of the dynamic training of SMVs. In contrast to the invasive conductance catheter method, the minimal invasive assessment of the mechanical performance within the ETD enables a intermittent quantification of the dynamic training.

HEDGEHOG AND THE CONTROL OF MUSCLE CELL DIVERSIFICATION

SM Hughes

MRC Muscle and Cell Motility Unit and MRC Centre for Developmental Neurobiology, 4th floor South, New Hunts House, King’s College London, London, UK

Hedgehog proteins have been implicated in the control of myogenesis in the medial vertebrate somite. In the mouse, normal epaxial expression of the myogenic transcription factor myf-5 is dependent on Sonic hedgehog. Here we examine in zebrafish the interaction between Hedgehog signals, the expression of myoD family genes, including the newly cloned zmyf5, and slow myogenesis. We show that Sonic hedgehog is necessary for normal expression of both zmyf5 and zmyoD in adaxial slow muscle precursors, but not in lateral paraxial mesoderm. Expression of both genes is initiated normally in rostral presomitic mesoderm in sonic you mutants, that lack Sonic hedgehog. Similar initiation continues during tailbud outgrowth when the cells forming caudal somites are generated. However, adaxial cells in sonic you embryos are delayed in terminal differentiation and caudal adaxial cells fail to maintain myogenic regulatory factor expression. Despite these defects, other signals are able to maintain, or re-initiate, slow muscle development in sonic you mutants. In the cyclops mutant, the absence of floorplate-derived Tiggywinkle hedgehog and Sonic hedgehog has no discernible effect on slow adaxial myogenesis. Similarly, the absence of notochord-derived Sonic hedgehog and Echidna hedgehog in mutants lacking notochord delays, but does not prevent, adaxial slow muscle development. In contrast, removal of both Sonic hedgehog and a floorplate signal, probably Tiggywinkle hedgehog, from the embryonic midline in cyclops.sonic you double mutants essentially abolishes slow myogenesis. We conclude that several midline signals, likely to be various Hedgehogs, collaborate to maintain adaxial slow myogenesis in the zebrafish embryo. Although Hedgehog signals can induce slow adaxial myogenesis, their major in vivo role in zebrafish adaxial myogenesis is maintenance and progression to terminal differentiation. Consistent with this view, Sonic hedgehog induces terminal differentiation and up-regulation of slow myosin expression in murine and chicken myoblasts in cell culture.

CHANGES OF SARCOLEMMA REGION FOLLOWING ECCENTRIC CONTRACTION

A Jakubiec-Puka (1), D Biral (2)

(1) Dept. of Cellular Biochemistry, Nencki Institute of Experimental Biology, Pasteura 3, 02-093 Warsaw, Poland and (2 ) C.N.R. Unit for muscle Biology and Physiopathology, University of Padova, viale G. Colombo 3, 35121 Padova, Italy

The skeletal muscle gets damaged much easier while work-overloaded in extension than a muscle overworked in a neutral or in a shortened position. In such a muscle, pain and dysfunction appear with concomitant signs of impairment of the muscle fibre membrane integrity. Damage and necrosis of muscle fibres are quite common. In ultrastructure of the contractile apparatus overstretched and disrupted sarcomeres, contraction bands and Z-line streaming are observed. The reason for muscle fibre injury following eccentric contraction is so far unknown. Several hypotheses have been put forward, concerning both metabolic and mechanical mechanisms [7].

However, no convincing evidence have been raised so far revealing the primary cause of muscle damage occurring after eccentric contraction. As recently found [1], in the muscle overworked in an extended position, numerous fibres of normal-looking shape and size lose dystrophin from the sarcolemma region, similar to what happens in the dystrophic muscle. They also lose (to a lesser degree) other "costamere" proteins: the dystrophin-associated complex proteins and the spectrin [2]. Besides, several other clinical and metabolic features are quite common to the dystrophin-deficient dystrophic muscle and also in the muscle following eccentric contraction [7]. In the present work we have observed the ultrastructure of sarcolemma region in the muscles overworked in an extended position.

The albino Wistar rat muscles, the slow soleus and the fast extensor digitorum longus (EDL), were maintained in an extended position by immobilization of the ankle joint in a plastic tube at an angle of 90^o and 160^o, respectively, or by cutting the synergistic muscles, as previously described [1,4]. The sciatic nerve was continuously stimulated for 2, 4, 6, 24, 48 and 72 h by pulses of 0.3 ms duration and 20 Hz frequency. As controls, we used muscles from untreated animals of the same population, muscles immobilized in extension without stimulation and muscles stimulated without joint immobilization. All procedures of muscle isolation and storage, as well as of samples preparation for immunofluorescence and electron microscopy study were applied as previously [1,4]. Monoclonal antibodies against dystrophin (1:300), b-dystroglycan (1:50), a-sarcoglycan (1:20) and spectrin (1:50) were used. Negative controls were performed by omitting the primary antibody.

As recently found [1,2], and confirmed in this work, in the muscle stimulated in an extended position certain regions were severely damaged containing necrotic fibres, while some other regions were nearly normal. Within the better preserved parts of the experimental muscles, numerous fibres of normal-looking shape and size were not stained on the surface, or stained only discontinuously, with the anti-dystrophin antibody. The same fibres often lose staining with b-dystroglycan antibody, and also with a-sarcoglycan, g-sarcoglycan and spectrin antibodies. Such fibres were found after 4, 6 and 24 h, but not after 2 h of experiment in the soleus and EDL muscles. They were never found in the control muscles, nor in the muscle maintained in extension without stimulation, being however occasionally seen in the muscle stimulated in a neutral position.

In some fibres of the muscles stimulated in extension, disruptions of sarcolemma were seen with concomitant fibre swelling, post-mortem-like changes of the contractile apparatus and damage of mitochondria. However, most muscle fibres were preserved, with normal-looking mitochondria and a lot of polysomes. Within well preserved fibres of the experimental muscles, numerous vesicles of about 50-100nm in size were seen in the sarcolemma region. Those vesicles, of round or elliptic shape, were often open to extracellular space. Such structures correspond to those known as caveolae, present in most types of cells, including the muscle fibres [5]. They were relatively frequent in the control soleus muscle, while in the control EDL they appeared rather scarcely. Caveolae became numerous in both muscles following stimulation in extension. They seem to appear more crowded within regions of sarcolemma close to the Z-line, i.e. in the "costamere region"", than at the A-band level.

The increased number of caveolae and the elevation of caveolin-3 ( the muscle caveolae specific protein) were found in the Duchenne and mdx dystrophic muscles [6]. So, our observation indicated the next feature of similarity between the muscle overworked in extension and the dystrophic muscle. In the dystrophic muscle the increased number of caveolae and the elevated content of caveolin-3 was supposed to be a pathogenetic event of dystrophy [6]. On the other hand, however, the genetic defect of caveolin-3 and its absence within the sarcolemma region, was recently identified as causing the autosomal dominant limb girdle muscular dystrophy ( LGMD-1C) [3]. Thus more likely the lack than the excess of caveolin-3 in the sarcolemma region is the pathogenetic one. Our observation may suggest that the increased amount of caveolae in the dystrophin-deficient muscles appears to be a compensatory phenomenon.

1) Biral D, Sandri M, Jakubiec-Puka A Basic Appl Myol 1998; 8:205-210.

2) Biral D, Jakubiec-Puka A, Ciechomska I, Sandri M, Rossini K, Carraro U, Betto R Acta neurophatol in press.

3) Galbiati F, Volonte D, Minetti C, Chu JB, Lisanti MP J Biol Chem 1999; 274: 25632-25641.

4) Jakubiec-Puka A J Muscle Res Cell Motil 1985; 6:385-401.

5) Okamoto T, Schlegel A, Scherer PE, Lisanti MP J Biol Chem 1998; 273:5419-5422.

6) Repetto S, Bado M, Broda P, Lucania G, Masetti E, Sotgia F, Carbone T, Pavan A, Bonilla E, Cordone G, Lisanti MP, Minetti C Biochem Biophys Res Commun 1999; 261:547-550.

7) Salmons S Muscle Damage Oxford, New York, Tokyo, Oxford University Press, 1997.

TROPONIN T ISOFORM REGULATION DURING MUSCLE DEVELOPMENT AND ADAPTATION, AND FUNCTIONAL SIGNIFICANCE

J-P Jin, Q-Q Huang, O Ogut, A Chen, J Wang

Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4970, USA

Troponin T (TnT) is the tropomyosin (Tm)-binding subunit of the troponin complex and plays a central role in the Ca^2±regulation of muscle contraction. Through interactions with troponin I (TnI), troponin C (TnC), Tm and actin, TnT functions as an organizer molecule in the thin filament-based regulatory system. Three separate genes have evolved in vertebrate to encode the cardiac, slow and fast skeletal muscle TnTs. Fiber type-specific and developmentally regulated alternative RNA splicing further generates multiple TnT isoforms.

The major structural difference among the cardiac, slow and fast skeletal muscle TnT’s, as well as among the alternatively spliced isoforms, is in the NH₂-terminal domain. The most striking feature distinguishing various TnT isoforms is their NH₂-terminal charge. Alternative splicing of one or more NH₂-terminal coding exons generates TnT isoform switches during the development of cardiac and fast skeletal muscles. The functional significance of the conserved acidic to basic TnT isoform transition is not well understood. Different TnT isoforms are also expressed in different types of muscle fibers, during the functional adaptation of muscle, and under pathological conditions. Studies of TnT structure-function relationship have revealed that the COOH-terminal T2 fragment of TnT interacts with TnI, TnC and Tm, whereas the central region of the TnT polypeptide interact with the head-to-tail overlapping region of Tm. In contrast, the alternatively spliced NH₂-terminal variable region of TnT has not been identified with direct interaction with other thin filament proteins.

In contrast, the structure of the NH₂-terminal domain may modulate TnT’s molecular conformation and function. Reconstituted thin filaments containing cardiac TnT isoforms differing in the NH₂-terminal variable region showed differences in the sensitivity to Ca²⁺ activation of actomyosin ATPase. The interactions of TnI and Tm with TnT isoforms containing a more acidic NH₂-terminal region are more tolerant to low pH compared to the basic TnT isoforms, suggesting a role of TnT isoform regulation in the functional adaptation of muscle fibers. Muscle fibers expressing more acidic TnT isoforms showed higher sensitivity to Ca²⁺ in the development of force. The binding of transition metal ions to a segment in the NH₂-terminal variable region of avian pectoral muscle TnT induces three-dimensional structure changes within the NH₂-terminal region of chicken breast muscle TnT can produce long range secondary effects on the conformation and function of the other domains of TnT.

The regulated TnT isoform switch during muscle development and adaptation suggests that the expression of TnT isoforms with structural differences in the alternatively spliced NH₂-terminal variable region may be a mechanism to fine tune the contractile property as an adaptation to the cellular environment and functional state of the muscle. On the other hand, the cardiac TnT expressed in embryonic and neonatal developing skeletal muscles also undergoes the same isoform switch as in the heart although their functional environments are different. Timing of the alternative splicing-generated cardiac TnT isoform switching is synchronized in the perinatal developing cardiac and skeletal muscles in a species-determined manner, indicating a determination by genetic program. This is important to the understanding that changes in TnT isoform expression is not a simple response to the functional demands and may contribute to both physiological adaptation and pathogenesis.

While the functional significance of TnT isoform diversity is not fully understood, pathological conditions, such as diabetic myopathy and myocardial hypertrophy, can result in switches of TnT isoform expression. The finding that various point mutations scattered in the cardiac TnT polypeptide chain cause a similar pathological phenotype in human familial cardiomyopathy suggests that changes in protein conformation, rather than the mutant site per se, are the key in the structure-function relationship of the TnT mutants or isoforms.

SORTING AND DYNAMICS OF MYOSIN ALKALI LIGHT CHAIN EXPRESSED IN CULTURED CARDIOMYOCYTES AND CARDIAC FIBROBLASTS

MM Khan, M Komiyama,Y Shimada

Department of Anatomy and Cell Biology, School of Medicine, Chiba University, Chiba 260-8670, Japan

Subcellular sorting of myosin alkali light chain (LC) isoproteins were examined within the same cells by co-expression of cDNAs of different LC isoforms tagged with different epitopes in cultured chicken embryonic cardiomyocytes and fibroblasts. Expressed LC isoforms were labeled with antibodies to the tags and their distribution was analyzed by confocal laser scanning microscopy. In cardiomyocytes, nonmuscle type (LC3nm) was distributed throughout the cytoplasm, while fast muscle types (LC1f and LC3f) were localized at A bands of myofibrils. Slow muscle type (LC1sa) exhibited a striated pattern when co-expressed with LC3nm, but this pattern became blurred upon co-expression with slow/ventricular (LC1sb) or the fast muscle types. LC1sb was distributed at A bands when co-expressed with LC3nm or LC 1sa, while its distribution to the cytoplasmic areas increased if it was co-expressed with the fast muscle types. Thus, sorting specificity of LC isoproteins to the sarcomeres increased in the order from LC3nm, to LC1sa, to LC1sb, and to LC1f and LC3f. In fibroblasts, their sorting specificity to stress fibers increased from LC3nm, to LC1sb, to LC1sa, and to LC1f and LC3f. This indicates that the order of the affinity of LC isoproteins for myosin heavy chain (MHC) varies depending on the MHC isoforms. Further, for both the cardiac and nonmuscle MHCs, the fast muscle LCs exhibited the highest affinity. This suggests that the fast muscle LCs may be evolved isoforms possessing the ability to associate tightly with a variety of MHC isoforms.

Various chimeric cDNAs between LC3nm and LC3f were constructed and expressed in order to decide domain(s) of LC isoforms responsible for their sorting. If most of the second EF-hand of LC3nm was replaced by the counterpart of LC3f, the chimeric protein exhibited the same sorting pattern as LC3f. This suggests that the difference of 12 amino acids in this domain (loop and its neighboring regions) is responsible for sorting of LC isoproteins.

In order to study how newly synthesized LC molecules are incorporated into myofibrils, cDNA of LC3f tagged with green fluorescence protein (LC3f-GFP) was transfected into cultured chicken cardiomyocytes, and the assembly of expressed LC3f-GFP was observed in living cells under a fluorescence microscope equipped with a cooled CCD camera. At 14-16 hours after transfection, LC3f-GFP was diffusely distributed in the cytoplasm of cardiomyocytes. In some cells, however, intense fluorescence spots of LC3f-GFP were found along myofibrils with a periodicity of 1.2 micrometers. Confocal microscopy of such cells stained with rhodamine-labeled phalloidin revealed that the fluorescence spots of LC3f-GFP were localized at both ends of the A-band. When these cells were further incubated, LC3f-GFP came to be localized at all levels of the A-bands by 26 hours

after transfection. These results indicate that myosin filaments are not replaced with newly synthesized myosin molecules at once along their entire length, but molecules in filaments are replaced individually from their ends.

VASCULAR DELAY AND INTERMITTENT STIMULATION: KEYS TO SUCCESS IN CARDIOMYOPLASTY

A Kashem, WP Santamore, BBY Chiang (1), AD Slater (1)

Cardiovascular Research Division, Temple University HSC, Pennsylvania, USA and (1) Division of Thoracic and Cardiovascular Surgery, University of Louisville, Kentucky, USA

In cardiomyoplasty (CMP), over-use leads to extensive muscle damage and conversion to slow, weak type I fibers, which may explain observed minimal systolic assistance with latissimus dorsi muscle (LDM) stimulation. We hypothesized that preserving LDM integrity by vascular delay and intermittent stimulation would greatly increase LDM cardiac assistance. In 12 dogs, left ventricular (LV) dysfunction was induced by intracoronary microsphere injections and a LDM vascular delay procedure was performed. Fourteen days later, CMP surgery was performed followed by progressive LDM conditioning. In half, LDM was stimulated 24 hours/day (CS). In other half, LDM was stimulated intermittently 10 hours on/14 hours off/day (IS). After 9 weeks, effects of LDM stimulation were evaluated by comparing LDM assisted (S) to non-stimulated (NS) beats. LDM stimulation increased (p<0.05) peak LV pressure (LVPmax), stroke volume (SV), stroke work (SW), stroke power (SP), and peak aortic flow (maxAoF) in both CS and IS. But, the increases were greater in IS. Quantative morphometry showed minimal LDM damage in IS, 7.5 ± 1.1%. Vascular delay causes revascularization andintermittent stimulation blocks full conversion to type I fibers. Thus, large hemodynamic increases can occur with LDM stimulation. Vascular delay plus intermittent stimulation are vital for this success and should be considered for clinical cardiomyoplasty.

Table.

% Changes LVP max SV SW Power MaxQ

(mm Hg) (ml) (gm.m) (gm.m/sec) (l/min)

CS 14.9±3.7* 25.1±9.7* 45.9±16.6* 57.4±19.5* 35.8±11.8*

IS 21.6±1.0* 34.2±9.8* 67.4±16.4* 99.0±12.7* 58.0±16.0*

*p<0.05 S vs NS;p<0.05 IS vs CS; Data: Mean ±SEM

DENERVATED MUSCLES IN HUMAN FIRST RESULTS OF TRAINING WITH ELECTRICAL STIMULATION

H Kern, Ch Hofer, M Strosshofer, W Mayr (1), M Mödlin, C Forstner, W Richter (2)

Department of Physical Medicine, Wilhelminenspital, (1) Department of Biomedical Engineering & Physics, University of Vienna and (2) Department of Radiology, Wilhelminenspital, Vienna, Austria

We examined the possibility of training denervated human muscles by means of functional electrical stimulation (FES) in complete denervated paraplegics.

In patients with complete conus cauda lesion (Frankel A) was examined if by using appropriate electrical stimulation muscular degeneration could be prevented and already degenerated muscles could be improved to an extent that tetanic contractions and functional movements could be triggered using FES. These patients started training with electrical stimulation 2 years after the lesion. Tetanic muscle contractions with knee extension moments of 16,5–38 Nm could be obtained after 16–26 months.

Muscle training was begun in the sitting position with single twitches (biphasic rectangular pulse, average pulse width 120 ms) followed by tetanic contraction cycles (on: 1-2 seconds, off: 2-4 seconds) initially without weight until nearly complete knee extension was achieved and later with weight cuffs 0,5 – 1 kg on the ankle (6–12 repetitions, 6 series, once later twice daily). Endurance training was performed by additional stimulation with 5–8 Hz with 10 s contraction time and 1 s break later followed by 5 s contraction time and 1 s break (pulse duration 60–70 ms).

Improvement in muscle force (53%–73%) and muscle cross-section area (22%–58%) within 1 year of tetanic muscle training was documented by means of torque measurement and computer tomography respectively.

As knee stabilisation in the standing position requires relatively low moment and standing up is supported by the upper body, standing training can be started early on. In our patients FES started 1–2 years after lesion (complete conus cauda). Standing training could be carried out 9–12 months after commencement of FES.

FIRST APPLICATION OF FUNCTIONAL ELECTROSTIMULATION FOR MUSCLE TRAINING ON BOARD OF MIR SPACE STATION

G Freilinger, W Mayr (1), R Rafolt (1), M Bijak (1), W Girsch, H Lanmuller (1), S Sauermann (1), E Unger (1), Y Koryak (2), B Shenkman (2), I Kozlovskaya (2), A Gregoriev (2)

Dept. of Plastic and Reconstructive Surgery, and (1) Dept. of Biomedical Engineering and Physics, University of Vienna Medical School, Austria and (2) Institute for Biomedical Problems, State Scientific Center, Moscow, Russia

The next step in orbit is the launching of ISS and its permanent occupation. A major concern for individuals living and working in space under microgravity conditions is the impairment of the musculo-skeletal system.

We designed an AUSTRO-RUSSIAN project, MYOSTIM to maintain skeletal musclemass and fiber composition with a passive training method, based on Functional Electrostimulation (FES), with minimal impairment for the cosmonaut.

With experience in terrestrial medicine, the system has been tested on bedrest candidates, in isolation studies and used for the first time by the cosmonautes in the MIR-station. Data of preflight, flight, and postflight investigations are now available. To document muscle condition tendometry, dynamometry, histo-morphological and histoenzimatic investigations (biopsy from M. vastus lateralis) as well as neuro-sensoric tests and reflex tests to investigate the neuromuscular coordination were included. In addition a locomotive exercise tolerance test was performed and the cosmonaut asked for his subjective remarks.

CARDIOMYOPLASTY-SKELETAL MUSCLE ASSIST RANDOMIZED TRIAL (C-SMART):
6 MONTH RESULTS

JK Kirklin, JB Young, RC Bourge, M Silver, E Loh, D Schmidt, S Raible, R Rodeheffer, G Torre, D Humen, AD Roettger, on behalf of the C-SMART Investigators

The University of Alabama at Birmingham, Birmingham, Alabama, USA

Dynamic cardiomyoplasty (DCMP) has been applied to the treatment of heart failure for several decades, but randomized studies demonstrating its efficacy have been lacking . C-SMART is the first and only randomized trial of DCMP vs medical therapy alone (MRx).

In the randomized trial of 103 NYHA III heart failure patients, 51 patients underwent DCMP. The medical and surgical cohorts were not significantly different in this study. The overall mean age was 56 years, illness duration 5.3 years, ejection fraction 24%, wedge pressure 18 mmHg, cardiac index 2.3 l/min/m², echo diastolic dimension 7 cm, peak VO₂/kg 16 ml/kg/min. The mean Minnesota Living with Heart Failure (MLwHF) score was 58 with 78% male patients. Ethiology of disease included 81% with dilated cardiomyopathy. Common co-morbid conditions included diabetes (33% of patients) and atrial fibrillation (16%). Virtually all were on digoxine, diuretics, and converting enzyme inhibitors (at least 93% each), while 17% were on beta blockers and 13% on amiodarone.

Results: Hospital death occurred in 2/51 (4%) DCMP patients (sepsis, worse failure) with 1 sudden death within 30 days. Six month survival was 86% for DCMP and 84% for MRx (p>0.05). Among patients surviving at least 6 months, 79% of DCMP vs 25% of MRx were NYHA I or II. DCMP cohort improved the primary efficacy endpoint 6 minute walk distance from 1084 ft to 1247 ft vs MRx (1137 to 1030 ft); p=0.002. MLwHF quality of life improved with DCMP from 55 to 36 vs the MRx change of 54 to 51; p=0.002.

In conclusions, the randomized C-SMART data demonstrates that DCMP, at 6 months follow-up, significantly improves NYHA class, exercise tolerance, and quality of life compared to drug therapy alone in symptomatic congestive heart failure patients. Despite surgical intervention, short-term survival is similar between groups. Twelve month follow-up data is currently being analyzed.

GENE EXPRESSION PROFILING OF HUMAN SKELETAL MUSCLE DURING AGEING
AND DIFFERENTIATION USING AN ARRAYED COLLECTION OF 3’-END MUSCLE cDNAs

G Lanfranchi, B Pacchioni, S Trevisan, S Toppo, N Cannata,C Ievolella, A Pallavicini, P Laveder, G Valle, S Imbeaud¹, S Bortoli¹, E Eveno¹, R Mariage-Samson¹,B Soury-Segurens¹, V Renault¹, NA Fayein¹, C Decraene¹, C Matingou¹, MD Devignes¹, GPiétu¹, C Auffray¹, V Mouly², G Piron-Hamelin² and G Butler-Browne²

CRIBI Biotechnology Centre, Università degli Studi di Padova, Padova, Italy, ¹ Genexpress, CNRS ERS1984, Villejuif, France, *CNRS URA 2115, Paris, France and ² CNRS URA 2115, 105 Boulevard de l’Hôpital, 75634 Paris, France

For several years, the muscular transcriptome has been investigated through the combined efforts of our teams by partial systematic sequencing of cDNA libraries [1, 2], gene mapping using radiation hybrids [3, 4, 5, 6] and expression profiling using cDNA arrays [7].

The CRIBI Group has systematically sequenced 52,000 clones from a 3’-specific cDNA library from human skeletal muscle. The corresponding sequences have been integrated in a proprietary database (http://www.grup.it) after extensive clustering and comparison to public databases using originally developed software [2, 3, 5]. From this data a catalogue of 4,200 unique muscle transcripts has been obtained. A first selection of 2,016 of such transcripts has been collected by isolation and re-sequencing of representative primary cDNA clones, creating the first release of the CRIBI muscle archive.

The arrayed clones were amplified and spotted at high density on nylon filters and used for hybridization with 14 complex cDNA targets derived from mRNA from adult and fetal tissues. Of 101 transcripts demonstrating a skeletal and/or cardiac muscle-restricted pattern of expression, 69 encode known muscular proteins, while the remainders are completely novel.

The same 3’-end cDNA arrays have been used to monitor gene expression patterns during muscular ageing and differentiation, and investigate the underlying molecular mechanisms. Complex targets were derived from mRNA of well-characterised human myogenic precursor cells, called satellite cells at 3 different proliferative states (21, 39 and 47 divisions), before (myoblast) and after (myotube) induction of differentiation [8]. Hybridisation signals were collected in quadruplicate on phosphor screens, analysed and quantitated using specific software as previously described [9].

Differential expression was observed for more than one hundred genes when comparing 3 physiological and metabolic states: ageing myoblasts, ageing myotubes, and myoblast to myotube transition. Based on sequence analysis, these genes can be classified in 3 categories: i) as expected, 15 genes already known to be involved in ageing, like ankyrin 1 and microtubule associated protein, or in differentiation, like troponin T, tropomyosin and myosin light chains; ii) 60 known genes with as yet non described roles in ageing and/or in differentiation; iii) 60 novel genes, which are candidates to study in relation with muscle function.

To confirm and further investigate the specificity of transcription of the genes corresponding to the selected clones, Northern blot analysis with RNA isolated from myoblasts and myotubes at the three proliferative steps are in progress.

References

1. Houlgatte et al., Genome Res. 5:272-304, 1995.

2. Lanfranchi et al., Genome Res. 6:35-42, 1996.

3. Pallavicini et al., Hum. Mol. Genet. 9:1445-50, 1996.

4. Schuler at al., Science 274:540-6, 1996.

5. Bortoluzzi et al., Genome Res. 8:817-25, 1998.

6. Deloukas et al., Science 282:744-6, 1998.

7. Piétu et al., Genome Res. 6:492-503, 1996 ; 9:195-209, 1999 ; 9:1313-20, 1999.

8. Mouly V., et al., Neuromuscular Disord. 3: 371-377, 1993.

9. Piétu G., et al., Genome Res. 6:492-503, 1996.

EFFECTS OF AGING ON THE REGULATION OF MUSCLE CONTRACTION AT THE MOTOR UNIT, MUSCLE CELL, AND MOLECULAR LEVELS

L Larsson

Noll Physiological Research Center & Department of Cellular and Molecular Physiology, School of Medicine, Hershey Medical Center, The Pennsylvania State University, U.S.A.

Falls are a major cause of morbidity and mortality in the growing population of elderly citizens, and the prevention of falls and gait instability is therefore a very important socioeconomic concern. The mechanisms underlying the increased risk for falls in the elderly are complex and involve aging-related changes in both the central and peripheral nervous system, and in the muscle tissue itself. In recent years, the strong negative influence of the age-related loss of muscle mass, strength and quality, i.e., sarcopenia, on impaired motor function in old age has become increasingly evident. Therefore, our long-term objective is to have a detailed understanding of the mechanisms underlying the deficits in neuromuscular function present in the elderly. In an attempt to improve our understanding of the mechanisms underlying the impaired muscle function in old age, a series of experimental animal studies have been conducted to study aging-related changes in the spatial organization of different motor unit types, and regulation of muscle contraction at the motor unit, muscle cell and molecular levels.

Regulation of muscle contraction at the motor unit, muscle cell and motor protein levels were studied in rodents (mice and rats) ranging in age from 2 to 30 months. Contractile properties (twitch and tetanus), myosin isoform expression, sarcoplasmic reticulum Ca2±uptake activity, and spatial organization of motor unit fibers were studied in single motor units of the fast- and slow-twitch type using the glycogen- depletion technique. Maximum velocity of unloaded shortening, force generation capacity, and morphological properties were measured in short single membrane permeabilized muscle cell segments expressing different myosin heavy and light chain isoforms and isoform combinations. The molecular interaction between specific myosin isoforms and fluorescent-labelled actin filaments were studied using a novel in vitro motility assay, i.e., actomyosin interactions were studied after extraction of myosin from short muscle fiber segments. In addition, results obtained from men and women ranging in age from 20 to 90 years will also be presented.

Significant aging-related changes in motor unit size, spatial organization of motor unit fibers and contractile properties were observed in both fast- and slow-twitch motor units. These changes were paralleled by an aging-related loss of -motoneurons. The prolongation of the isometric twitch, observed in both fast- and slow-twitch motor units, was not related to a myosin isoform transition.

The impaired Ca2±uptake activity of the sarcoplasmic reticulum observed in muscle cells expressing fast myosin isoforms is suggested to be an important factor underlying the slowing of the isometric twitch in fast-twitch motor units. The dominating observations from the cellular and molecular physiological experiments were the slowing of the maximum velocity of unloaded shortening in muscle cells expressing the /slow myosin isoform and the decreased motility speed of actin filaments propelled by /slow myosin, indicating altered enzymatic and functional properties of the molecular motor protein myosin in old age. Underlying mechanisms and aging-related changes in human skeletal muscle will be discussed

This study was supported by grants from the Gerontology Center at Penn State, the European Commission (BMH4-CT96-0174 ), the Swedish Medical Research Council (08651), the Muscular Dystrophy Association, and NIH (M01-RR10732-03).

G PROTEIN-COUPLED RECEPTORS, CALCIUM DEREGULATION AND APOPTOSIS IN THE HEART

J Ligutti, N Nakajima, A Cavalli, R Betto (1), D Biral (1), R Sabbadini

Heart Institute and Department of Biology, San Diego State University, San Diego California, U.S.A. 92182-4614, (2) C.N.R. Unit for Muscle Biology and Physiopathology, Department of Biomedical Sciences, University of Padova, Italy

The naturally occurring lipid mediator, sphingosine-1-phosphate (S1P), is emerging as an important first messenger that controls cell growth in many tissue types. Sphingosine-1-phosphate is released into the serum by activated blood platelets. Since platelet activation is involved in the thrombus of Acute Myocardial Infarction (AMI) and since over 70% of AMIs involve thrombus formation, we reasoned that extracellular S1P could contribute to the pathogenic and remodeling processes of the heart. Recently, surface membrane receptors for S1P had been identified as belonging to the Endothelial cell Differentiation Gene (Edg) family. We have discovered the expression of S1P-specific Edg receptors (EdgRs) on the surfaces of cardiomyocytes and cardiofibroblasts and we initiated a study of S1Pís role in cardiac function. Indirect immunofluorescence and confocal microscopy were used to identify EdgR protein expression and we initiated a study of potential S1P- and EdgR-mediated modulation of cardiac cell function. Using primary cultures of rat neonatal cardiomyocytes, we have found that S1P has cardiotoxic effects that include calcium deregulation and apoptotic cell death. Apoptosis and calcium deregulation are mediated by the Edg family of G Protein-Coupled receptors (GPCRs) as judged by the ability of PTX to mitigate the apoptotic response and by the ability of antisense Edg constructs to diminish the calcium responses to S1P. In contrast to the myocytes, cardiofibroblasts are not driven into apoptosis by S1P, but are stimulated to proliferate as judged by 3H-thymidine incorporation studies. We speculate that S1P is produced by the heart as a consequence of thrombus formation during infarction and that S1P is responsible, in part, for the cell death of myocytes and the stimulation of fibroblasts during myocardial remodeling and scar formation. Supported by Medlyte Diagnostics and NIH/NIGMS MBRS Grant # 1 R25 GM58906-01, NIH Fogerty MIRT #50106D, and by Italian CNR and MURST funds.

CARDIOMYOPLASTY AND IMPLANTABLE DEFIBRILLATOR IN HEART FAILURE PATIENTS: POSITIVE IMPACT ON PATIENT SURVIVAL.

R Lorusso, C Werling, H Kaulbach, T Mesana, PS Gerometta, G Bolotin, V Chekanov

Brescia, Ludwigshafen, Koblenz, Marseille, Milan, Haifa, Milwaukee on behalf of the CIDEF Group

Objective. To review the patient outcome after cardiomyoplasty in the presence of a combined implantation of an implantable defibrillator and to preliminarily analyse the impact on long-term patient survival.

Methods. The clinical series of 7 different Centres were reviewed. Cardiomyoplasty procedures were performed in 90 patients. Cardiomyoplasty as sole procedure was performed in 64 patients whereas implantable defibrillators were implanted in 26 patients (as part of a combined procedure in 16 patients, previously positioned in 5 and subsequently implanted in 5).

Results. Follow-up ranged from 2 years to 9 years. There were 30 deaths in the cardiomyoplasty group, whereas only 4 deaths occurred in the cardiomyoplasty-ICD group. In Group A the majority of deaths occurred because of sudden death whereas in Group B only 1 death occurred due to ICD malfunction. Deaths due to arrhythmic events were not related to hemodynamic conditions.

Conclusions. These preliminary results indicate that the combination of ICD in cardiomyoplasty patients significantly reduced postoperative mortality due to prevention of sudden death.

THE EFFECTS OF NITRIC OXIDE ON THE CONTRACTION OF SKELETAL MUSCLE

Department Physiol Pharmacol. Fac. Med. U.C.L., Brussels, Belgium

Review of the literature suggests to classify the effects of nitric oxide NO on skeletal muscles fibres in two groups. In a first group, the effects of NO are direct, due to nitrosation or metal nitrosylation of target proteins: depression of isometric force, shortening velocity of loaded or unloaded contractions, glycolysis and of mitochondrial respiration. The effect on calcium release channels varies, being inhibitory at low and stimulatory at high concentration of NO. The general consequence of the direct effects of NO is to "brake" the contraction and its associated metabolism. In the second group, the effects of NO are mediated by cGMP: increase of the shortening velocity of loaded or unloaded contractions, maximal mechanical power, initial rate of force development, frequency of tetanic fusion, glucose uptake, glycolysis and mitochondrial respiration; decreases of half relaxation time of tetanus and twitch, twitch time-to-peak, force maintained during unfused tetanus and of stimulus associated calcium release. There is negligible effect on maximal force of isometric twitch and tetanus. The general consequence of cGMP mediated effects of NO is to improve mechanical and metabolic muscle power, similar to a transformation of slow-twitch to fast-twitch muscle, an effect that we may summarize as a "slow-to-fast" shift.

References

G. Maréchal & Ph. Gailly (1999). The effects of nitric oxide on the contraction of skeletal muscle. Cel. Mol. Life Sci., 55, 1088-1102.

SPECIES-SPECIFIC BIOCHEMICAL PROPERTIES AND ADAPTATIVE CHANGES OF THE SARCOPLASMIC RETICULUM OF HUMAN SKELETAL MUSCLE UNDER PATHOLOGICAL CONDITIONS

A Margreth, E Damiani, A Pallanca

NRC Unit for Muscle Biology and Physiopathology, Department of Experimental Biomedical Sciences, viale G. Colombo 3, 35121 Padova, Italy.

There is still incomplete biochemical knowledge of sarcoplasmic reticulum (SR) fast and slow phenotypes in human skeletal muscle. Study of such problems on the isolated SR has been seriously hampered by the mixed fiber composition of human muscles. On account of studies on representative muscles of experimental animals of large size (dog, rabbit), i.e. muscles almost homogeneously composed of a single type of fibers, several generalizations have been made. The first is that in adult animals a single SR Ca2±ATPase isoform is expressed in muscle fibers, either SERCA1 (fast-twitch isoform) or SERCA2 (slow-twitch isoform), but not both. The second is that SERCA2 only is under control by regulatory protein phospholamban (PLB), depending on phosphorylation by SR-bound Ca2±CaM protein kinase (CaM K II) and which disrupts its inhibitory interaction with the Ca2±pump.

While the segregation of SERCA1 or SERCA2 within the corresponding type of motor unit seems to be a general rule, there is emerging evidence from our own studies on human adult skeletal muscle, using immunofluorescent techniques, that, as an apparently species-linked property, PLB is not confined to slow-twitch fibers. In agreement with this finding, immunoblot analysis of the isolated, sucrose-density purified SR from adult human skeletal muscle has indicated that the protein level of expression of PLB, both is significantly high in comparison to rabbit slow-twitch muscle and is not directly related to the level of SERCA2. Furthermore, we have found that in the isolated SR PLB is intensely phosphorylated by endogenous CaM K II. Human SR-bound CaM K II, although differing slightly in apparent molecular size, seems to have subunit type composition and biochemical functional properties similar to those of rabbit fast twitch SR CaM K II, including the ability to phosphorylate also tradin (Damiani et al., Biochem. Biophys. Res. Commun. 209, 457-465, 1995). These several findings seem to argue for a critical role of CaM K II-mediated phosporylation of PLB, in modulation of SR Ca2±transport in human skeletal muscle, somewhat regardless of fiber type. In the case of human mixed skeletal muscle, therefore, one can envision how adaptive changes in the ability to handle Ca2+, in response to changes in the pattern of usage of the muscle, and to some extent independent of the preferential recruitment either of fast or slow motor units, may involve the fine tuning of SR Ca2±ATPase activity through the action of the PLB-CaM K II regulatory system. Long-term adaptation might entail a remodeling of motor units, the significant cause being a change in the pattern of firing of motor neurons. Isoform transitions both of myosin and other myofibrillar proteins and of SERCA are well documented by cross reinnervation studies of fast and slow muscles, and by the effects of chronic electrical stimulation at low frequency of fast-twitch muscles in experimental animals. Hinting to the possibility of analogous changes in human skeletal muscle, is the evidence of intermediate fibers (with respect to isomyosin composition) in muscle of patients with Steinertís disease (noticeable a little degenerative primary myopathy), and which were interpreted as fibers en route from one type to the other type (Salviati et al., Neurology 36, 693-697, 1986). In some although not all cases of Steinertís disease, as well as interestingly, in one case of myotonia (Thomsen’s disease), we analogously found a significant proportion of muscle fibers expressing both SERCA1 and SERCA2, to the best of our knowledge that had not been reported previously. Our data seem to be compatible with the interpretation of an ongoing transition of SERCA2 into SERCA1 within the originally homogeneous slow-twitch fiber population and which does not appear to be coupled to a repression of synthesis of PLB, but rather to be associated with the presence of PLB in a highly phosphorylated form, and which would potentiate the effect of the function of Ca2±ATPase isoforms.

Thus, there seems to be a wealth of interactive mechanisms by which the rate of relaxation may become accelerated in human mixed muscles, and more than it could be inferred from studies of pure fast and slow muscles of experimental animals. The control mechanism of SR Ca2±transport system, like the modulation of SR Ca2±release by endogenous effectors, such as Ca2+ and CaM, either directly or through the action of CaM K II, are all problems central to a better understanding of the biopathology of human skeletal muscle, including the ageing process.

Work supported by funds from Telethon Italy (project . n. 848) and MURST (COFIN-99).

WALKING ENERGY COST IN CHILDREN WITH NEUROGENIC MOTOR IMPAIRMENTS

A Martinuzzi, E Trevisi, P Zamparo (1), PE Di Prampero (1).

IRCCS "E. Medea" Polo Regionale di Conegliano, (1) Dip. Scienze e Tecnologie Biomediche, Università di Udine

Disability in subjects with neuromuscular disorders is primarily determined by the underlying defect in muscle or motor neuron function, which seldom is amenable of efficacious therapeutic intervention. Other factors can however positively or negatively affect the level of performance, and these could be more readily influenced by therapeutic or rehabilitative protocols.

Energy expenditure of daily motor activity can be regarded as one of the variables which could strongly influence the final motor capability of a subject, and in patients with primary neurogenic defects such as cerebral palsy, could add further disability linked to early muscle exhaustion to the underlying impairment. We have measured the energy cost of level walking in children affected by cerebral palsy (diplegia) and age matched controls. All subjects were able to walk unassisted for at least 4-5 min. Normal controls were asked to walk at self selected speed (sss) (1.15±0.12 m/s) and additionally at lower speed, to allow comparison with the slower walking speed (0.85±0.26 m/s)? of patients. Oxygen uptake was measured at rest and during walking using a portable telemetric system (K4, Cosmed, I). Energy cost of walking (Cw) was calculated from the ratio of the net (above rest) steady state VO2 (ml O2/min/Kg) to the effective speed of progression (m/sec) and converted in J/m/Kg on the assumption that 1ml O2 consumed yields 20.9 J.

Results: Cw in patients was significantly higher than that observed in normal control (5.74±3.42 J/m/Kg vs 3.00±0.66 J/m/Kg). The Cw of normal children walking at the same speed as disabled children was slightly higher but still much lower than that observed in patients (3.23±0.82 J/m/Kg). The variable found to associate strongly with Cw in patients was the degree of spasticity as measured by the Ashworth scale.

In conclusion, patients with neurogenic motor impairments (cerebral palsy) face the double trouble of defective motoneuronal activity and increased energy cost of muscle activation such as that entailed by walking. The additive effect of neurogenic impairment, decreased movement efficiency, and muscle fatigability, leads to further motor limitation, and needs to be addressed as primary target of rehabilitative strategy

DYSTROPHIC PROCESSES CAN BE SEPARATED INTO TWO DISTINCT STAGES IN MDX SKELETAL MUSCLE

R Matsuda

Dept. of Life Sci., Univ. of Tokyo, Meguro-ku, Japan

We have shown that degenerating muscle fibers undergo apoptosis in dystrophin-deficient mdx skeletal muscle using Evans Blue-vital staining coupled with TUNEL (Matsuda, et al. (1995)). The apoptotic nuclei were detected exclusively in Evans Blue-positive fibers. Strub, et al. (1997) reported that Evans Blue-positive fibers had plasma membrane with increased permeability which allowed dye-albumin complex pass through the membrane since the dye tend to bind serum albumin. Using anti mouse serum albumin antibody staining coupled with Evans Blue vital staining and TUNEL, we could differentiate the muscle degeneration processes into two distinct stages. Stage 1, unbound Evans Blue and Ca2+ passed through the plasma membrane, followed by stage 2, the dye-albumin complex passed through the membrane. Muscle hypercontraction and TUNEL-positive nuclei appeared simultaneously at the stage 1. Deterioration of cell shape and necrotic processes became apparent at the stage 2. If one wants to interfere the apoptotic processes in dystrophic fibers by chemotherapy, the inhibitor molecules have to be water-soluble and smaller than the Evans Blue dye molecule (mol wt=960.8).

FUNCTIONAL ELECTROSTIMULATION AS A COUNTERMEASURE AGAINST MUSCULAR ATROPHY
IN LONG-TERM SPACE FLIGHTS

W Mayr, G Freilinger, R Rafolt, M Bijak, W Girsch, H Lanmuller, M Reichel, S Sauermann, E Unger, V Gratchev, Y Koryak, I Kozlovskaya, A Gregoriev

Dept. of Biomedical Engineering and Physics, and Dept. of Plastic and Reconstructive Surgery, University of Vienna Medical School, Austria and Institute for Biomedical Problems, State Scientific Center, Moscow, Russia

Long-term flights in microgravity cause atrophy and morphological changes of skeletal muscles. Training with mechanical devices is insufficient regarding time to exercise and space for devices.

The objective of the project MYOSTIM is to develop a training method, based on Functional Electrostimulation (FES) to preserve muscle mass and fiber composition with minimal

impairment to the cosmonaut.

For a pilot experiment on board of the MIR station a suitable 8-channel FES equipment was developed. It consists of electrode trousers, that carry surface electrodes and cables, two

interconnected 4-channel stimulators, and a laptop-PC for stimulator programming and processing compliance data. An automatic extensive training of 4 muscle groups of the lower extremities is performed for 6 hours per day, with 1 s on / 2 s off tetanic contractions at 20 to 30% of maximum tetanic muscle force. Synchronous activation of antagonists of thigh and lower leg prevents from uncoordinated movements.

The first successful test on board of MIR was performed by 2 cosmonauts between December 1998 and February 1999 respectively March 1999 and August 1999.

EFFECTS OF TENOTOMY ON RAT SLOW MUSCLE REGENERATION

A Megighian, D Danieli Betto, E Germinario, M Midrio

Department of Human Anatomy and Physiology, University of Padova, Italy

The muscle after an acute degeneration is a useful experimental model for studying factors, which affect or direct muscle differentiation. Indeed, regeneration occurs with a sequence of events similar, although not identical to fetal myogenesis. Regeneration starts from undifferentiated cells and through the myoblast and myotube stages it ends with the adult, differentiated cells. The soleus (slow) muscle of the rat restores its metabolic-histochemical fiber pattern about in a month, but the terminal phenotype is easily changed, if conditions under which regeneration occurs are changed, for instance following denervation (Carraro U et al Exp Neurol 79:106, 1983; Whalen RG et al Dev Biol 141:24, 1990) or in absence of normal postural load (Bigard AX et al Acta Physiol Scand 161:23, 1997).

In a previous work (Midrio M et al Muscle & Nerve 21:226, 1998) we reported that neuromotor impulses are required for the appearance of myosin heavy chains (MHC) isoforms which are typically expressed in oxidative fibers (types 1 and 2A). Some data of the same paper, and other still unpublished data, support also the hypothesis that the nerve inhibits the expression of 2A MHC isoform independently of motor discharge, possibly through the release of chemical factors.

The aim of the present work was to investigate the effects of tenotomy on soleus muscle regeneration and the interaction between tenotomy and denervation. As other forms of muscular disuse (e.g. following hindlimb suspension), tenotomy causes both a reduction of neuromotor discharge and a reduction of muscle load, and it would be conceptually important to discriminate the effects of the two changes.

In our experimental model, tenotomy is caused by section of the Achilleus tendon of soleus muscle and degeneration by injection of bupivacaine in the muscle. The effects of tenotomy in control regenerating muscles will be compared with the effects in surgically or functionally denervated muscles. Functional denervation will be performed by blocking the sciatic nerve impulse conduction with tetrodotoxin (TTX). The possible presence of neurotrophic component in the influence of innervation on muscle regeneration will be tested by blocking axoplasmic flow in the sciatic nerve with vinblastine. Preliminary results show that tenotomy, similarly to hindlimb suspension causes an increased expression of 2A MHC isoforms. The vinblastine block apparently does not affect this effect of tenotomy. The effects of TTX block are under investigation.

DEMAND DYNAMIC CARDIOMYOPLASTY AND LD WRAP MECHANOGRAPHY: NEW CHARACTERISTICS OF A MYOSTIMULATOR (DEMAND LD-CARDIO PACER)
TO PHYSIOLOGICALLY ACTIVATE LD WRAP AND CLINICALLY DETERMINE ITS DYNAMIC CHARACTERISTICS

F Monese, F Di Gregorio, U Carraro (1)

MEDICO s.p.a., Rubano (Padova), (1) Department of Biomedical Sciences, University of Padova, Italy

In Dynamic Cardiomyoplasty the fully transformed Latissimus Dorsi (LD) is highly fatigue-resistant, but slower and weaker than myocardium. To have fatigue-resistance at higher muscle power, we introduced the daily activity-rest stimulation [Muneretto C, et al. 1997. Basic Appl Myol 7: 55-56; Barbiero M, et al. 1999. Basic Appl Myol 9: 192-204; Carraro U, et al. 2000. The Annals of Thoracic Surgery 2000, in press], a sound result of burst-intermittent stimulation by animal experiments [Arpesella et al 1996. Basic Appl Myol 6: 341-350; Arpesella et al. 1998. Ann Thorac Surg 66: 1983-1990; Duan C, et al. 1998 Basic Appl Myol 1998; 8: 35-40; Kashem M, et al. 1998. Basic Appl Myol 8: 231-236; Duan C, et al. 1999. Am J Physiol 276: R1534-1540]. Clinically, Demand Dynamic Cardiomyoplasty (DDyC) guarantees the activity-rest regime by a cardiac rate-based demand stimulation. Every day LD wrap rests during periods of hours, e.g., during day and night while the patient is asleep. A new non invasive diagnostic tool (LD mechanogram) determines dynamic characteristics of LD wrap by a combined polygraph-echocardiography analysis of tetanic fusion frequency threshold (TFF), a functional index of LD fibre-type transformation [Carraro U, et al. 1998. J Cardiovasc Diagn P 15: 115-125]. We are developing a dedicated cardiomyostimulator with new options for both demand stimulation and testing of the LD wrap. MEDICO spa of Rubano (Padova) collaborates with human and financial resources to develop the Demand LD-Cardio Pacer. An accessory for echocardiography, a phono/pressure transducer optimised for the LD wrap recordings, will be also developed to non-invasively analyse dynamic characteristics of the LD wrap.

With commercial cardiomyostimulators, the activity-rest stimulation is obtained using a cardiac frequency cut-off, which could only be programmed in steps of ten beat per minute (bpm). To avoid the worst case of almost no daily stimulation, the cardiostimulator ought to be either set to a quasi-continuous stimulation or to a demand stimulation with rare periods of rest. The solutions to this problem are either to decrease to 1 bpm the setting steps or add clock and counter to a new device, so that to limit to 30,000 the electrical impulses delivered per day. Characteristics of the new cardiomyostimulator (Demand LD-Cardio Pacer) will be: 1. Interpulse intervals from 0 to 700 msec; 2. Number of impulses per train from one to six; 3. Intercalated-recovery stimulation during activity periods: cycles of 3 min activity followed by 6 or 9 min rest; 4 Activity-rest periods programmable in steps of one hour; 5. Maximum of impulses per day: 30,000.

Stimulator characteristics have been bench-tested. To test effects on the LD power and trophism of the new, physiological stimulation protocol, prototypes of the Demand LD-Cardio Pacer are implanted in a simplified sheep model of DDyC, in which the LD is mobilised 7/14 day-after a pre-operation of vascular delay [Santamore WP, et al. 1999. Basic Appl Myol 95-100; Chiang BB, et al. 1999. ASAIO J 45: 350-355; Ali AT, et al. 1999. An Thorac. Surg 67: 1304-1311].

The options of the Demand LD/Cardio Pacer would also include sequential settings, which allow to automatically perform mechanography of LD wrap during clinical follow-up. It would be possible to: i) automatically analyse stimulation threshold; ii) optimise synchronisation of LD wrap contraction-relaxation during ventricular-systole; and iii) determine tetanic fusion frequency of LD wrap, to monitor over time dynamic contractile characteristics of the LD wrap.

Supported by Italian M.U.R.S.T. Contract n. 98-06192428 - "Italian Trial of Demand Dynamic Cardiomyoplasty (TiCCD)"

IS INJURY TO THE TORACODORSAL NERVE PRESENT AFTER LONG-TERM DYNAMIC CARDIOMYOPLASTY IN GOATS?

E Monnet, EC Orton, G Chield, (1) D Getzy, (2) G Jacobs, L Metelman

Department of Clinical Sciences, College of Veterinary Sciences, Colorado State University, Fort Collins, Co; (1) IDEXX Veterinary Services, Broomfield, Co; (2) Teletronics Pacing Systems, Inc, Englewood, Co, USA

Long term dynamic cardiomyoplasty is associated with atrophy, fibrosis, and fatty infiltration of the Latissimus Dorsi muscle (LDM). Ischemia, decreased muscle preload and chronic electrical stimulation have been recognized as factor for muscle deterioration after cardiomioplasty. Transposition of the LDM around the heart might be associated with damages to the thoracodorsal nerve that can contribute to LDM deterioration. The purpose of the study was to evaluate the neuromuscular function of the thoracodorsal and LDM after dynamic cardiomyoplasty. WE performed neuromuscular functional analysis and histology on the LDM and thoracodorsal nerve of 6 normal goats and 6 goats after 6 month of dynamic cardiomioplasty. Electromyographic analysis showed positive sharp waves and fibrillation potentials in the LDM of 3 goats from the dynamic cardiomioplasty group. Conduction velocity of the thoracodorsal nerve of goats from the dynamic cardiomioplasty group (58.32±9.80 m/s) was reduced compared to the goats from the control group (71.48±5.71 m/s, p=0.02).

Loss of myelin sheaths, collapse of endoneural connective tissue, and solitary foci of axonophagia and myelinophagia further documented severe injury to the thoracodorsal nerve in goats from the dynamic cardiomioplasty group. The LDM wrap denervated after long term dynamic cardiomyoplasty.

TROPONINE C PLASTICITY IN UNLOADING CONDITIONS

Y Mounier, P Kischel, B Bastide, L Stevens

Laboratoire de Plasticité Neuromusculaire, Université des Sciences et Technologies de Lille. F 59655 Villeneuve d’Ascq

Troponin C (TnC) plays a key role in the regulation of muscle contraction, thereby modulating the Ca^+±activation characteristics of skinned muscle fibers. This study was performed to assess the effects of hindlimb unloading (HU) on TnC expression and the related functional changes observed in the slow postural muscles.

We investigated the TnC isoform expression by immunoblotting in whole soleus muscles, and in single fibers also checked for their Ca⁺⁺ activation characteristics (maximal force P₀, Tension/pCa relationships) and for their pharmacological reaction to bepridil, a Ca²⁺ sensitizer targeting TnC and known to exert a differential effect on slow and fast fibers.

In the whole soleus muscle, the slow and fast TnC isoforms, TnCs and TnCf, were expressed. The low amount of TnCf found in control conditions clearly increased after HU while the TnCs content decreased. In single fibers from control muscles, TnCs was either expressed alone (slow fibers) or coexpressed with TnCf in hybrid fibers (hybrid slow or hybrid fast fibers, depending on the predominant isoform). TnCf was never expressed alone. After HU, the increase in the TnCf proportion was due to the increase in the proportion of hybrid fast fibers, to the detriment of the pure slow fiber population. These hybrid fast fibers were functionally identical to the control ones : similar pCa threshold, pCa50, Hill coefficient nH and bepridil-sensitivity. In the slow (pure or hybrid) fibers, no TnC modifications were found after HU when compared with control slow fibers although some functional changes were revealed.

Therefore, the TnC isoform could account for the functional shift in the transformed fibers, but results obtained on the remaining slow fibers suggested changes in other regulatory proteins.

This work was supported by grants from the Centre National d’Etudes Spatiales and the Fonds Européen de Développement Régional (FEDER n° F007).

EXERCISE LACTATE ANAEROBIC THRESHOLD IN HEREDITARY SPASTIC PARAPLEGIA

L Pasquali, ML Manca, E Pastorini, FM Santorelli, G Siciliano

Department of Neurosciences, Neurological Clinics, University of Pisa

Hereditary spastic paraplegia (HSP) is a group of familial neurodegenerative disorders characterized by progressive lower limb spasticity and weakness due to degeneration of corticospinal axons. These disorders are classified both genetically, according to the mode of inheritance, and clinically, as pure and complicated forms. These latter are characterized, in addition to progressive lower limb spasticity, by other neurological abnormalities such as optic neuropathy, retinopathy, extrapiramidal disturbance, dementia, ataxia, ichthyosis, mental retardation and deafness. Recently gene mutations of a mitochondrial protein, the paraplegin, have been reported in some HSP patients.

The aim of our study was to evaluate exercise lactate anaerobic threshold (LAT) in 5 patients (4 M and 1 F, mean age 45.6 ? 16.3 yrs) affected by HSP, both autosomal dominant or sporadic. During intermittent submaximal cycling at increasing power output levels, we also assessed plasma epinephrine and norepinephrine as well as cardiorespiratory parameters.

Analysis of venous lactate curve showed that lactate anaerobic threshold was reached at 50% of predicted maximal power output in patients vs 60–70% in normal controls. Furthermore peak values of examined parameters were significantly higher in HSP individuals with respect to controls: 379 vs 271 mmol/L for lactate, 461 vs. 210 pg/ml for epinephrine, 369 vs. 299 pg/ml for norepinephrine, respectively.

Analysis of lactate data could suggest a possible mitochondrial involvement in HSP patients. Furthermore, the abnormal lactate production appears not independent of catecholaminergic response to exercise.

THE TRANSCRIPTIONAL PROFILE OF THE AGING PROCESS IN SKELETAL AND CARDIAC MUSCLE

TA Prolla

University of Wisconsin, Madison, WI, Usa

The gene expression profile of the aging process was analyzed in skeletal muscle of mice. Use of high-density oligonucleotide arrays representing over 20,000 genes revealed that aging resulted in a differential gene expression pattern indicative of a marked stress response and lower expression of metabolic and biosynthetic genes. Most alterations were either completely or partially prevented by caloric restriction, the only intervention known to retard aging in mammals. Transcriptional patterns of calorie-restricted animals suggest that caloric restriction retards the aging process in skeletal muscle by causing a metabolic shift toward increased protein turnover and decreased macromolecular damage. We are currently applying the same technique to cardiac muscle.

A NEW TECHNIQUE TO EVALUATE SYSTOLIC ASSISTANCE IN DEMAND DYNAMIC CARDIOMIOPLASTY USING AORTIC FLOW VELOCITY MEASURED BY INTRAVASCULAR DOPPLER FLOW WIRE

G Rigatelli, M Zanchetta (1), M Barbiero (2), L Pedon (1), P Maiolino (1), U Carraro (3), S Dalla Volta

Cardiology Chair, University of Padova; (1) Division of Cardiology, Cittadella General Hospital, Padova; (2) Division of Cardiology and Centre for Advanced Heart Failure, Legnago General Hospital, Verona; (3) Department of Biomedical Sciences, University of Padova

There are no certain data regarding real systolic assistance that cardiomyoplasty could offer to patient with progressive heart failure. The main reason is the results of transthoracic echocardiography of aortic flow velocity, a good index of cardiac function. We describe a new technique which measures aortic flow velocity using intravascular Doppler flow wire in order to test systolic cardiac assistance during stimulated best in dynamic cardiomioplasty.

Methods: the technique has been tested in five coronopathic patients (M/F=4/1; age±61-71; Atrial fibrillations/sinus rhythm=1/4; VTD=76,6±8,26 ml/mq; EF=67,4±8,56) with no aortic valve stenosis and reflow or aortic ectasia and aneurysmas shown by transthoracic echocardiography. The analyses were performed during normal cardiac left catherization for coronarography, in order to study stability of the wave and repeability of velocity flow data. Through a 5F introducer femoral arterial access a Flex™ Doppler flow wire of 018 inch was advanced under fluoroscopy control into a coronary 4F Judking Right catheter an positioned in thoracic aorta just below the istimic portion. Stimulated beast were obtained with a right ventricle stimulator catheter through a femoral venous access. Then the flow wire was connected with its digital display and a registration of flow velocity during normal beat and after a stimulated beat was performed. The data expressed in cm/s and calculated as mean±SD, were analysed with T test and a vakue of P<0.05 was considered significant.

Results: Four series of measurement, composed by 10 normal beats and 1 stimulated beat, were recorder: we measured the maximal peak velocity of the beat n.9 (normal) and beat n.1 (post-stimulated beat) of the subsequent series. The values respectively for normal beats and for post-stimulated beats were: -first series: 50.4±13.5 and 63.4±17.5 cm/s (P=0.0091); - second series:49.8±8.4 and 62.4±17.7 cm/s (P=0.0249); -fourth series: 56.8±15.4 and 63.4±13.3 cm/S (P=0.0024). No complication were observed after the procedure.

Discussion: Statistical analysis shows significant increasing in peak flow velocity after the stimulated beat. Therefore that technique may be sensitive enough to detect a similar phenomenon related to a systolic assistance due to contraction of Latissimus Dorsi wrap. The technique is safe and effective. Flow velocity data are repeatable and their acquisition is simple. These potentials and the possibility to perform the measures through a radial or brachial arterial access, may allow achieving by a relatively simple approach final data on a true systolic assistance to a failing cardiac function in demand dynamic cardiomioplasty.

MYOBLAST-MEDIATED DELIVERY OF TIGHTLY REGULATED THERAPEUTIC GENES

FMV Rossi, CR Ozawa, M Springer, HM Blau

Department of Molecular Pharmacology, Stanford University, Stanford, CA 94305-5332, USA

We have developed protocols that allow the isolation of primary myoblasts from muscle biopsies of a variety of species, including mouse and human. These cells can be easily expanded in vitro to obtain large numbers of myoblasts that can be further manipulated. Using efficient retroviral delivery methods, therapeutic genes can be introduced and expressed at high levels in polyclonal populations of myoblasts. Following in vitro characterization, these populations can then be re-introduced into syngeneic living subjects by simple intramuscular injection. Due to their inherent plasticity, a large proportion of the injected myoblasts will survive and fuse into the resident muscle fibers surrounding the injection site, thus delivering their genetic material to the endogenous tissue. This results in expression and appropriate processing of the exogenous therapeutic protein in the host muscle.

The injected myoblasts show no detectable tendency to migrate to sites of muscle injury. This, combined with the practical hurdles associated with delivering large number of cells to poorly accessible sites (such as the diaphragm), makes it unlikely that cell-autonomous genetic defects such as muscular dystrophies could be corrected using this methodology. However, myoblasts can be used to efficiently deliver secreted factors with either local or systemic therapeutic effects. Indeed, several indications suggest that myoblast-mediated delivery is among the most efficient methodologies to express exogenous genes in vivo, yielding both high levels of expression and long term persistence.

While the high efficiency of myoblasts-mediated gene delivery has several advantages, it is now clear that efficient gene expression presents new problems. For example, we have recently shown that when myoblasts were used to deliver vascular endothelial growth factor (VEGF), the high levels of VEGF obtained induced disorganized proliferation of the host endothelial progenitors and led to the formation of large hemangiomas. Thus, it is of critical importance that the timing and the levels of expression of the exogenous gene can be efficiently and tightly regulated.

Over the past two years we have developed retroviral vector systems that allow the integration of tetracycline (tet) regulated expression cassettes in the genome of the infected cells (Retrotet system). With these vectors, polyclonal populations of myoblasts expressing tet-controlled exogenous genes can readily be obtained. Our method eliminates the need for clonal selection, which often results in cells with altered growth and differentiation characteristics. When fully induced, this system yields expression levels comparable to those obtained with wild type retroviral constructs. Basal expression in non-induced conditions is low, and can be further reduced by expressing both the traditional tet-controlled transcriptional activator (rtTAb) and a novel tet-regulated transcriptional repressor (tTRg) in the same cells. In this regulatory network, tTRg binds the inducible promoter in the absence of inducer and actively represses expression of the gene of interest. In the presence of the tet analog doxycycline, tTRg binding is inhibited and rtTAb can now occupy the inducible promoter and activate expression. Using this novel system, up to six orders of magnitude induction can be obtained in primary myoblasts in vitro.

Preliminary experiments aimed at assessing the efficiency of this system in vivo upon transplantation of the inducible myoblasts are currently ongoing, and will be presented.

TIME-COURSE OF EXERCISE AND APOPTOSIS IN THE MDX MICE

K Rossini, A Donà, M Sandri, U Carraro

C.N.R. Unit for Muscle Biology and Physiopathology, Department of Biomedical Sciences, University of Padova, viale Colombo, 3 35121 Padova, Italy

The runners were housed in a cage with an exercise wheel and allowed to run spontaneously for two hours or for an entire night (about 12 hours) and, the morning after, Tibialis Anterior of both hindlimbs removed. We checked the activity of mice by computer monitoring the covered distance, the average speed, and the time when the wheel was moving. Apoptosis was assessed by the terminal deoxynucleotidyl transferase assay and expressed as number of apoptotic nuclei for mm of muscle tissue, and by electron microscopy for morfological features.

In 12-hours running groups mdx mice present a significant minor activity (18±0.7) and in comparison with 2-hours mdx runners (36±3.5 p<0.001). Consequently, it seems that normal mice work constantly while mdx mice have an activity peak after 2-hours exercise and then it decreases maybe because mdx muscular fibers are frailer, more fatigable and susceptible at exercise-induced damage than normal ones.

Control non-runner mdx mice present 40±13 apoptotic myonuclei/mm³, 2-hours runner mice 84±13 (p=0.04 against control) and 12-hours mice 158±32 (p=0.04 against control; p=0.23 against 2-hours), while in 12-hours mdx runners interstitial nuclei/mm³ (188±46) significantly decrease in comparison with 2-hours group (348±50). Electron microscopy confirm that apoptotic myonuclei increase after 2-hours running in comparison with sedentary and some more after 12-hours running. Besides it show that apoptotic process involve satellite cells.

Beside confirming that apoptosis present in mdx mice at rest dramatically increases after exercise, results suggest that inflammation and interstitial apoptosis increase during a short-time exercise while the apoptotic process in myofibers (doubled in 2-hours runners in comparison with sedentary group) becomes more manifested after a long-term exercise, even if mdx mice activity decreases.

IS A HIGH INTRACELLULAR LIPID CONTENT AN ADAPTATIONAL RESPONSE TO A LOW CAPILLARY SUPPLY IN SKELETAL MUSCLE?

AM Saenger

Institute of Zoology, University of Salzburg, Austria

Slow (red, aerobic) axial muscle of three species of cyprinid fish was examined for intracellular lipid under three different conditions: laboratory-reared fish with and without locomotory exercise training, and fish living in the wild. Under each life regime, there was the same striking species-specific difference regarding intracellular lipid storage suggesting that accumulation of intracellular lipid within slow muscle is partly under the influence of factors other than environmental ones. A similar difference could be observed with volume densities of mitochondria. In contrary, the capillary supply of slow axial muscle was inverse what means: those fish with high mitochondrial content had low capillary supply indicating that the oxygen supply does not match the oxygen need. The negative correlation of intracellular lipid and capillary density strongly supports the idea that intracellular lipid facilitates oxygen transport from capillaries to mitochondria within the muscle fibres in those fish with a relatively low capillary density but high lipid content.

Saenger, A.M. (1999). Morphometric analysis of axial muscle in cyprinid fish provides support that intracellular lipid plays a role in slow fibre oxygen supply. J.Fish Biol. 54, 1029-1037.

ENDURANCE TRAINING AFFECTS WHITE AXIAL MUSCLE IN THE CYPRINID SPECIES CHALCALBURNUS CHALCOIDES MENTO (AGASSIZ, 1832), CYPRINIDAE, TELEOSTEI

AM Saenger, U Putscher

Institute of Zoology, University of Salzburg, Austria

The effect of an endurance training programme lasting 17 weeks was studied in the cyprinid species Chalcalburnus chalcoides mento (Agassiz, 1832), the Danube bleak. Red, intermediate and white axial muscle were investigated by means of histochemistry, electronmicroscopy and morphometry. Compared to other cyprinid species an endurance training regime affects more fast white muscle than slow red muscle. Qualitative and quantitative analyses revealed that training induced significant increases in fibre diameters of intermediate and white muscle fibres and capillary supply in red and white muscle whereas volume densities of mitochondria, lipid and myofibrils, and mATPase- and SDH-activity were unaffected.

APOPTOSIS AND MUSCULAR DYSTROPHIES

M Sandri, AH El Meslemani, C Sandri, K Rossini, R Betto, D Sandona, U Carraro

C.N.R. Unit for Muscle Biology and Physiopathology, Department of Biomedical Sciences, University of Padova, viale Colombo 3, 35121 Padova, Italy.

Apoptosis was detected in different muscular diseases, including severe dystrophin deficiency. However apoptotic regulatory mechanisms in skeletal muscle cells have not been studied in detail. Caspases, which are involved in apoptotic cell death of various cell systems, are sequentially activated through a processing by other members of caspases. We studied five patients affected by Duchenne Muscular Dystrophy (DMD) and eight patients affected by facio-scapulo-humeral dystrophy (FSHD), the results were compared with data obtained by skeletal muscle of normal subjects. We showed a significant increase of apoptotic myonuclei, of bax and bcl-2 positive myofibers especially in DMD patients. Moreover a positive correlation was detected between apoptotic nuclei and bax expression (p<0.01) while bcl-2 level didn’t correlate with skeletal muscle apoptosis (p=NS). The expression of 9 different caspases was analysed by RNase protection assay in dystrophic patients with highest apoptotic myonuclei and in normal subjects. We didn’t detect any caspase transcript in normal skeletal muscle. DMD muscles expressed caspase 8, 3, 5, 2, 7 and Granzyme B mRNAs. Caspase 6, 3 and Granzyme B transcripts were upregulated in FSHD. Since caspase 3 was commonly expressed in dystrophic muscles we studied caspase 3 localization by immunohistochemical analysis. Immunohistochemistry displayed a significant upregulation of caspase 3 in dystrophin deficient myofibers according to mRNA findings. Tissue levels of caspase 3 significantly correlated with apoptotic myonuclei (p<0.05) and with bax expression (p>0.01). Furthermore lysates of skeletal muscle showed that activity of caspase 3 increased in dystrophin deficient samples. In vitro we studied staurosporine-treated C2C12 myotubes to produce apoptosis and to evaluate the caspases involved in the process. A broad-spectrum caspase inhibitor significantly reduced the staurosporine-induced cell death. Inhibitors of caspase-1, -2, -5, and -6 were almost ineffective in protecting myotubes from apoptosis, Conversely, inhibitors of caspase-3, -8 and -9 gave 75% of protection and inhibit DNA fragmentation detected both by TUNEL and ladder assay. Furthermore, the activity of caspase-3 in cell lysates was significantly increased after staurosporine, an effect that was prevented in the presence of the specific inhibitor. Western blot analyses revealed processing of caspase-3 and of its nucleic protein target, PARP. These data indicate that human skeletal muscle fibers, during disease, modulate the expression of caspases and that caspase 3 is involved in myofiber cell death, opening new perspective in the pharmacological treatments of muscular dystrophies, such as pancaspase inhibitors.

NERVE ACTIVITY-DEPENDENT REGULATION OF THE MUSCLE PHENOTYPE: A NEW ROLE FOR RAS

S Schiaffino, M Murgia, AL Serrano, E Calabria, G Pallafacchina

Department of Biomedical Sciences, University of Padova, Italy

Nerve activity is known to affect the growth and diversification of skeletal muscle fibres, however the signal transduction pathways that couple depolarization at the muscle cell surface with transcriptional

changes in muscle nuclei are still unknown. To identify the mechanisms responsible for excitation-transcription coupling we have used an in vivo muscle regeneration model. In the absence of the nerve regenerating fibres in the rat soleus muscle express fast-type but not slow-type MyHC

transcripts. In contrast, innervation induces rapid up-regulation of MyHC-slow and concomitant down-regulation of fast MyHC isogenes. The MyHC changes induced by the nerve can be reproduced by electrostimulation of denervated muscle with a continuous low-frequency pattern that resembles the activity pattern of normal soleus motor units. We have transfected regenerating muscles with constitutively active or dominant interfering Ras mutants. Activated Ras up-regulates MyHC-slow and down-regulates fast-type MyHC genes in regenerating denervated muscle, thus mimicking the effect of slow motor neurons. Conversely, the up-regulation of MyHC-slow induced by slow motor neurons is inhibited by dominant negative RasN17. Selective activation of different pathways downstream of Ras was found to affect differentially muscle fibre growth and fibre type differentiation. RasV12S35, that activates the MAPK (ERK) pathway was able to induce

MyHC-slow but not muscle growth, whereas RasV12C40, that activates the PI3K pathway, affects muscle growth but not MyHC gene expression. Ras has been traditionally associated with the inhibition of myoblast fusion and muscle cell differentiation in culture, however the present study points to a new role of Ras in the differentiation of the muscle phenotype by nerve activity.

Murgia M, Serrano AL, Calabria E, Pallafacchina G, Lømo T, Schiaffino S. (2000) Ras is involved in nerve-activity-dependent regulation of muscle genes. Nature Cell Biol. 2:142-147.

ASSOCIATION OF CELLULAR CARDIOMYOPLASTY WITH MULTISITE CARDIAC PACING

A Shafy, JC Chachques, P Argyriadis, L Shen, C Rajnoch, A Berrebi, JN Fabiani, A Carpentier

Department of Cardiac Surgery, Broussais Hospital, Paris, France

Current possibilities in cell therapy for heart failure is the transplantation into the myocardium of autologous myoblasts (originated from skeletal muscle), fetal cardiomyocytes, cells derived from bone marrow stem cells, and smooth muscle cells. Adult myocardium can not repair after infarction due to absence of stem cells. Cell transplantation strategies for heart failure have been designed to replace damaged cells with cells that can perform cardiac work.

Electromechanical coupling of the transplanted myoblasts with the cardiomyocytes is still not clear, but there is a possibility that transplanted myoblasts could be paced in synchrony with the cardiac cycle. Pacing would require sufficient voltage to activate all the transplanted myoblasts. In this study we explored the possibility to electrostimulate the ventricles following autologous skeletal cell implantation as a potential therapeutic option for heart failure.

Materials and Methods

Twenty sheep were included in this study. A model of myocardial infarction was created by ligation of 2 coronary branches (LAD and D2). Three weeks later we ejected 2,5 * 10 of autologous skeletal cultured myoblasts in the centre and in the periphery of infarcted areas. Multisite pacing of the right atrium + right and left ventricles was performed using a two chamber pulse generator ( Medtronic model 4710).

Four groups were studied:

Group 1 (n=5) Infarction ( control)

Group 2 (n=5) Infarction + Electrical stimulation ( control)

Group 3 (n=5) Infarction + Myoblast transplantation

Group 4 (n=5) Infarction + Myoblast transplantation + Electrical stimulation

Serum troponin was used to evaluate the infarction, one day before the procedure and for five postoperative days. Echocardiographic and immuno-histological studies were performed at 2 months.

Results: A model of a left ventricular infarction was established which is reproducible and applicable for investigation of therapeutic options in chronic heart failure.

20 animals underwent creation of left ventricular infarction, of which 2 died at extubation due to irreversible ventricular fibrillation. Serum troponin I started to rise 4 hours after creation of infarction, reach the peak at 48 hours (125.4±69.7 ng/ml) then returned to normal 4 days later. In 10 sheep primary myoblasts culture was established, 25 millions cells were obtained after 3 weeks of culture. Myoblasts pacing was established in 10 sheep with successful sensing of the right atrium followed by synchronous pacing of both ventricles after a short (70 ms) AV delay (normal delay at rest = 110 ms). Preliminary echocardiographic results seems to demonstrate a significant improvement in LV function and a reduction of the infarcted area in the Infarction + Myoblast transplantation + electrostimulation group.

In conclusion, combined cellular transplantation with multisite electrical stimulation could be a potential option to improve left ventricular function. Cell distribution, development of myotubes into the myocardium and the connection between cardiomyocytes and myoblasts will potentially be improved by associating cell therapy with cardiac pacing.

A POSSIBLE ROLE OF SPARC (SECRETED PROTEIN-ACIDIC AND RICH IN CYSTEINE) IN MYOGENESIS

M Shiozuka

Department of Basic Human Sciences, School of Human Sciences, Waseda University, Saitama, Japan

Our previous studies have demonstrated that follistatin enhanced myogenic differentiation in culture (Zool. Sci. 14 : 327-330 1997). This stimulatory effect was also elicited by its related molecules such as SPARC (Secreted Protein-Acidic and Rich in Cysteine, also known as osteonectin or BM-40). SPARC has been demonstrated to affect cellular interaction with matrix proteins, modulate cellular growth, bind to and/or regulate the activity of growth factors, and be expressed in many tissues including skeletal muscle.

In this study, we are using primary cultures of chick skeletal myoblasts from embryonic pectoral muscle and somites to study the independent and combined effects of SPARC and other myogenesis-regulatory factors by means of morphological, biochemical and immunocytochemical methods. Concomitantly, we examined the effect of alpha 2-macrogrobulin that is another activin-binding protein. Alpha 2-macrogrobulin has no effect on myogenesis and myogenesis-inhibitory activities of activin A and TGF-beta1. Application of exogenous SPARC to cultures resulted in the counteraction to myogenesis-inhibiting effect of activin A and the stimulation of myogenic differentiation.

Further, the spatiotemporal distribution of SPARC protein in the developing chick embryo and myogenic cells in culture was examined by immunohistochemical techniques. This protein was detectable in myoblasts and myotubes.

In addition, the stimulative effects of PDGF -AB and -BB on myogenesis were suppressed by SPARC. It is potentially important that only PDGFs that comprise B chain can stimulate myogenesis.

These in vitro findings suggest that SPARC is a possible important regulator of follistatin/activin and PDGFs systems in skeletal muscle development.

METABOLIC AND MUSCLE ADAPTATION TO AEROBIC TRAINING IN MITOCHONDRIAL MYOPATHIES

G Siciliano, ML Manca, M Renna, E Pastorini, L Murri

Department of Neurosciences, Neurological Clinics, University of Pisa

Mitochondrial myopathies (MM) with respiratory chain defects are multisystem diseases characterised by heteroplasmic, primary or secondary nucleus-driven, mitochondrial DNA mutations responsible for impairment of aerobic cell metabolism. At the skeletal muscle level involvement of respiratory chain function is the cause of insufficient ATP production and deranged metabolism, a main effect of which is represented by abnormal production of lactate. Recently, it has been reported beneficial effects of training on muscle performance in MM.

Aim of this study was to evaluate in 12 MM patients functional adaptation of skeletal muscle to aerobic training by evaluating metabolic and catecholaminergic responses to exercise. Data from histological and mitochondrial DNA (mtDNA) analysis of muscle biopsies from MM patients were related to metabolic and catecholaminergic parameters obtained during a constant-workload exercise performed at near lactate threshold (LT), before and after the supervised 10-week course of aerobic training.

A significant decrease in exercise peak values of lactate (38.6%, p < 0.01) was observed after training in MM patients; on the contrary the decrease was not significantly different from controls for catecholamines.

No relationship was found between the decrease in lactate and catecholamine peak values and number of ragged red fibers or cytochrome c oxidase negative fibers at muscle biopsy. On the contrary an inverse trend with the proportion of deleted mtDNA was observed.

The results show that lactate accumulation during exercise is decreased after aerobic training in MM, and that this decrease is not dependent from catecholaminergic adaptation to the training. Furthermore, the level of mutated mtDNA in muscle biopsy could be a useful predictor for the effectiveness of aerobic training program, suggesting some gene shifting mechanisms in mediating muscle adaptation to training itself.

THE CALCIUM-CALPAIN HYPOTHESIS FOR NEUROMUSCULAR DEGENERATION

A Stracher, MA Badalamente

Department of Biochemistry and Orthopaedics, State University of New York at Brooklyn and Stony Brook, NY, USA

It is well know that there is an abnormal calcium accumulation in DMD myofibers, as well as in murine mdx myofibers. The mechanism by which increased intracellular calcium in dystrophic myofibers causes muscle damage may be the activation of calcium-dependent proteases, now known as calpains, and has give rise to the formulation of a more common cytotoxic pathway known as Ca²⁺ /calpain hypothesis, which may be responsible for a number of related neurodegenerative disorders.

Inhibition of muscle degeneration by the tripeptide calpain inhibitor, leupeptin, was tested in vivo in a dystrophin-deficient mdx murine model. Our prior studies in rat and primate models, testing leupeptin as a calpain inhibitor for denervation atrophy, consistently indicated that this tripeptide inhibited myofiber degeneration after complete peripheral nerve severance with microsurgical repair. In a short-term control study intramuscular administration of leupeptin for 30 days into mdx mice inhibited muscle degeneration as assessed by histologic analysis. Calpain inhibition could be correlated with retention of myofiber size and our results suggest that this may be a promising treatment modality in human Duchenne muscular dystrophy.

CHRONIC AORTIC COUNTERPULSATION WITH LATISSIMUS DORSI: CLINICAL FOLLOW-UP. CARDIOMYOPLASTY COMPARISON

J Trainini, JL Barisani, J Mouras, E Cabrera Fisher, A Christen

Presidente Peron Hospital, Buenos Aires, Argentina

Dynamic aortomyoplasty is an alternative technique to heart transplantation. The goal of our study was to evaluate the benefits of aortic counterpulsation obtained by dynamic aortomyoplasty in patients with heart failure refractory to pharmacological treatment and contraindications to heart transplant or cardiomyoplasty.

In this study we compared preoperative and postoperative data of selected patients submitted who were treated with dynamic thoracic aortomyoplasty. This surgical technique wrap the right latissimus dorsi muscle flap around the ascending aorta. This muscle flap was electrically stimulated during diastole, following a muscle-conditioning protocol, to obtain diastolic augmentation. At twelve months follow-up period we evaluated, invasively and noninvasively, the hemodynamic and clinical effects of aortomyoplasty.

We observed a significant decrease in the number of hospitalizations (p<0.001), NYHA functional class (p<0.001), left atrial diameter (p<0.05), wedge pressure (p<0.05), left ventricular diameter (p<0.05) and pulmonary artery systolic pressure (p<0.05); and a significant increase in the 6-minute walking test (p<0.001), cardiac index (p<0.01), noninvasive evaluation of diastolic augmentation (p=0.01), left ventricular shortening fraction (p<0.05), and radioisotopic left ventricular ejection fraction (p<0.05). We also found a non significant decrease in peak oxygen consumption (p=0.05).

This nine patients operated with aortomyoplasty was compared with sixteen patients treated with cardiomyoplasty at twelve months of follow-up.

Dynamic biologic assistance with latissimus dorsi in heart failure, either aortomyoplasty or cardiomyoplasty in selected patients with severe heart failure, resulted in an important improvement of hemodynamic parameters, heart functional data and clinical functional, without significant differences between the results of both techniques.

A DATABASE OF TRANSCRIPTS EXPRESSED IN HUMAN SKELETAL MUSCLE

G Valle, S Toppo, N Cannata, A Pallavicini, P Laveder, C Ievolella, F Stanchi, B Pacchioni, S Trevisan, M Salamon, G Bortoletto, R Dioguardi, P Scannapieco, E Frigimelica, R Zimbello, B Simionati, G Lanfranchi

CRIBI Biotechnology Centre, University of Padova, Italy

The aim of this study is the systematic identification and characterization of most transcripts expressed in human skeletal muscle. Firstly, transcripts are discovered and identified by systematic sequencing of muscle cDNA libraries that are specifically produced for this purpose (Lanfranchi et al. 1996). The resulting Expressed Sequence Tags (ESTs) are the starting point of our work. Very often, in other similar projects, ESTs are not only used for the identification of new transcripts, but also for their preliminary characterization based on the analysis of the coding sequences. For our project the choice was to avoid any compromise and to produce cDNA libraries really optimized for the identification of transcript tags. Therefore, we have developed a protocol for the production of cDNA libraries containing short inserts, corresponding to the 400-500 bases at the 3’-end of the mRNAs. These regions of the mRNA are very little informative in terms of coding sequence; nevertheless, we believe that they are excellent transcript tags. Furthermore, since all the cDNA inserts are short and similar in size, we avoid any bias due to different lengths of the mRNAs and we can simplify the procedures for sample preparation, as well as the clustering procedures. The full-length sequence of the transcripts is produced either after screening and sequencing full-length cDNA inserts, or more recently, by searching the human genomic DNA sequence and inferring the putative coding sequence of each gene.

Currently we have identified more than 4000 independent transcripts. About 50% were found only once in our EST database, probably because their very low level of expression. The remaining 50% of the transcripts (more than 2000 sequences) were found two or more times. About 1300 muscular transcripts have been obtained as full length sequences either in our lab or by assembly sequences available in the public domain databases. Of the 2000 annotated sequences about 50% corresponds to transcripts with unknown function.

Several studies are currently under way in our laboratory for finding out the function of some of these genes, using several approaches including array hybridization, yeast two hybrids, recombinant proteins, immunocitochemistry and immunoelectromicroscopy. In particular we have focused our attention on several proteins of the Z-band, which were discovered in our laboratory such as telethonin, ZASP, HSPD355 and HSPD 2860 (Valle et al. 1997, Faulkner et al. 1999). Recently, in collaboration with several other group we found that telethonin is responsible for the autosomal recessive Limb Girdle Muscular Dystrophy of type 2G (Moreira et al. 2000).

The accession of the muscle transcript database is http://grup.bio.unipd.it/muscle.

References

1. Faulkner, G., Pallavicini, A., Formentin, E., Comelli, A., Ievolella, C., Trevisan, S., Bortoletto, G., Scannapieco, P., M. Salamon, Mouly, V., Valle , G. and Lanfranchi, G. (1999). ZASP: a new Z-band alternatively spliced PDZ-motif protein: Journal of Cell Biology 146, 465-475.

2. Lanfranchi, G., Muraro, T., Caldara, F., Pacchioni, B., Pallavicini, A., Pandolfo, D., Toppo, S., Trevisan, S., Scarso, S. and Valle, G. (1996). Identification of 4,370 expressed sequence tags from a 3’-end-specific cDNA library of human skeletal muscle by DNA sequencing and filter hybridization. Genome Research 6, 35-42.

3. Moreira, E. S., Wiltshire, T. J., Faulkner, G., Nilforoushan, A., Vaizof, M., Suzuki, O. T., Valle, G., Reeves, R., Zatz, M., Passos-Bueno, M. R. and Jenne, D. E. (2000). Limb-grindle muscular dystrophy type 2G (LGMD 2G) is caused by mutations in the gene encoding the sarcomeric protein telethonin. Nature Genetics 24, 163-166.

4. Valle, G., Faulkner, G., De Antoni, A., Pacchioni, B., Pallavicini, A., Pandolfo, D., Tiso, N., Toppo, S., Trevisan, S. and Lanfranchi, G. (1997). Telethonin, a novel sarcomeric protein of the human heart and skeletal muscle. FEBS Letter 415, 163-168.

BENEFICIAL EFFECTS OF THE ATII BLOCKER IRBESARTAN ON THE SKELETAL MUSCLE
OF RATS WITH CONGESTIVE HEART FAILURE

G Vescovo, B Ravara, M Sandri, L Dalla Libera

Internal Medicine Venice-Adria, CNR Unit for Muscle Physiopathology Padua, Italy

It is known that CHF is accompanied by a skeletal muscle myopathy characterized by and increased expression of fast MHCs and fibres type, myocyte atrophy, increased fatigability and decreased endurance. Skeletal muscle atrophy is secondary to myocyte apoptosis that is probably triggered directly or indirectly by the high levels of circulating TNF-?. ATII receptors are thought to play a role in the control of apoptosis. We tested the hypothesis that ATII receptor blockade could prevent skeletal muscle apoptosis and therefore the development of the myopathy in rats with CHF.

CHF was induced by injecting 30 mg/kg monocrotaline in 18 80-100 gr Sprague Dawley rats. 5 rats were injected with saline and served as controls. After 2 weeks 11 out the 18 rats were treated with 7 mg/kg/die Irbesartan, delivered through Alzet osmotic minipumps. After 2 additional weeks rats were sacrificed and Tibialis Anterior muscle and heart were excised.

Cross Sectional Area (CSA) was measured on computerized planimetry, MHCs composition was determined electrophoretically, myocyte apoptosis was detected with the TUNEL method, Bcl2 and Caspase3 were determined with immunoblotting, and TNF? with immunoenzymatic assay.

BW LV/RV M/V CSA MHC2a MHC2b TUNEL+ CASP3 BCL2 TNF

CONTROL 113+5 3.5+0.6 1.12+0.7 211+30 26
+1.7 74+1.7 1.2
+2.8 100
+8 97
+12 19.0
+1.7

CHF 116+7 2.7+0.6 0.25+0.15 137+43 18
+3.0 81+3.0 39.6
+42.7 206
+80 70
+16 25.2
+1.3

IRBES 121+6 2.2+0.5 0.39+0.06 173+31 25
+6.3 75+6.3 12.3
+12.7 188
+71 94
+35 22.1
+2.2

Irbesartan, though was unable to modify the severity of CHF as demonstrated by the LV Mass Volume ratio, was able to protect from the development of myocyte apoptosis and therefore from muscle atrophy. The MHCs composition with Irbesartan treatment did not shift toward the fast isoform. Circulating levels of TNF? were also decreased in the Irbesartan group, suggesting once again that apoptosis could be reduced by blocking TNF?. It can be postulated that ATII receptor blockade could protect from the development of skeletal muscle apoptosis.

DYNAMIC CARDIOMYOPLASTY IN A GROWING ORGANISM

B Voss, M Thielmann, M El-Mehsen, PA Schnabel (1), S Hagl (1), R Lange

Department of Cardiac Surgery, Deutsches Herzzentrum München, and (1) Departments of Cardiac Surgery and Pathology, University Hospital Heidelberg, Germany

Until now cardiomyoplasty has been a treatment option for adults only. However, there may be a demand for cardiomyoplasty in children. The purpose of this study was to investigate the possibility to apply the method of cardiomyoplasty before growth is completed.

Methods: The latissimus dorsi muscle (LD) was wrapped around the heart of 20 "Göttinger Minipigs", (9.1 ? 1.2 kg/BW). The LD was fixed to the pericardium in group 1 (n=11) and to the epi-myocardium in group 2 (n=9) and stimulated with burst-impulses. After 5.6 ? 1.8 months hemodynamic and histological follow-up-examinations were carried out in 13 surviving animals (weight 32.4 ? 5.3 kg).

Results: In group 1 (n=6) only the left ventricle was covered by the LD. In 4 animals the LD contracted strongly, in 2, the outer border of the muscle was atrophied. In group 2 (n=7) both ventricles were covered by the LD in all animals and showed strong contractions. In 2 animals the outer border of the muscle was atrophied. In both groups the contracting parts of the LD showed an intact muscle structure, but compared to the contralateral LD, there was a higher percentage of interstitial fat and connective tissue. Hemodynamic measurements and the well-being of the animals suggest, that restriction of cardiac chamber diameter did not occur. The electrical stimulation of the LD caused a minimal increase of left ventricular pressure and aortic peak flow in group 2.

In conclusion, Cardiomyoplasty can be applied in a growing organism. There is growth of the LD with the heart. The muscle structure remains intact. To prevent dislocation of the LD, it seems to be important to fix the LD directly onto the epi-myocardium.

MUSCLE-SPECIFIC TRANSCRIPTION OF b-MYOSIN HEAVY CHAIN TRANSGENE REQUIRES
AN A/T-RICH ELEMENT

DR Vyas (2), JJ McCarthy (2), GL Tsika (2), RW Tsika (1,2,3)

(1) Department of Biochemistry, College of Medicine, (2) Department of Biomedical Sciences, College of Veterinary Medicine, (3) Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, Missouri

The expression of the endogenous bMyosin Heavy Chain (bMyHC) gene is restricted primarily to slow-type I skeletal muscle fibers of small adult rodents and its expression pattern can be changed dramatically in response to altered neuromuscular activity such as, mechanical overload (MOV) or non-weight bearing (NWB) (1, 2). Previously we have demonstrated that a transgene comprised of 293-base pair of bMyHC promoter (b293) mimics the expression pattern of the endogenous bMyHC gene during development and in response to MOV (3). The specific intracellular signaling pathway(s) underlying fiber-type and/or malleable bMyHC transcription has not been defined as yet, although recent efforts have led to a working model of intracellular signaling and skeletal muscle fiber-type-specific gene transcription. This model postulates that slow type I fiber-specific gene transcription occurs via activation of a calcium/calmodulin-regulated calcineurin-dependent intracellular signaling program that involves interactions between nuclear factor of activated t-cells (NF-AT) and myocytes enhancer factor-2 (MEF2) proteins. Activation of the cellular phosphatase; calcineurin, by chronically elevated intracellular calcium levels mediates nuclear translocation of NF-AT transcription factor(s) and subsequent transcription of NF-AT dependent target genes (4, 5). Interestingly, slow-type I skeletal muscle fibers have been reported to have 2 to 6–fold higher basal levels of intracellular calcium than fast-type II fibers. Since MOV is associated with an increased proportion in slow-type I fibers while NWB leads to reduced numbers of slow-type I fibers (1, 2), we have examined the involvement of consensus NF-AT and A/T-rich elements in modulating b293 transgene transcription in response to these two diverse stimuli. Previously, we have shown by direct, competition and supershift electrophoretic mobility shift assays (EMSA) that the highly enriched specific MOV binding complex formed at the bA/T-rich element (-269 5’-GGAGATATTTTT-3’ –258) was antigenically distinct from GATA-4, MEF2A-D, SRF, and Oct-1 (6). We extend these findings herein by showing that the MOV-P/bA/T-rich binding complex is antigenically distinct from NF-AT proteins (NF-AT p, c, 3, and 4) and that ³⁵S-methionine-labelled in vitro translated NF-AT isoproteins bind an interleukin-4 NF-AT element, but not the bA/T-rich element. In contrast, when either the IL-4 (5’-TGGAAAAT-3’) or bMyHC NF-AT (bNF-AT: -179 5’-TGTTTCCT-3’ –171) elements are reacted with either control plantaris (CP), MOV-P, control soleus (CS) or NWB-S nuclear extract, multiple complexes formed; one that was immune-depleted by rel homology domain antisera, and supershifted by NF-ATc specific antisera only. Both NF-AT elements demonstrated binding to ³⁵S-methionine-labelled in vitro translated NF-AT isoproteins. In contrast to the slight but non-significant difference in NF-AT binding activity observed between CP and MOV-P nuclear extracts, NF-AT binding activity was significantly reduced in NWB-S nuclear extract when compared to CP, MOV-P and CS nuclear extracts. Independent mutation of the bA/T-rich element within the context of transgene b293 resulted in the complete loss of transgene expression in the soleus muscle adult transgenic mice. Analysis of b293NF-ATmut transgenic lines is currently under way. These data clearly show that the independent input from the bA/T-rich element is required for basal slow-twitch muscle-specific expression of chromosomally located transgene b293 in adult transgenic mice. Further, in vitro analysis suggests a role for the bNF-AT element in transgene b293 expression under conditions of altered neuromuscular activity. Additional work will be required to clarify whether the bA/T-rich and bNF-AT element collaborate to direct slow fiber-specific expression and responsiveness to altered mechanical loads.

References:

1. Tsika, G. L., J. L. Wiedenman, L. Gao, J. J. McCarthy, K. Sheriff-Carter, I. D. Rivera-Rivera and R. W. Tsika. Induction of b-MHC transgene in overloaded skeletal muscle is not eliminated by mutation of conserved elements. Am. J. Physiol. 271 (Cell Physiol. 40): C690-C699, 1996.

2. J. J. McCarthy, A. M. Fox, G. L. Tsika, L. Gao and R. W. Tsika. b-MHC transgene expression in suspended and mechanically overloaded/suspended soleus muscle of transgenic mice. Am. J. Physiol. 272 (Regulatory Integrative Comp. Physiol. 41): R1552-R1561, 1997.

3. McCarthy, J. J., D. R. Vyas, G. L. Tsika and R. W. Tsika. Segregated regulatory elements direct b-myosin heavy chain expression in response to altered muscle activity. J. Biol. Chem. 274(20):14270-14279, 1999.

4. Chin, E. R., E. N. Olson, J. A. Richardson, Q. Yang, C. Humphries, J. M. Shelton, H. Wu, W. Zhu, R. Bassel-Duby, and R. S. Williams. A calcineurin-dependent transcriptional pathway control skeletal muscle fiber type. Genes and Devel. 12:2499-2509, 1998.

5. Naya, F. J., B. Mercer, J. Shelton, J. A. Richardson, R. S. Williams and E. N. Olson. Stimulation of slow skeletal muscle fiber gene expression by calcineurin in vivo. J. Biol. Chem. 275(7):4545-4548, 2000.

6. Vyas, D. R., J. J. McCarthy and R. W. Tsika. Nuclear protein binding at the ?-myosin heavy chain A/T-rich element is enriched following increased skeletal muscle activity. J. Biol. Chem. 274(43):30832-30842, 1999.

DYNAMIC CARDIOMYOPLASTY: CLINICAL EXPERIENCE AFTER SEVEN YEARS

C Werling, W Saggau

Department of Cardiac Surgery, Herzzentrum Ludwigshafen, Germany

Since 1993 Dynamic Cardiomyoplasty (CMP) was performed in 23 patients aged between 33 and 68 years. Two patients suffered from ischemic and 21 patients from dilated cardiomyopathy. Mean preoperative NYHA class was III, LVEF 22%, CI 2,0, LVEDP 22mmHg, PAPsys 44 mmHg, VO2max 15,4 ml/kg/min and LVEDD 69mm. Sinus rhythm was found in 15 patients, 8 patients had an atrial fibrillation, 4 of them intermittent. Two patients had documented sustained VT and two patients VF with successful reanimation. Therefore 4 patients had an preoperative indication for an ICD implantation

CMP was performed without concomitant surgery using the left latissimus dorsi muscle. For training of the muscle the standard protocol was used, in 3 cases the progressive pulse number protocol was followed. Chronical stimulation was performed with 6 pulses every second beat.

As seen by other groups, there was only a moderate change of conventionally measured hemodynamics, whereas clinical status improved significantly. Perioperatively one patient with ischemic cardiomyopathy died immediately after the procedure because of acute myocardial infarction, a second one after 5 days because of VF and one patient after 12 days because of a sepsis after an acute infection of the gall bladder. In the follow up period of 86 months 8 late deaths occurred. Three patients had a sudden death after 3, 6 and 25 months, 2 patients died of heart failure after 1,5 and 14 months and 3 patients had a noncardiac cause of death, two after 17 and one after 22 months.

At the beginning of our experience, patients were applied to us, who not only had physical contraindications for transplantation as age, but also psychosocial contraindications. In 1996 four of ten of our patients lived in psychosocial unstable conditions, for instance were homeless or were alcohol or drug abusers. All cardiac diseased belonged to this risk group. From 1996 we saw unsolved psychosocial problems as a relative contraindication for cardiomyoplasty. Since then only 2 of 13 patients from this risk group were operated and one of them also had a cardiac death.

Until now we provided 5 patients with ICD and cardiomyostimulator, 3 of them before and 2 after CMP. All of them had episodes of VT or VF in the follow up period. On the other hand we lost 4 patients because of sudden death. Our total survival rate amounts to 52% after 7 years. If you only take in account the cardiac deceased the survival rate is 70% and if you exclude the patients, who had a sudden death and were not provided with an ICD, the survival rate would be 87% and probably could be highly improved by combining an ICD with an cardiomyostimulator in every patient.

LOCOMOTOR (LAUFBAND) THERAPY IN SCI PERSONS

A Wernig, A Nanassy and S Mueller

Dept. Physiology Univ. Bonn and Klinikum Karlsbad-Langensteinbach, Germany

Locomotor training on the treadmill (Laufband-therapy) focuses on intensive walking in upright position, facilitated by body weight support via a harness, the moving band of the treadmill and initial limb setting by two therapists if necessary. These principles are derived from recent observations in spinal animals on activity-related "learning" of the isolated spinal cord and on the "rules of spinal locomotion" in lower vertebrates.

First, results of a 5-year study are reported in which 89 incompletely paralyzed (44 chronic and 45 acute) patients exercised on the Laufband are compared with a total of 64 patients treated conventionally for comparable periods of time (median 10.5 weeks). Laufband therapy achieved significantly better results in all comparisons (Wernig et al., Europ.J.Neurosci.7,823-829, 1995).

A number of chronically wheelchair-bound patients (not capable of raising from the wheelchair or walking without help from other persons) became independent and walked with help of a rollator or two canes for distances of at least 100 meters. Most chronic patients not capable of stair case walking learned to do so either by themselves or with help from another person following Laufband therapy. Also acute patients treated on the Laufband achieved better results than conventionally treated patients.

The results of a follow-up evaluation are reported, in which walking capability of Laufband treated patients, immediately following therapy, is compared with that after 1-6 years in domestic surrounding. All but one originally chronic patients and all originally acute patients maintained or even improved their walking capability (Wernig et al., Spinal Cord, 36, 744-749,

Supported in parts by Deutsche Stiftung Querschnittlähmung

HUMAN MUSCLE TISSUE GROWN IN MICE

A Wernig, R Schäfer, U Knauf, A Irintchev, M Zweyer, LVB Anderson* and E Kremmer#

Department of Physiology, University of Bonn, Wilhelmstrasse 31, D-53111, Bonn, Germany, * Neurobiology Department, University Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK, # GSF, Institute of Clinical Molecular Biology and Tumor Genetics, München, Germany

In order to create a test model for human muscle we xenotransplanted human myoblasts into muscles of immune competent and incompetent (SCID, SCID-b) adult mice. Immunosuppression of normal C57bl/10 mice consisted of daily cyclosporin A (CsA) administration (50 mg/kg s.c.) plus weekly injection of a T-cell depleting monoclonal antibody against Thy-1.2 (mouse IgG2a, 300 mg/animal). Host muscles were pretreated by cryodamage with or without x-irradiation (16 Gy). Four weeks after implantation nuclei in small and large diameter muscle fibers labelled with the human specific ALU-DNA probe. These muscle fibers also expressed human proteins as indicated by immunostaining with human specific antibodies against dystrophin (Dy10/12B2) and neural cell adhesion molecule (NCAM). Such cells were innervated as judged from the presence of Ach receptors and Ach esterase. Few fiber profiles expressed both human and mouse proteins. Human tissue could also be demonstrated employing quantitative ELISA. For dystrophin and chemiluminescence coupled with the ALU-DNA probe. The amount of donor-derived tissue was lowest without pretreatment, higher after mild cryodamage and highest after irradiation plus severe cryodamage of host muscles. Thus, tissue formation by human myoblasts, similar to mouse myoblasts, is dependent on pretreatment of muscle.

SKELETAL MUSCLE VENTRICLES IN THE PIG: LEFT VENTRICULAR OFF-LOADING DEMONSTRATED BY OBSERVATION OF PRESSURE-VOLUME LOOPS

SM West, JP Blackhurst, S Salmons and JC Jarvis

Department of Human Anatomy and Cell Biology, The University of Liverpool, New Medical School, Ashton Street, Liverpool L69 3GE, UK

Skeletal muscle ventricles (SMVs) were made from conditioned latissimus dorsi (LD) muscles in anaesthetised pigs. They were lined with artificial or biological elastic linings to make them blood-tight, and connected to the descending aorta via a single conduit. The posterior border of the LD muscle formed the mouth of the SMV. The SMV was activated in every third cardiac cycle, at various times within the cardiac cycle that were determined by the delay from the previous R-wave and the duration of SMV activation. Left ventricular (LV) work was monitored in real time by means of a pressure transducer and an impedance catheter placed within the LV. The volume of the SMV was monitored by three pairs of sonomicrometer crystals. The pressure within, and the flow out of, the SMV were also monitored.

Suture lines were designed to accommodate the variable thickness of the porcine LD so that the formed SMV had an adequate outlet tract and contracted consistently along its length. The use of an internal elastic lining reduced the effective preload on the muscle wall with only minor reduction in its ability to generate pressure and flow.

SMVs were able to produce stroke work larger than that of the native LV and substantial diastolic aortic pressure augmentation was observed in the assisted cycles. The LV pressure-volume loops showed the expected reduction in area in the beat following SMV activation, but also showed a global progressive reduction in area developing over several cycles when the SMV was activated after a period of quiescence.

Thus single-limbed SMVs connected as diastolic counterpulsators not only augment diastolic pressure but significantly reduce the workload of the LV.

The support of the British Heart Foundation is gratefully acknowledged.

CALCINEURIN-DEPENDENT SIGNALING EVENTS IN MUSCLE PLASTICITY

RS Williams

University of Texas Southwestern Medical Center, Dallas, TX USA

Different patterns of motor nerve activity drive distinctive programs of gene transcription in skeletal muscles, thereby establishing a high degree of metabolic and physiological specialization among myofiber subtypes. Recently, we proposed that the influence of motor nerve activity on skeletal muscle fiber type is transduced to the relevant genes by calcineurin, a serine/threonine protein phosphatase. Calcineurin acts, in turn, to control the functional activity of NFAT and MEF2 transcription factors. Calcineurin-dependent gene regulation in cultured skeletal myocytes is integrated with additional calcium-regulated signaling inputs, specifically calmodulin-dependent protein kinase activity, to drive selective transcription of genes representative of the slow (Type I) or oxidative (Type 1 and Type IIa) myofiber programs. In adult transgenic mice, the transactivating function of MEF2 proteins is detectable selectively in Type I and Iia myofibers, and both NFAT and MEF2 binding sites are necessary for properly regulated function of a slow fiber-specific enhancer. Forced expression of activated calcineurin in skeletal muscle of transgenic mice up-regulates a MEF2-dependent reporter gene, and increases the proportion of Type I and Iia fibers. Finally, we have identified two novel muscle-specific proteins termed MCIP1 and MCIP2 that function as endogenous inhibitors of calcineurin signaling, and appear to function in a negative feedback loop to restrain responses to activated calcineurin. These results provide new insights into the molecular mechanisms by which specialized characteristics of skeletal myofiber subtypes are established and maintained.

DEVELOPMENTAL RELATIONSHIP OF MYOSIN BINDING PROTEINS (MYOMESIN, CONNECTIN
AND C-PROTEIN) TO MYOSIN IN CHICKEN SOMITES AS STUDIED BY IMMUNOFLUORESCENCE MICROSCOPY

Y-G Yang(1), T Obinata(2), Y Shimada(1)

(1)Department of Anatomy and Cell Biology, School of Medicine, Chiba University, Chiba 260-8670, Japan and (2)Department of Biology, Faculty of Science, Chiba University, Chiba 263-8522, Japan

The developmental relationship of myosin binding proteins (myomesin, connectin and C-protein) to myosin was studied in chicken cervical somites by immunofluorescence microscopy. Initially, muscle and non-muscle myosins appeared as slender rods at the same sites. Then, they fused to form non-striated fibrils. As muscle myosin formed a striated structure (A bands), non-muscle myosin disappeared from this structure.

Myomesin (reactive with monoclonal antibodies MyB4 and MyBB78) and connectin (carboxy terminal region, reactive with monoclonal antibody T51) were seen as dots in the center of these myosin rods. Then these proteins formed characteristic mature striations on non-striated fibrils of myosin. Earlier alignment of these myosin binding proteins than myosin indicates that the correct assembly of these proteins seems to be related to the formation of initial myosin rods as well as subsequent linear and periodic alignment of myosin molecules to form early A bands. Around myosin rods/myomesin dots/connectin T51 dots, connectin spots reactive with 9D10 were scattered.

These spots may represent radiating connectin filaments from these rods/dots to link myosin rods to I-Z-I structures of myofibrils to be incorporated. Since the slow isoform of C-protein formed its characteristic bands ("doublets") prior to H zone formation within A bands by myosin, this isoform may assist to align precisely myosin filaments within the A band region. The presence of the slow, then the slow and the cardiac, and finally the co-existence of the slow and the fast isoforms of C-protein may interfere with the incorporation and co-polymerization of non-adult isoforms into myofibrils

	Fibroblasts	Smooth Muscle Cells	Endothelial Cells
Control	100	100	100
10 µM DEF	386	201	152
100 µM DEF	782	341	182
1 mM DEF	1054	532	213

	Distal (%)	Middle (%)	Proximal (%)
Control	3.04±0.46	3.36±0.25	4.15±0.15
ABG only	5.17±0.29	5.40±0.81	5.93±0.37
ABG+DEF	7.93±0.80	9.03±0.81	9.76±1.03

Nonischemic myocardium before CMP	0.594±0.067
Nonischemic myocardium after CMP, no DEF	0.651±0.102
Nonischemic myocardium after CMP + DEF	0.814±0.141
Ischemic myocardium before CMP	0.083±0.019
Ischemic myocardium after CMP, no DEF	0.151±0.036
Ischemic myocardium after CMP + DEF	0.369±0.042

	Thoracic Aorta – top	Thoracic Aorta – bottom	Abdominal Aorta – top	Abdominal Aorta – bottom
Control (11 wk HCD)	3.8	3.0	3.0	3.3
30 CPM, 2 V, HCD	3.6	2.4	2.5	2.6
30 CPM, 3 V, HCD	3.4	1.0	0.6	0.5
30 CPM, 4 V, HCD	3.5	1.0	0.6	0.5
60 CPM, 3 V, HCD	3.5	0.7	0.5	0.6
120 CPM, 3 V, HCD	3.1	2.2	2.7	2.1

	BW	LV/RV	M/V	CSA	MHC2a	MHC2b	TUNEL+	CASP3	BCL2	TNF
CONTROL	113+5	3.5+0.6	1.12+0.7	211+30	26 +1.7	74+1.7	1.2 +2.8	100 +8	97 +12	19.0 +1.7
CHF	116+7	2.7+0.6	0.25+0.15	137+43	18 +3.0	81+3.0	39.6 +42.7	206 +80	70 +16	25.2 +1.3
IRBES	121+6	2.2+0.5	0.39+0.06	173+31	25 +6.3	75+6.3	12.3 +12.7	188 +71	94 +35	22.1 +2.2