BAM 10 (5), 2000

Table of Contents

Articles       209        Determination of the oligomeric status of the sarcoplasmic reticulum Ca²⁺-ATPase using optimized chemical crosslinking
                                  NJ Lennon and K Ohlendieck     [Full text pdf 790Kb]

                    217        Electromyographic activity of rat ankle extensors during treadmill locomotion after hindlimb unloading
                                  C Langlet, M-H Canu, and M Falempin     [Full text pdf 65.3Kb]

                    225        Relationship between force and histological changes in mouse anterior crural muscles following eccentric exercise
                                  M Newton, K Nosaka, and P Sacco     [Full text pdf 101Kb]

                    231        Improvement in walking distance after rehabilitation in patients with peripheral arterial disease is associated
                                  with changes in skeletal muscle myosin heavy chains
                                  GB Ambrosio, G Scannapieco, G Vescovo, B Ravara, R Parisi, C Bortoluzzi, and L Dalla Libera
                                  [Full text pdf 290Kb]

                     237        Persistent gene transfer to skeletal muscle mediated by stably transfected early myogenic progenitor cells
                                   S Kimura, M Ikezawa, R Pruchnic, L Balkir, Z Qu, J Lowenstein, S Takeda, C Gates, B Cao, T Miike, and J Huard    
                                   [Full text pdf 647Kb]

                    249        Correlations between time, Latissimus Dorsi wrap properties and systolic assistance in demand dynamic cardiomyoplasty?
                                  G Rigatelli, U Carraro, M Barbiero, and G Rigatelli    [Full text pdf 59.2Kb]
 

Determination of the Oligomeric Status of the Sarcoplasmic Reticulum Ca²⁺-ATPase Using Optimized Chemical Crosslinking

Niall J. Lennon and Kay Ohlendieck

Department of Pharmacology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland

In determining protein-protein interactions in native muscle membranes biochemically, bifunctional crosslinking agents of various length and solubility have been established as effective tools. Gel electrophoretic and immunoblotting methodology have proven to be highly suitable for the analysis of crosslinked products. Here, we have analysed the oligomeric status of a well established muscle membrane complex, the sarcoplasmic reticulum Ca²⁺-ATPase, employing a rapid optimization procedure of chemical crosslinking. Both, the fast SERCA1 and the slow SERCA2 isoforms of this enzyme appear to exist predominantly as a tetrameric complex under native conditions. Direct protein-protein interactions are postulated to be important for cooperative kinetics and protection against proteolytic degradation. In order to keep artifacts of random crosslinking and hydrolysis of crosslinkers to a minimum, an efficient and swift experimental scheme is described to improve reaction conditions with respect to concentration ratios between crosslinkers and biomembranes, length of incubation time, pH and temperature. Employing the 11.4-Å probe bis-sulfosuccinimidyl-suberate and a mini-gel system for the analysis of microsomal vesicles, highly reproducible and optimal results could be obtained with relatively small amounts of skeletal muscle tissue. Thus, the reaction scheme of optimization of crosslinking described in this study is generally suitable for the analysis of supramolecular complexes in biomembranes and should improve the initial determination of the quaternary protein structures within muscle membrane microdomains.

Key words: bissulfosuccinimidyl-suberate, Ca²⁺-ATPase, chemical crosslinking, oligomerization, sarcoplasmic reticulum.

Basic Appl Myol 10 (5): 209-216, 2000

Address correspondence to:

Dr. Kay Ohlendieck, Department of Pharmacology, University College Dublin, Belfield, Dublin 4, Ireland, tel. 353 1 706 1557, fax 353 1 269 2749, Email kay.ohlendieck@ucd.ie.

Electromyographic Activity of Rat Ankle Extensors During Treadmill Locomotion after Hindlimb Unloading

Cécile Langlet, Marie-Hélène Canu, and Maurice Falempin

Laboratoire de Plasticité Neuromusculaire, Université des Sciences et Technologies de Lille, Villeneuve d’Ascq, France

The aim of this work was to study the electromyographic characteristics of two ankle extensors during treadmill locomotion after hindlimb unloading. The studied muscles were the soleus which contains mainly slow fibres, and three parts (red, mixed and white) of the heterogeneous gastrocnemius. Our results showed that the cycle duration was significantly increased (+26%) after 14 days of unloading; the burst duration was increased in the soleus (+18%) and in the red gastrocnemius (+20%). The mean EMG (burst area divided by burst duration) was decreased when treadmill speed was increased in the soleus (-11%) and the red part (-5%) of the gastrocnemius after unloading; in contrast, it was greatly increased in the white part (+46%). These data suggest that hindlimb unloading shifts the normal speed-related increase in muscle effort from the red to the white compartment of the muscle.

Key words: ATPase, fibre-type, gastrocnemius, microgravity, soleus.

Basic Appl Myol 10 (5): 217-224, 2000

Address correspondence to:

M.H. Canu, Laboratoire de Plasticité Neuromusculaire, Université des Sciences et Technologies de Lille, Bâtiment SN4, F-59655 Villeneuve d’Ascq cedex, France, tel. (33) 3 20 33 70 87, fax (33) 3 20 43 68 88, Email canu@univ-lille1.fr.

Relationship between Force and Histological Changes in Mouse Anterior Crural Muscles Following Eccentric Exercise

Mike Newton⁽¹⁾, Kazunori Nosaka^{(1, 2)}, and Paul Sacco⁽¹⁾

(1) School of Biomedical and Sports Science, Edith Cowan University, Western Australia, Australia, and (2) Exercise and Sports Science, Department of Environmental Science, Yokohama City University, Yokohama, Japan

   Abstract

The purpose of this study was to determine the relationship between mononuclear cell number and maximal isometric force (P_o) following active lengthening exercise. The tibialis anterior muscles (TA) of mice were actively lengthened 120 times from a dorsiflexed to plantarflexed position in 300 ms while stimulating the peroneal nerve (100 Hz, 310 ms). P_o at 150 Hz was measured immediately before and after,10 min, 3 and 10 days after exercise and the total number of mononuclear cells per field of view (MN) for the TA muscles were counted. P_o declined to 55% of the baseline value both immediately and 10 min after exercise, declined further to 25% at 3 days, and was still significantly reduced at 10 days following exercise. TA muscles at 3 and 10 days post-exercise showed marked increases in MN, however, the only significant relationship between P_o and MN was at 3 days following exercise (r = -0.64). The results of this study do not support the use of P_o following active lengthening exercise to either predict the extent of subsequent injury, or estimate the concurrent degree of histological damage.

Key words: eccentric exercise, E-C coupling failure, maximal isometric force, mononuclear cells, muscle damage.

Basic Appl Myol 10 (5): 225-229, 2000

Address correspondence to:

Mike Newton, School of Biomedical and Sports Science, Edith Cowan University, 100 Joondalup Drive, Joondalup, WA 6027, AUSTRALIA, tel. 61 8 9400 5171, fax 61 8 9400 5717, Email m.newton@ecu.edu.au.

Improvement in Walking Distance after Rehabilitation in Patients with Peripheral Arterial Disease is Associated with Changes in Skeletal Muscle Myosin Heavy Chains

Giovanni Battista Ambrosio, Gianluigi Scannapieco⁽²⁾, Giorgio Vescovo, Barbara Ravara⁽¹⁾, Roberto Parisi, Cristiano Bortoluzzi, and Luciano Dalla Libera⁽¹⁾

Internal Medicine I, Venice City Hospital, Venice, (1) CNR Unit for Muscle Biology and Physiopathology, University of Padua, Padua, and (2) Internal Medicine Ca’ Foncello Hospital, Treviso, Italy

Intermittent claudication in peripheral arterial disease limits exercise capacity. Rehabilitation can produce an improvement in absolute walking distance. Very often changes in skeletal muscle, rather than changes in blood flow, are responsible for the improved exercise capacity. We studied 8 patients with intermittent claudication (Fontaine 2 class) that underwent a 4-week treadmill mild-moderate aerobic endurance training. We analyzed the Myosin Heavy Chain (MHC) composition of the gastrocnemius muscle before and after rehabilitation by taking needle microbiopsies. The absolute walking distance increased from 394 ± 188 m to 580 ± 215 (p<0.05). No changes in peak VO₂ were found. This improvement was accompanied by a significant increase in the percent expression of the slow aerobic MHC1 isoform, which was paralleled by a decrease of the anaerobic, fast twitch MHC2b.

This finding suggests that the increased exercise capacity obtained with this specific rehabilitation protocol may be related to favorable changes in skeletal muscle composition.

Key words: claudication, exercise, myosin heavy chains, peripheral arterial disease, rehabilitation, skeletal muscle.

Basic Appl Myol 10 (5): 231-235, 2000

Address correspondence to:

Dr Luciano Dalla Libera, CNR Unit for Muscle Biology and Physiopathology University of Padua, 35100 Padua, Italy, tel. +39 049 8276031, fax +39 049 8276040, Email ldl@civ.bio.unipd.it.

Persistent Gene Transfer to Skeletal Muscle Mediated by Stably Transfected Early Myogenic Progenitor Cells

Shigemi Kimura, Makoto Ikezawa, Ryan Pruchnic, Levent Balkir, Zhuqing Qu, Jason Lowenstein, Shin’ichi Takeda⁽¹⁾, Charley Gates, Baohong Cao, Teruhisa Miike⁽²⁾, and Johnny Huard

Growth and Development Laboratory, Departments of Orthopaedic Surgery and Molecular Genetics & Biochemistry, Children’s Hospital of Pittsburgh and University of Pittsburgh, Pittsburgh, (1) Department of Molecular Genetics, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Ogawa-Higashi, Kodaira, Tokyo, Japan, and (2) Department of Child Development, Kumamoto University School of Medicine, Honjo, Kumamoto, Japan

Abstract

Duchenne muscular dystrophy is a progressive muscle weakness characterized by a lack of dystrophin expression in the sarcolemma of muscle fibers. Both myoblast transplantation and gene therapy based on direct and ex vivo gene transfer techniques have been investigated as ways to deliver dystrophin in dystrophic muscle. Although the myoblast-mediated ex vivo gene transfer approach has been found capable of improving viral gene transfer to skeletal muscle, the poor survival rate of the injected cells as well as the immune response against the virally transduced cells has significantly hindered the success of this technique. For this paper, we investigated the use of non-viral vectors through the ex vivo approach based on early myogenic progenitor cells, which have been found highly capable of surviving post-implantation, to improve both the cell survival rate and the long-term persistence of gene transfer to skeletal muscle. We transfected a population of early myogenic progenitor cells derived from mdx mice with a plasmid encoding b-galactosidase, mini-dystrophin and the neomycin resistance gene. The selected muscle cells were capable of expressing b-galactosidase and differentiating into myotubes expressing dystrophin in vitro. More importantly, the transplantation of the transfected cells can be used to deliver b -galactosidase and dystrophin in skeletal muscle of adult mdx mice. The infiltration of CD4+ and CD8+ activated lymphocytes at the injected site suggests that the persistence of the transfected myofibers is limited by the immune response. On the other hand, the persistent transgene expression observed with the same approach in transgenic mdx mice that expressb-galactosidase (mdx/b-gal) suggests that immune response is triggered by theb-galactosidase reporter gene.

Key words: Duchenne muscular dystrophy, dystrophin, early myogenic progenitor cells, immune responses, mdx mouse, myoblast-mediated ex vivo gene transfer, plasmid DNA, b -galactosidase, transgenic mdx/b -gal.

Basic Appl Myol 10 (5): 237-248, 2000

Address correspondence to:

Johnny Huard, Ph.D., Director, Growth and Development Laboratory, Depts. of Orthopaedic Surgery and Molecular Genetics & Biochemistry, Children’s Hospital of Pittsburgh and University of Pittsburgh, Pittsburgh, PA, 15213, Email jhuard+@pitt.edu.

Correlations between Time, Latissimus Dorsi Wrap Properties and Systolic Assistance in Demand Dynamic Cardiomyoplasty?

Gianluca Rigatelli, Ugo Carraro⁽¹⁾, Mario Barbiero, and Giorgio Rigatelli

Division of Cardiology, Cardiomyoplasty Project, Legnago General Hospital, Verona, and (1) Department of Biomedical Sciences, University of Padua, Padua, Italy

A certain systolic assistance in Demand Dynamic Cardiomyoplasty (DDCMP) has been suggested. We here evaluate relations between muscular properties of Latissimus Dorsi wrap (LDW) and systolic function using the doppler flow wire combined with LDW mechanogram. On the basis of previous study, we divided the patients in two groups on the time between intervention and start of demand protocol (group 1: 12 months and group 2: 42 ± 21.6 months). A comparative study with a correlation analysis including time variables and the increase in maximal peak aortic velocity (MPAV) among not assisted and assisted beats was made. Time from the intervention (dyn-time) is similar. NYHA class, time between intervention and start of demand protocol (dyn/dem-time) and time from start of demand protocol (dem-time) were statistically different (p < 0.05). The increase in MPAV was significatively higher in group 1. A linear correlation has been found between the increase in MPAV and dem-time (r = 0.82), NYHA class and dyn/dem-time (r = 0.76), tetanic fusion frequency and dyn/dem-time (r = 0.99), increase in MPAV and tetanic fusion frequency (r = 0.73). In DDCMP systolic assistance is correlated to speed of LDW rapidity and time of demand stimulation. Long time continuous stimulation alters functional performance of LDW and lowers cardiac assistance. Demand stimulation protocol maintains over time contractile properties of the wrap and increases muscle performance.

Key words: Dynamic Cardiomyoplasty, heart failure, intravascular ultrasound.

Basic Appl Myol 10 (5): 249-251, 2000

Address correspondence to:

Dr. G. Rigatelli, Department of Cardiology, Advanced Heart Failure Center, Legnago General Hospital, via Giannella, 37040 Legnago, Italy, Email jackyheart@katamail.com.