BAM 8 (3), 1998
Table of Contents
Editorial & Letters 181 [Full text pdf 129 Kb]
Articles
185 Ultrastructural
changes
in the skeletal muscle of senile rats with
significant age-dependent
motor
deficits
AM
Kaminska, A Fidzianska, G Schulze, Coper,K Ossowska, S Wolfarth
and I
Hausmanowa-Petrusewicz
[Full text pdf 716 Kb]
191
Dynamic
cardiomyoplasty.
Clinical follow-up in Argentina
JC
Trainini, JL Barisani, JH Mouras, E Cabrera Fischer and B
Elenc [Full
text pdf 128 Kb]
197 Muscle
differentiation of normal and double-muscled bovinefoetal
myoblasts in primary culture
B Picard, F Depreux and Y
Geay [Full text
pdf 627 Kb]
205
The
fate
of dystrophin and some signs of apoptosis in the skeletal
muscle work- overloaded in
extension
D Biral, M Sandri, A Jakubiec-Puka [Full text pdf 990 Kb]
211
Autologous
biological
glue and aprotinin prevent ischemia in latissimus dorsi
muscle
after mobilizatio
VS Chekanov, VV Nikolaychik, MA Rieder, GV
Tchekanov, I Hernandez, R Eisenstei, D Francischelli,
PI Lelkes
and DH Schmidt [Full
text pdf 1.24 Mb]
Myology News
237 BAM '98 - International
Conference on MUSCLE PLASTICITY.Damage, Aging,
Cardiomyoplasty and Gene
TherapyAbano Terme (Padova) Italy, June 14-16, 1998
In this issue the BAM News present Program and Abstracts of the "Fifth Abano Terme Meeting on Rehabilitation, BAM'98 - International Conference on Muscle Plasticity" which will be held in Abano Terme and Padova (Italy), June 14 - 16, 1998.
The Conference Topics range
from Activity-induced Muscle Damage to Muscle-related Gene
Therapy, but Muscle Plasticity and its Medical Applications are
main goals.
Local Organizers and the
Members of the International Scientific Committee hope that
numerous participants will meet in Abano Terme to: 1) exchange
information on the latest developments in four interconnected
sub-fields (Activity-induced muscle damage, Contribution of
skeletal myopathies to Chronic Heart Failure, Dynamic
Cardiomyoplasty and Skeletal Muscle Circulatory Assistance, and
Muscle-related Gene Therapy), 2) organise a European Study Group
on Skeletal Muscle Circulatory Assistance, and 3) extend the
Italian Trial on Demand Dynamic Cardiomyoplasty.
Beside others topics, clinical
effectiveness of Dynamic Cardiomyoplasty will be discussed and
some exciting news will be presented. Indeed, using a skeletal
muscle to support a failing heart is the result of a
multidisciplinary approach, which asks much more than surgery
and a trivial device. Among new developments in this surgical
therapy of the cardiac heart failure world-class leader in the
field will present their expertise in: i) Minimally invasive
video assisted Cardiomyoplasty, ii) Vascular delay before LD
wrapping, iii) Monitoring of cardiac function, iv) Protocols for
conditioning and regime stimulation of LD wrap, v) Bedside
monitoring of the dynamic characteristics of LD flap, and vi)
Demand Dynamic Cardiomyoplasty.
Organizers gratefully
acknowledge Siemens and Hewlett Packard which, even at such a
short notice, were so kind to provide, respectively, a Megacart
polygraph, and a cardiac Echo Doppler with Tissue Imaging to be
used on patients during the Tutorial on LD Flap Monitoring.
Let we hope that the
perspectives open by the Conference will attract new interested
and interesting scientists, surgeons and doctors to developments
and implementations of the new approaches.
Ugo Carraro, C.N.R. Unit for
Muscle Biology and Physiopathology, University of Padova, Italy
Giorgio Vescovo, Internal
Medicine I, Venice General Hospital, Venice, Italy
Mario Barbiero, Division of
Cardiology, Legnago General Hospital, Legnago (Verona), Italy
Dino Casarotto, Cardiovascular
Surgery, University of Padova, Italy
Claudio Muneretto,
Cardiovascular Surgery, University of Brescia, Italy
Basic Appl Myol 8 (3), 181, 1998
Letter to Basic and Applied Myology
In their interesting paper [3]
Duan and his colleagues examine the chronic effects of
stimulating rabbit latissimus dorsi muscle with a burst pattern.
They report that peak isometric force had fallen to half the
control level after 6 weeks of stimulation for 24 hours/day, but
had risen to twice that of control after 12 weeks of stimulation
for 12 hours/day.
Their seemingly remarkable
findings nevertheless present a significant problem. The ability
of a muscle to generate force is directly proportional to its
cross-sectional area, yet the morphological changes reported by
Duan et al. for the intermittent pattern (Figure 5) are minimal.
If we multiply the percentage of SO, FOG and FG fibres by their
respective mean cross-sectional areas and add the 3 results, we
can use the data to estimate the change in the overall
cross-sectional area of the muscle. For the intermittent pattern
there is actually a reduction of 5% in cross-sectional area
(mainly because of the smaller proportion of the larger FG
fibres) compared to a reported increase of 105% in peak
isometric force. How do we reconcile these conflicting results?
The simple answer is that 'peak
isometric force' measured in this study does not equate to the
maximum force-generating capability of the muscle. To explain
this statement we need to refer back to protocols worked out
many years ago for measuring the rate of tension development and
amplitude of tetanic contraction in fast and slow muscles [1, 2,
7].
When a fast rabbit muscle is
stimulated at successively higher frequencies, a fused
contraction is not normally achieved until about 100 Hz and peak
isometric tetanic tension continues to increase up to at least
150 Hz. Duan et al. state that they made their measurement of
peak isometric force with the same pattern used for conditioning
- that is to say, with a maximum frequency of only 25 Hz. This
would not elicit maximum isometric tension from the control
muscles.
The important corollary is that
with such a stimulation protocol 'peak isometric force' would be
a sensitive function of the contractile speed of the
experimental muscles. Duan et al. give no figures for
contractile speed, but in another study the time to peak twitch
contraction of muscles subjected to a similar aggregate amount
of activity increased by about 100% [6]. The greater degree of
fusion produced by this slower contractile speed could easily
generate the doubling of tension reported by the authors, even
in the absence of any concomitant change in the mass or
cross-sectional area of the muscle.
What of the results for
'continuous' stimulation? For this group there is an estimated
reduction of 34% in overall cross-sectional area, somewhat
smaller than that expected from the reported decrease of 48% in
peak isometric force. Muscles subjected to this amount of
stimulation would typically show substantial fusion, even at 25
Hz [5, 7]. However, because of a very slow time course of rise
of tetanic tension they would achieve full contraction only
after about 500 ms of tetanic stimulation [1, 2, 7]. Duan et al.
measured peak isometric tension with tetani of 250 ms duration,
and a tetanus of this short duration would underestimate the
tension-generating capacity of the muscle.
Duan and his colleagues are in
all probability correct when they argue that the capacity to
produce force is better preserved in the long term by a less
continuous pattern of stimulation. Indeed, Ferguson et al. [4],
whom they cite, came to a similar conclusion. However, for the
reasons stated here, it is unfortunately not possible to place
any reliance on the figures they report for the size of this
effect.
Stanley Salmons and Jonathan
C. Jarvis
British Heart Foundation,
Skeletal Muscle Assist Research Group, Department of Human
Anatomy and Cell Biology,
University of Liverpool,
Liverpool L69 3GE, UK.
References
[1] Buller AJ, Lewis DM: The
rate of tension development in isometric tetanic contractions of
mammalian fast and slow skeletal muscle. J Physiol 1965; 176:
337-354.
[2] Close R: Neural influences on physiological properties of
fast and slow limb muscles, in Podolsky RJ (ed): Contractility
of Muscle Cells and Related Processes. New Jersey, Prentice
Hall, 1971, pp 175-188.
[3] Duan C, Trumble DR, Christlieb IY, Magovern JA, Magovern GJ:
Improved function in muscles trained via interval stimulation.
Basic Appl Myol 1998; 8: 35-39.
[4] Ferguson AS, Stone HE, Roessmann U, Burke M, Tisdale E,
Mortimer JT: Muscle plasticity: comparison of a 30-Hz burst with
10-Hz continuous stimulation. J Appl Physiol 1989; 66:
1143-1151.
[5] Jarvis JC: Power production and working capacity of rabbit
tibialis anterior muscles after chronic electrical stimulation
at 10 Hz. J Physiol 1993; 470: 157-169.
[6] Jarvis JC, Sutherland H, Mayne CN, Gilroy SJ, Salmons S:
Induction of a fast-oxidative phenotype by chronic muscle
stimulation: mechanical and biochemical studies. Am J Physiol
1996; 270: C306-312.
[7] Salmons S, Vrbová G: The influence of activity on some
contractile characteristics of mammalian fast and slow muscles.
J Physiol 1969; 201: 535-549.
Response
Professor Salmons raises some
interesting issues concerning our recent report on muscle
training via interval burst stimulation (BAM 1998; 8 (1):
35-39). He is, of course, quite right in asserting that our
measurements of peak isometric force do not equate to the
maximum force-generating capability of the muscle. This however,
was not our intent. The principal goal of this work was to
determine whether periodic intervals of rest can improve the
functional capacity of fatigue-resistant muscle stimulated under
conditions generally accepted for clinical use. While we
recognize the importance of characterizing skeletal muscle
function under a wide range of stimulation and loading
conditions, such extensive testing would likely have fatigued
the control muscle and impaired our ability to assess chronic
work capacity. Moreover, in practical terms, preserving a given
fiber type distribution over the long term requires that
skeletal muscle be activated with the same pulse pattern used
during training. We therefore chose to limit testing to a single
stimulation regimen (25 Hz, 250 ms burst duration, 5
contractions/min) which had been used to train the muscle and
has been proven safe for clinical use.
We concur with Professor
Salmons that the force-generating capacity of muscle is directly
proportional to its cross-sectional area (CAS), but must
disagree with his contention that changes in whole muscle CSA
can be estimated from the fiber distribution and fiber areas
reported in Figures 4 and 5 respectively. In our paper, percent
fiber distribution refers to relative number of each fiber type
in a given CSA of muscle tissue (note that the percentages
always add up to 100). These figures do not indicate the
absolute number of fibers present. Thus, it is not possible to
estimate the change in whole muscle CSA in the manner suggested
without assuming that the total number of muscle fibers remains
constant whit training (an assumption for which there is little
basis). It therefore remains quite possible that the muscles
trained via interval stimulation actually increased CSA by
increasing the total number of muscle fibers in a manner
analogous to exercise training (1).
Admittedly, measurements of
whole-muscle mass and CSA would have been helpful in determining
the physiologic basis for the large power increases seen in the
interval stimulation group but the amount of tissue needed to
perform electrophoresis, histochemical, and biochemical analyses
made this impossible. We can only hypothesize that the improved
contractile performance described in our report was due to some
combination of muscle hypertrophy (seen qualitatively),
increased number and recruitment of FOG motor units (2), and
reduced contractile speed (as Professor Salmons has suggested).
However, this does not diminish the fact that interval training
has been shown to improve chronic muscle performance under
clinically-appropriate stimulation conditions. This is the
central tenet of this work, a message which we hope has been
clarified through this correspondence.
Chanping Duan, Dennis R.
Trumble, Ignacio Y. Christlieb, and James Magovern
Cardiothoracic Surgical
Research, Allegheny University of the Health Sciences,
Department of Surgery, Allegheny General Hospital, Pittsburgh,
Pennsylvania, USA
References
[1] Gonyea WJ, Sale DG, Gonyea
FB, and Mikesky A. Exercise induced increases in muscle fiber
number. Eur J Appl Physiol 1986; 55: 137-141
[2] McArdle WD, Katch FI, and Katch VL. Neural control of human
movement, in: Exercise Physiology: Energy, Nutrition and
HumanPerformance (2nd Ed.). Philadelphia, PA, Lea & Febiger,
1986, pp 305-319
Basic Appl Myol 8 (3), 181-183, 1998
Letter to Basic and Applied Myology
I read with interest Professor
Salmons' letter [Basic Appl myol 1998; X (x): 0-0] commenting
our latest BAM publication [4]. Professor Salmons is correct
regarding the difficulty of reconciliating the 105% increase in
peak isometric force in the 12-week interval stimulation group
over control muscles. However, recognizing that the aim of this
study was not to prove or disprove basic principles [1, 3, 6],
established long before the start of biomechanical
cardiocirculatory assist with skeletal muscle, but to research
into the new ones that must regulate muscle physiology under the
novel electrostimulating patterns utilized for clinical
purposes, such reconciliation becomes easier.
In our clinically oriented work
we did not intend to elicit maximum isometric tension or to
generate maximum isometric force. Indeed, Professor Salmons is
right in stating that in our study 'peak isometric force' does
not equate to the maximum force-generating capability of the
muscle. Our protocol did not call for it to be so. A close
review of our manuscript reveals the use of the term maximum in
only two occasions, in: Results, paragraph 1, lines 2 and 3. We
assumed, as the reviewers and most readers probably did, that
maximum under the testing protocol was understood, as opposed to
absolute maximum, incompatible with clinical use in circulatory
assist. We apologize for the inconvenience. Elsewhere throughout
the paper, the term peak isometric force is used.
We recognize with Professor
Salmons the ability of a whole muscle to generate force is
directly proportional to its cross-sectional area (CSA), and
that a fast muscle (fast oxidative [FO] fibers) does not
normally achieve a fused contraction until about 100 Hz, and
continues to increase up to around 150 Hz. Recognizing however
that peak isometric contraction, regardless of generated force,
is dependent upon the number (percentage) of fiber recruited at
one time, we elected to utilize a stimulation pattern equal to
that used for muscle conditioning. Our specific aim was to
demonstrate what difference had made the conditioning pattern in
treated muscles as compared to controls. A 25 Hz burst of 250 ms
duration, with pulse width of 210 msec was chosen for its
closeness to clinical electrostimulation programs. Frequencies
higher than 35 Hz and burst durations longer than 150 ms have
proven to be deleterious for long-term application of muscle
powered circulatory assist [5].We cannot agree with Professor
Salmons' interpretation of figures 4 and 5. An estimation of the
change in the overall CSA of the muscles in each group is
irrelevant to the purpose of our research. Other observations
related to non-clinically applicable basic principles are
irrelevant as well. Fast glycolytic (FG) fibers are abundant in
the latissimus dorsi muscle and take the largest portion of the
CSA. They are strong and respond well to high frequencies (100
Hz +), but for our purpose are useless in account of being very
prone to fatigue. Distribution of slow oxidative (SO) fibers
increases dramatically with continuous stimulation but occupy
the smallest CSA in so stimulated muscles. They respond well to
low frequencies (10 Hz +) and are resistant to fatigue, but for
our purpose are also useless because muscles conditioned in this
manner become significantly weaker and slower. Fast oxidative
glycolytic (FOG) fibers on the other hand, retain much of the
best of the other two types over the long periods of time.
Muscles trained under an interval stimulation protocol of 12
weeks, with burst at a frequency of 25 Hz and pulses of 210 msec
amplitude, proved to develop the highest peak isometric force
with this set of parameters and, following an initial decrease,
to maintain the highest contractile function for as long as 170
minutes, and that is an undisputable fact. The explanation is
found in the proper interpretation of figures 4 and 5 of our
paper. Under the interval stimulation protocol, fiber
distribution (%) of FOG fibers increased significantly over
controls, and FOG fibers were the only ones to reach
significance in fiber CSA increase. These findings suggest that
FOG fibers are more energy-efficient from the thermodynamic
point of view [2], which possibly makes them responsible for
better long-term contractile performance and fatigue resistance,
and that interval stimulation may be able to yiels and sustain a
larger proportion of FOG fibers of greater CSA, in an otherwise
healthy working muscle.
Prof. Ignacio Y. Christlieb,
M.D.
Cardiothoracic Surgical
Research, Allegheny University of the Health Sciences,
Department of Surgery. Pittsburgh, Pennsylvania, U.S.A.
References
[1] Buller AJ, Lewis DM: The
rate of tension development in isometric tetanic contractions of
mammalian fast and slow skeletal muscle. J Physiol 1965; 176:
337-354.
[2] Christlieb IY, Cesarman E: Thermodynamics of skeletal muscle
fiber: do we need to redefine "active" and "resting" states?
Basic Appl Myol 1996; 6 (3):199-202.
[3] Close R: Neural influence on physiological properties of
fast and slow limb muscles, in Podolsky RJ (ed): Contractility
of Muscle Cells and Related Processes. New Jersey, Prentice
Hall, 1971, pp 175-188.
[4] Duan C, Trumble DR, Christlieb IY, Magovern JA, Magovern GJ:
Improved function in muscles trained via interval stimulation.
Basic Appl Myol 1998; 8 (1): 35-39.
[5] Magovern JG, Simpson KA: Clinical cardiomyoplasty: review of
the 10-year United States experience. Ann Thorac Surg 1996; 61:
413-419.
[6] Salmons S, Vrbova G: The influence of activity on some
contractile characteristics of mammalian fast and slow muscles.
J Physiol 1969; 535-549.
Response
The estimation was based on published evidence that the number of fibres does not increase in chronically stimulated muscle [1, 2]. In our view, experimental data that provide an insight into mechanism will ultimately be of the most value clinically, particularly when those data are derived from small laboratory animals.
[1] Pette D, Muller W, Leisner
E, Vrbová G: Time dependent effects on contractile properties,
fibre population, myosin light chains and enzymes of energy
metabolism in intermittently and continuously stimulated fast
twitch muscles of the rabbit. Pflugers Archiv 1976; 364:
103-112.
[2] Salmons S, Henriksson J: The adaptive response of skeletal
muscle to<W0><C5,5,0,0,0,0> increased use. Muscle
Nerve 1981; 4: 94-105.
Stanley Salmons and Jonathan
C. Jarvis
British Heart Foundation,
Skeletal Muscle Assist Research Group, Department of Human
Anatomy and Cell Biology,
University of Liverpool,
Liverpool L69 3GE, UK.
Basic Appl Myol 8 (3), 183-184, 1998
Ultrastructural Changes in the Skeletal Muscle of Senile Rats with Significant Age-Dependent Motor Deficits
Anna M. Kaminska(1, 2), Anna Fidzianska(1, 2), Gert Schulze(3), Helmut Coper(3), Krystyna Ossowska(4), Stanislaw Wolfarth(4) and Irena Hausmanowa-Petrusewicz(2)
Neurological Department, Medical Academy, Warsaw, Poland, (2) Neuromuscular Unit, Medical Research Center, Polish Academy of Sciences, Warsaw, Poland, (3) Institute for Neuropsychopharmacology, Free University, Berlin, Germany and (4) Department of Neuropharmacology, Institute of Pharmacology of the Polish Academy of Sciences, Cracow, Poland
The anterior tibial (AT) muscle of 6 female Wistar rats aged 35-44 months, was examined by an electron microscopy. Previously, significant age-dependent functional and morphological deficits were found in these rats including [1] a strongly decreased muscle mass and force, [2] muscle stiffness, [3] spontaneous, tonic electromyographic activity, and [4] light-microscopic features of chronic denervation atrophy. In the present study diverse ultrastructural changes were found in muscle which correspond to chronic denervation atrophy. A number of already described abnormalities could be demonstrated in our material. However, sarcoplasmic reticulum (SR) tubular formations and extensive muscle fiber fragmentation have not been previously associated with senile changes in muscle. This can be explained by a very advanced age of our experimental animals and, in consequence, a more advanced denervation atrophy. Therefore it would appear that no single abnormality or set of morphological changes are characteristic of senile skeletal muscle.
Key words: skeletal muscle,
ultrastructure, denervation atrophy, aging.
Basic Appl. Myol. 8 (3):
185-190, 1998
Address correspondence to:
A.M. Kamimska, Neurological Department, Medical Academy, 1A Banacha Str., 02-097 Warsaw, Poland, phone (48 22) 659 75 05, fax (48 22) 668 85 12, Email amkamlamwaw.edu.pl.
Dynamic Cardiomyoplasty. Clinical Follow-Up in Argentina
Jorge C. Trainini, José L. Barisani, Jorge H. Mouras, Edmundo Cabrera Fischer and Benjamín Elencwajg
Department of Cardiovascular Surgery, President Perón Hospital, Anatole France 770, (1870) Avellaneda, Provincia de Buenos Aires, República Argentina
The aim of the present study is to evaluate results obtained after applying dynamic cardiomyoplasty to patients with dilated cardiomyopathy and severe ventricular dysfunction (Functional Class III-IV, New York Heart Association).A dynamic cardiomyoplasty procedure was performed in 15 patients with a mean age of 59.2 ± 6.4 years old. Despite the medical treatment with inhibitors of the converted enzyme or vasodilators, all of these patients required 2.2 ± 0.7 hospitalizations/patient/year owing to congestive heart failure in the year before dynamic cardiomioplasty was applied. In 8 patients the etiology of the cardiomyopathy was idiopathic, ischemic-necrotic in 6 and Chagas' disease in the other.Hemodynamic studies were done preoperatively in all patients and every six months postoperatively.Twelve patients had a follow-up for two years. The following values related to two-years evaluation improved significantly in comparison with baseline: functional class (1.7 ± 0.6 versus 3.06 ± 0.2); radionuclide left ventricular ejection fraction (29.7 ± 5% versus 23.6 ± 3%); fractional shortening (20.6 ± 5% versus 15.6 ± 4%). Walking test values increased from 332 ± 127 meters to 421 ± 102 meters. Left ventricular diastolic diameter remained unchanged (72.7 ± 7 mm versus 72.3 ± 8 mm).Improvement was observed in functional capacity and left ventricle systolic function parameters two years after cardiomyoplasty was applied.
Key words: congestive heart failure, cardiomyopathy, latissimus dorsi muscle, cardiomyoplasty, skeletal muscle.
Basic Appl. Myol. 8 (3):191-195, 1998
Address correspondence to:
Dr. Jorge Trainini, Department of Cardiovascular Surgery, President Perón Hospital, Anatole France 770, (1870) Avellaneda, Provincia de Buenos Aires, República Argentina, home phone 541 302 3810, business phone 541 204 1021128, business fax 541 302 3810.
Muscle Differentiation of Normal and Double-Muscled Bovine Foetal Myoblasts in Primary Culture
Brigitte Picard, Frédéric Depreux and Yves Geay
Laboratoire Croissance et Métabolismes des Herbivores, Equipe Croissance Musculaire, INRA, Theix, Saint-Genès Champanelle, France
Bovine muscle differentiation has been mainly studied in vivo. The comparison during foetal growth of two genetic types; which display muscle hypertrophy, and normal, have shown a delay in the evolution of the different myosin heavy chain isofoms in double-muscled compared to normal foetuses. To investigate these observed differences we studied the growth in primary cultures of myoblasts taken from double-muscled and normal foetuses at 110 days post-conception. These cultures were analysed at 2, 4, 6, 8, 12 and 14 days. The cell proliferation phase was monitored by means of corresponding marker antibodies, and the differentiation phase was characterised using a broad set of criteria. The total myofibrillar proteins were extracted and assayed. The proteins considered as early markers of muscle differentiation, such as desmin and connectin, were identified using antibodies. The different myosin heavy chain isoforms; foetal, alpha cardiac, slow and fast, were identified immunohistochemically and by immunoblotting. Creatine phosphokinase activity, another marker of muscle differentiation, was assayed at the different stages. The findings confirm the delayed differentiation of double-muscled foetuses and show this to be the consequence of a more intense cell proliferation phase.
Key words: myoblast, bovine, muscle hypertrophy, myosin.
Basic Appl. Myol. 8 (3): 197-203, 1998
Address correspondence to:
Picard Brigitte, Laboratoire Croissance et Métabolismes des Herbivores, Equipe Croissance Musculaire, INRA, Theix, 63122 Saint-Genès Champanelle, France, phone 04 73 62 40 56, fax 04 73 62 46 22,picard@clermont.inra.fr.
The Fate of Dystrophin and Some Signs of Apoptosis in the Skeletal Muscle Work-Overloaded in Extension
Donatella Biral, Marco Sandri, Anna Jakubiec-Puka(1)
Department of Biomedical
Sciences, The University of Padova, Padova Italy and (1)
Department of Cellular Biochemistry, Nencki Institute of
Experimental Biology, Warsaw, Poland
Rat muscles, soleus and extensor digitorum longus (EDL), were eccentrically electrostimulated at low frequency for 4-24h. The cryostat-sections of these muscles were examined by immunohistochemistry. Numerous muscle fibres were damaged and necrotic. Simultaneously, several preserved fibres were not stained, or stained only discontinuously, with the anti-dystrophin and anti-b-dystroglycan antibody. Apoptotic nuclei (TUNEL-positive) were occasionally noticed within the muscle fibres. Those changes appeared earlier in the EDL than in the soleus.
Key words: apoptosis, dystrophin, eccentric contraction, electrostimulation, muscle damage.
Basic Appl. Myol. 8 (3): 205-210, 1998
Address correspondence to:
Dr Anna Jakubiec-Puka, Dept. of Cellular Biochemistry, Nencki Inst. of Experimental Biology, Pasteura 3, 02-093 Warsaw, Poland, phone (48 22) 659 85 71, fax (48 22) 822 53 42.
Autologous Biological Glue and Aprotinin Prevent Ischemia in Latissimus Dorsi Muscle after Mobilization
Valeri S. Chekanov, Victor V. Nikolaychik, Michelle A. Rieder, Guennady V. Tchekanov, Irene Hernandez, Rueben Eisenstein, David Francischelli(1), Peter I. Lelkes and Donald H. Schmidt(2)
Milwaukee Heart Project, Sinai Samaritan Medical Center, Milwaukee, Wisconsin, (1) Medtronic, Inc., Minneapolis, Minnesota and (2) University of Wisconsin Medical School, Milwaukee, Wisconsin
The hemodynamic results of cardiomyoplasty, a promising form of surgical treatment for end-stage heart failure, do not support the subjective improvements seen clinically. We hypothesized that this disparity might be due to ischemia-reperfusion injury to the latissimus dorsi muscle (LDM) after mobilization. Having tested autologous biological glue (ABG) as a protective layer around traumatized muscle, as a means for facilitating revascularization, and as a drug depot to reduce local ischemia-reperfusion lesions, we wanted to determine if this protective and revascularization effect could be enhanced by adding aprotinin, a natural inhibitor of serine proteinases with the potential for preventing proteolytic degradation. To test for muscle damage and angiogenesis, we created pockets out of ischemic and nonischemic LDM. The control group had pockets without additives; the second group had pockets with ABG only; and the third had pockets with ABG and aprotinin. Light microscopy revealed that pockets treated with ABG, either alone or with aprotinin, had less leukocyte margination, fibrosis, calcified necrosis, and fibrous degeneration than in controls. In control pockets, after 56 days, capillaries occupied 4.1 ± 0.3% of the area in nonischemic LDM and 3.6 ± 0.7% in ischemic LDM (p > 0.05). In pockets treated with ABG only, capillaries occupied 5.5 ± 0.2% (p < 0.05) of the area in ischemic LDM; in pockets treated with ABG and aprotinin, 8.5 ± 1.1% (p < 0.05) area was occupied with capillaries. This data confirmed our hypothesis that aprotinin added to ABG prevents ischemia-reperfusion lesions after muscle mobilization, and enhances capillary ingrowth in both the ischemic muscle and the interlayer between ischemic and nonischemic muscle.
Key words: cardiomyoplasty, LDM ischemia, autologous biological glue, aprotinin.
Basic Appl. Myol. 8 (3):211-219, 1998
Address correspondence to:
Valeri S. Chekanov, M.D., Ph.D., Sinai Samaritan Medical Center, Milwaukee Heart Project, W419, P.O. Box 342, 945 N. 12th Street, Milwaukee, WI 53201-0342, phone 414 219 7899, fax 414 219 6266,Email cwchrist@facstaff.wisc.edu..
Induction of DNA Fragmentation in Rat Small Intestinal Smooth Muscle Cells by Ischemia
Hiroshi Ikeda, Yasuhiko Suzuki, Chao-Long Yang, Jie Tong and Gen Itoh
First Department of Pathology, Aichi Medical University, Nagakute-cho, Aichi-gun, Aichi-ken 480-1195, Japan
Injuries caused by ischemia in the small intestine have been widely accepted as resulting in necrosis. The aim of this study was to ascertain whether apoptosis of intestinal smooth muscle cells (ISMCs) also occurs. For this purpose rat small intestine subjected to ischemia was studied. Apoptosis was assessed by the TUNEL method and by the electrophoretic detection of DNA laddering. Necrosis was evaluated by a -smooth muscle actin monoclonal antibody labeling.ISMCs of the longitudinal layer (LL) were damaged by ischemia, whereas those of the circular layer (CL) were not. ISMCs showing fragmented DNA were first observed after 1.0 hour. Damaged cells showing both DNA laddering and actin labeling were first observed after 3 hours. Agarose gel electrophoresis of DNA confirmed these observations by showing both ladder and smear patterns. Finally, the expression of Bax but not Bcl-2 and Fas in ISMCs increased after ischemia.The present study demonstrated that rat ISMCs subjected to ischemia exhibit both DNA laddering and actin labeling. Apoptosis appears as the initial form of cell death, followed by necrosis. Enhanced expression of Bax may be implicated in this activation of apoptosis.
Key words: apoptosis, necrosis, Bax, smooth muscle cell, small intestine, rat.
Basic Appl. Myol. 8 (3): 221-229, 1998
Address correspondence to:
William P Santamore, Ph.D.,
500 S. Floyd St., Jewish CVR Institute, Dept of Surgery,
University of Louisville, Louisville, Kentucky 40202 USA, phone
502 852 4345, fax 502 852 1795.
Preliminary Report on Continuous Stimulation vs. Intermittent Stimulation of Latissimus Dorsi Muscle in Chronic Canine Model of Cardiomyoplasty
M. Abul Kashem, Ben Y. Chiang, Ahsan Ali, David Slater and William P. Santamore
Jewish Hospital Cardiothoracic Surgical Research Institute, Division of Thoracic and Cardiovascular Surgery, University of Louisville, Louisville, Kentucky, U.S.A.
In dynamic cardiomyoplasty (CMP), over-utilization leads to extensive muscle damage, which may explain the observed minimal systolic assistance with latissimus dorsi muscle (LDM) stimulation. Thus, we compared intermittent to continuous stimulation in CMP. In mongrel dogs, myocardial dysfunction was induced by intracoronary microsphere injections and vascular delay of the LDM was performed. After two weeks, a standard CMP was performed. LDM was progressively conditioned using continuous stimulation (Group CS; n = 3) or intermittent stimulation (Group IS; n = 3, 10 hours on/14 hours off per day). After 9 weeks, the effects of the LDM stimulation were examined. Significant hemodynamic increases were observed in both groups with LDM stimulation. In group CS, LDM stimulation increased peak left ventricular systolic pressure (8.7 ± 1.4) mmHg, peak aortic systolic pressure (10.1 ± 0.5 mmHg), stroke volume (2.3 ± 1.0 ml), stroke work (5.2 ± 1.6 gm·m), and peak aortic flow (1.5 ± 0.5 ml/min). In group IS, LDM stimulation increased peak left ventricular systolic pressure (19.1 ± 1.4 mmHg), peak aortic systolic pressure (15.9 ± 2.2 mmHg), stroke volume (7.5 ± 2.7 ml), stroke work (14.2 ± 4.4 gm·m), and peak aortic flow (5.9 ± 1.8 ml/min). However, these changes were significantly greater in the IS group compared with the CS group. Preliminary report suggests that intermittent stimulation can increase the cardiac augmentation with LDM stimulation.
Key words: cardiomyoplasty, vascular delay, continuous stimulation, intermittent stimulation, left ventricular dysfunction.
Basic Appl. Myol. 8 (3):
231-236, 1998
BAM '98 - International Conference on Muscle Plasticity
DAMAGE, AGING, CARDIOMYOPLASTY AND GENE THERAPY
Abano Terme (Padova) Italy,
June 14-16, 1998
SPONSORED BY
The University of
PadovaDepartment of Biomedical Sciences, C.N.R. Unit for Muscle
Biology and Physiopathology
The Working Group of "Cellular
Biology of the Heart" of The European Society of Cardiology
INTERNATIONAL SCIENTIFIC COMMITTEE
President: U Carraro, Padova, Italy; GB Ambrosio, Venice, Italy; G Arpesella, Bologna, Italy; M Cantini, Padova, Italy; 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; A Coats, London, UK; L Dalla Libera, Padova, Italy; S Dalla Volta, Padova, Italy; M Fiszman Paris, France; JA Faulkner, Ann Arbor, MI, USA; C Franceschi, Modena, Italy; M Grounds, Perth, Australia; G Itoh, Aichi, Japan; R Lorusso, Brescia, Italy; R Matsuda, Tokio, Japan; C Muneretto, Brescia, Italy; D Pette, Konstanz, Germany; P Poole-Wilson, London, UK; S Salmons, Liverpool, UK; LD Tomei, Richmond, CA, USA; R van der Veen, Maastricht, The Netherlands; G Vescovo, Venice, Italy
LOCAL COMMITTEE:
U Carraro, G Vescovo, M Barbiero, D Casarotto and C Muneretto
BAM'98 SECRETARIAT:
Department of Biomedical
Sciences, Viale Giuseppe Colombo 3, I-35121 Padova, Italy Tel:
+39 49 8276030; fax: +39 49 8276040; e-mail:
bam@civ.bio.unipd.it
Welcome Address
On behalf of the International
Scientific Committee we invite you to the "Fifth Abano Terme
Meeting on Rehabilitation, BAM'98 - International Conference on
Muscle Plasticity" which will be held in Abano Terme and Padova
(Italy), June 14 - 16, 1998.
The Conference Topics range
from Activity-induced Muscle Damage to Muscle-related Gene
Therapy, but Muscle Plasticity and its Medical Applications are
main goals.
Local Organizers and the
Members of the International Scientific Committee hope that
numerous participants will meet in Abano Terme to: 1) exchange
information on the latest developments in four interconnected
sub-fields (Activity-induced muscle damage, Contribution of
skeletal myopathies to Chronic Heart Failure, Dynamic
Cardiomyoplasty and Skeletal Muscle Circulatory Assistance, and
Muscle-related Gene Therapy), 2) organise a European Study Group
on Skeletal Muscle Circulatory Assistance, and 3) extend the
Italian Trial on Demand Dynamic Cardiomyoplasty.
Though carefully focused, the
Conference is attracting a wealth of speakers and a qualified
attendance from medical and industrial communities. Beside
others topics, clinical effectiveness of Dynamic Cardiomyoplasty
will be discussed and some exciting news will be presented.
Indeed, using a skeletal muscle to support a failing heart is
the result of a multidisciplinary approach, which asks much more
than surgery and a trivial device. Among new developments in
this surgical therapy of the cardiac heart failure world-class
leader in the field will present their expertise in: i)
Minimally invasive video assisted Cardiomyoplasty, ii) Vascular
delay before LD wrapping, iii) Monitoring of cardiac function,
iv) Protocols for conditioning and regime stimulation of LD
wrap, v) Bedside monitoring of the dynamic characteristics of LD
flap, and vi) Demand Dynamic Cardiomyoplasty.
Organizers gratefully
acknowledge Siemens and Hewlett Packard which, even at such a
short notice, were so kind to provide, respectively, a Megacart
polygraph, and a cardiac Echo Doppler with Tissue Imaging to be
used on patients during the Tutorial on LD Flap Monitoring.
Let we hope that the
perspectives open by the Conference will attract new interested
and interesting scientists, surgeons and doctors to developments
and implementations of the new approaches.
Ugo Carraro
C.N.R. Unit for Muscle Biology
and Physiopathology, University of Padova, Italy
Giorgio Vescovo
Internal Medicine I, Venice General Hospital, Venice, Italy
Mario Barbiero
Division of Cardiology, Legnago General Hospital, Legnago (Verona), Italy
Dino Casarotto
Cardiovascular Surgery, University of Padova, Italy
Claudio
MunerettoCardiovascular Surgery, University of Brescia, Italy
PROGRAM
SUNDAY June 14, 1998
Abano Terme Conference
Auditorium
09.00 - 09.10 Opening Ceremony
9.10-13.00 Activity-induced muscle damage
Chairmen: D. PETTE and J.A.
FAULKNER
09.10 - 09.50 M.J. JACKSON (Liverpool, UK): Skeletal muscle responses to exercise-induced oxidative stress
09.50 - 10.10 V. KOVANEN (Jyvaskyla, Finland): Forced lengthening contraction induced muscle damage in rats at different ages. Activation of collagen synthesis related gene expression
10.10 - 10.30 A. JAKUBIEC-PUKA (Warsaw, Poland): The immunohistochemical analysis of dystrophin and some myofibrillar proteins (desmin, a-actinin and tropomyosin) in skeletal muscle electrically stimulated in extension
10.30 - 11.00 P. LOUGHNA (London, UK): Are Lim-domain containing proteins involved in the regulation of muscle phenotype?
Coffee
11.30 - 12.00 L. LARSSON (University Park, USA): Acute quadriplegia and loss of muscle myosin in patients treated with non-depolarizing neuromuscular blocking agents and corticosteroids. Underlying cellular and molecular mechanisms
12.00 - 12.30 S. SALMONS (Liverpool, UK): Cardiac assistance from skeletal muscle: avoiding ischaemia in the latissimus dorsi muscle graft
12.30 - 13.00 C. CHISARI (Pisa, Italy): Increased susceptibility to muscle damage in nephrotic patients without renal failure
Lunch
15.00 - 19.00 Age-related muscle atrophy and plasticity
Chairmen: C. FRANCESCHI and L.
LARSSON
15.00 - 15.30 J.A. FAULKNER, (Ann Arbor, USA): Force and power output of skeletal muscles from young, adult and old mdx mice
15.30 - 16.00 A. MARGRETH (Padova, Italy): Functional behaviour of native ryanodine receptor / Ca2+ - release channel in slow skeletal muscle SR of young and old rats
16.00 - 16.30 V.S. CHEKANOV (Milwaukee, USA): Age-related muscle plasticity as a response to long-term electrical stimulation
Coffee
17.00 - 17.30 A.M. KAMINSKA (Warsaw, Poland): Regenerative capability of senile rat skeletal muscle
17.30 - 18.00 A. McARDLE (Liverpool, UK): The role of heat shock proteins in cytoprotection of adult and aged skeletal muscle
18.00 - 18.20 R. SCELSI (Pavia, Italy): Effects of ageing on the morphology of the human skeletal muscle
18.20 - 18.40 R.S. HIKIDA (Athens, USA): Is hypertrophy limited in elderly muscle fibers? A comparison of elderly and young strength-trained men
18.40 - 19.00 C. D'ALESSANDRO
(Pisa, Italy): Muscle function decline in the elderly:
biochemical indices of evaluation
21.00 - 22.00 Meeting of BAM
Editorial Board
MONDAY June 15, 1998
Abano Terme Conference
Auditorium
9.00 - 11.00 Skeletal Muscle in Heart Failure
Chairmen: P. POOLE-WILSON and
G.B. AMBROSIO
09.00 - 09.45 P. POOLE-WILSON (London, UK): Control of blood flow to skeletal muscle in heart failure
09.45 - 10.15 A. MORTARA (Montescano - Pv, Italy): Arterial baroreceptors function in CHF: Clinical and physiopathological implications
10.15 - 10.45 A. COATS (London, UK): Muscle wasting in CHF
10.45 - 11.15 G. VESCOVO (Venice, Italy): Skeletal muscle microbiopsies for assessing MHC composition: a marker of severity in CHF
Coffee
12.00 - 13.00 Apoptosis in cardiac and skeletal muscle
Chairmen: M.J. JACKSON and R.
FERRARI
12.00 - 12.30 C. CECONI (Brescia, Italy): Immunological disorders, apoptosis and cardiovascular injuries
12.30 - 13.00 H. IKEDA (Aichi, Japan) Disruption of plasma membrane in rat myocardial infarcted cells as monitored by use of lanthanum ions
13.00 - 15.00 Lunch and Poster discussion
V.S. CHEKANOV et al. (Milwaukee, WI, USA): Hemodynamic results after cardiomyoplasty in a chronic heart failure model with cessation of electrical stimulation for 12 hours daily
C. CHISARI et al. (Pisa, Italy): Exercise-induced muscle damage in muscular dystrophies: evaluation by serum CPK determination
Z. PODLUBNAYA et al. (Pushchino, Russia): The role of the plasticity of skeletal and cardiac muscles and polymorphism of myosin on surviving mammals in hibernation
C. RIZZI et al. (Padova, Italy): Monitoring muscle trophism and damage/regeneration in experimental and clinical muscle transplants
K. ROSSINI et al. (Padova, Italy): Time-course of exercise and apoptosis in dystrophin-deficient muscle of mice
M. SANDRI et al. (Padova, Italy): Expression of FasL in skeletal muscle cells
M. SMINK et al. (Maastricht,
The Netherlands): Ventricular reduction surgery in goats with
dilated hearts
15.00 - 17.00 Apoptosis in skeletal and cardiac muscle
Chairman: M.J. JACKSON and R.
FERRARI
15.00 - 15.30 D.S. TEWS (Mainz, Germany): Apoptotic muscle fiber loss in spinal muscular atrophies
16.00 - 16.30 M. SANDRI (Padova, Italy): Apoptosis of myofibers in Duchenne Muscular Dystrophy
16.30 - 17.00 L. DALLA LIBERA
(Padova, Italy): Skeletal muscle myopathy in CHF: the role of
apoptosis in determining atrophy and changes in MHCs.
17.00 Bus to Padova
MONDAY June 15, 1998 - Aula
Magna del Bò at The University of Padova
18.00 Visit to The Anatomical Theatre of The University
18.30 - 19.30 Aula Magna del Bò at The University of Padova:
BAM Lecture on Muscle
Plasticity: D. PETTE (Konstanz, Germany)
20.00 Concert
21.00 Banquet
23.00 Bus to Abano Terme
TUESDAY June 16, 1998 -
Abano Terme Conference Auditorium
09.00 - 11.00 Muscle Gene Therapy
Chairmen: J. CHAMBERLAIN and
M.Y. FISZMAN
09.00 - 09.20 G. FERRARI (Milan&Rome, Italy): Muscle regeneration by bone marrow-derived myogenic progenitors
09.20 - 09.40 J. CHAMBERLAIN (Ann Arbor, USA): Adenoviral mediated delivery of full-length dystrophin to immunocompetent adult mdx muscle
09.40 - 10.00 L. AUSTIN (Melbourne, Australia): The influence of leukaemia inhibitory factor (LIF) on the dystrophic process in muscle and its potential in myoblast transfer therapy
10.00 - 10.20 M. CANTINI (Padova, Italy): Human macrophages release a myoblast-selective mitogen
10.20 - 10.40 R.J-C. CHIU (Montreal, Canada): Milieu-dependent differentiation of skeletal myoblast implanted into the myocardium: current evidence
10.40 - 11.00 M.Y. FISZMAN (Paris, France): How to improve in vivo cardiac gene transfer
Coffee
11.30 - 13.30 Dynamic Cardiomyoplasty
Chairmen: A. CARPENTIER and R.
J-C. CHIU
11.30- 12.00 J.C. CHACHQUES (Paris, France): New developments in Dynamic Cardiomyoplasty
12.00 - 12.20 J. TRAININI (Buenos Aires, Argentina): Dynamic Cardiomyoplasty and Aortomyoplasty: the Buenos Aires experience
11.20 - 12.40 I.Y. CHRISTLIEB (Pittsburgh, USA): Intermittent stimulation and vascular delay enhance function of conditioned muscle
12.40 - 13.00 C. MUNERETTO (Brescia, Italy): Demand Dynamic Cardiomyoplasty: 18-month follow-up
Lunch
14.45 - 15.30 Tutorial: Mechanogram of Latissimus Dorsi Wrap At Bed-Side
U. Carraro, M. Barbiero, G.
Docali, C. Brunazzi, A. Cotogni
15.30 - 18.00 Dynamic Cardiomyoplasty and biological cardiac assistance
Chairmen: S. SALMONS and U.
CARRARO
15.30 - 15.50 F.H. VAN DER VEEN (Maastricht, The Netherlands): Validation of latissimus dorsi wrap mechanogram with fluoroscopic contraction analysis in three patient groups
15.50 - 16.10 V.S. CHEKANOV (Milwaukee, USA): How to evaluate long-term hemodynamic results in Cardiomyoplasty
16.10 - 16.30 R. LORUSSO (Brescia, Italy): Fatal rhythm disturbances after cardiomyoplasty procedure: is combined AICD implantation the optimal solution?
Coffee
17.00 - 17.20 J.C. JARVIS (Liverpool, UK): Numerical prediction and experimental measurement of cardiac assistance from skeletal muscle ventricles
17.20 - 17.40 G. BOLOTIN (Haifa, Israel): Coronary blood flow augmentation and afterload reduction: a comparison between intra-aortic balloon and descending aortomyoplasty counterpulsation
17.40 - 18.00 G. ARPESELLA (Bologna, Italy): Activity-rest stimulation regimes for skeletal muscle cardiac assist
18.00 - 18.30 F.H. VAN DER
VEEN (Maastricht, The Netherlands): Alternative stimulation and
conditioning protocol to transform skeletal muscle for
cardiomyoplasty
18.30 - 19.30 Meeting of the European Task Force for New Developments in Dynamic Cardiomyoplasty and Skeletal-Muscle-Powered (Sk-Ms-Pw) Cardiac Assists
CONTRIBUTORS
Ahtikoski A.M.
Biology of Physical Activity,
University of Jyvaskyla, Finland
Akopova I.
Institute of Theoretical and
Experimental Biophysics RAS, Pushchino, Russia
Amalfitano A.
Human Genetics, University of
Michigan, Ann Arbor, USA
Ambrosio G.B.
Internal Medicine I, Venice
City Hospital, Venice, Italy
Anker S.
Cardiac Medicine NHLI London ,
Dovehouse Street, London, UK
Argentini C.
Department of Biomedical
Sciences and C. N. R. Unit for Muscle Biology &
Physiopathology, University of Padova, Italy
Arpesella G.
Cardiovascular Surgery,
University of Bologna, Italy
Arslan P.
Institute of Experimental and
Laboratory Medicine, University of Padova, Italy
Austin
L.
Melbourne Neuromuscular
Research Centre, St Vincent's Hospital, Fitzroy, Australia
Barbiero M.
Division of Cardiology, Legnago
General Hospital, Verona, Italy
Barjot C.
Department of Human Genetics,
University of Michigan, Ann Arbor, Michigan, USA
Barsotti G.
Department of Medicine,
University of Pisa, Italy
Barylski N.
Department of Biological
Sciences and College of Osteopathic Med., Ohio University,
Athens, USA
Bennet T.
Bernard O'Brien Microsurgery
Research Institute, St Vincent's Hospital, Fitzroy, Australia
Berrebi A.
Department of Cardiac Surgery,
Broussais Hospital, Paris, France.
Bianchetti F.
Cardiology Department, Brescia,
Italy
Biral D.
C.N.R. Unit for Muscle Biology
and Physiopathology and Dept. of Biomedical Sciences, The
University of Padova, Italy
Bolotin G.
The Department of
Cardiothoracic Surgery, Carmel Medical Center, The Department of
Biological Engineering, I.I.T. Haifa, Israel
Bortoloso E.
C.N.R. Unit for Muscle Biology
and Physiopathology, Department of Biochemical Sciences,
University of Padova, Italy
Bower J.
Melbourne Neuromuscular
Research Centre, St Vincent's Hospital, Fitzroy, Australia
Bresci M.
Department of Neuroscience,
Section of Neurology, University of Pisa , Italy
Brunazzi C.
Cardiology, Pieve di Coriano
Hospital, Mantua, Italy
Calnek D.
Dept. of Human Genetics,
University of Michigan, Ann Arbor, Michigan, USA
Campos G.
Dept. of Biological Sciences
and College of Osteopathic Med., Ohio University, Athens, USA
Cantini M.
C.R.I.B.I. Center, and
Department of Biomedical Sciences, University of Padova, Italy
Carpentier A.
Department of Cardiac Surgery,
Broussais Hospital, Paris, France.
Carraro U.
CNR Unit for Muscle Biology and
Physiopathology, Department of Biomedical Sciences, University
of Padova, Italy
Casarotto D.
Cardiovascular Surgery,
University of Padova, Italy
Catani C.
CNR Unit for Muscle Biology and
Physiopathology, Department of Biomedical Sciences, University
of Padova, Italy
Ceconi C.
Chair of Cardiology, University
of Brescia, Italy
Chachques
J.C.
Department of Cardiac Surgery,
Broussais Hospital, Paris, France.
Chamberlain J. S.
Department of Human Genetics,
The University of Michigan, Ann Arbor, MI, USA
Chekanov
V.
S.
Milwaukee Heart Project,
Milwaukee, WI, USA
Chekanov
V.
S
Milwaukee Heart Project,
Milwaukee, WI, USA
Cheng Q.
Milwaukee Heart Project,
Milwaukee, WI, USA
Chisari
C.
Department of Neuroscience,
Section Neurology, University of Pisa, Italy
Chisari
C
Department of Neuroscence,
Section Neurology, University of Pisa, Italy
Chiu R. C-J.
The Montreal General Hospital,
Montreal, Quebec, Canada
Christlieb I.Y.
Cardiothoracic Surgical
Research, Department of Surgery, Allegheny University Hospitals,
Pittsburgh, Pennsylvania
Coats A.
Cardiac Medicine NHLI London,
Dovehouse Street, London, UK
Coletti G.
Cardiac Surgery, Brescia, Italy
Coper H.
Institute for
Neuropsychopharmacology, Free University, Berlin, Germany
Cossu G.
Department of Medical
Embryology and Histology, University of Rome, Italy
Cotogni A.
Division of Cardiology, Legnago
General Hospital (Verona)
Cupisti A.
Department of Medicine,
University of Pisa, Italy
Curello
S.
Chair of Cardiology, University
of Brescia, Italy
Curnis A.
Cardiology Department, Brescia,
Italy
D'Alessandro
C.
Department of Neuroscience,
Section of Neurology, University of Pisa, Italy
Dalla Libera L.
CNR Unit for Muscle Biology and
Physiopathology , Padova;
Damiani E.
C.N.R. Unit for Muscle Biology
and Physiopathology, Department of Biochemical Sciences,
University of Padova, Italy
DelloRusso C.
Departments of Human Genetics
and Physiology, University of Michigan, Ann Arbor, Michigan, USA
Docali G.
Division of Cardiology, Legnago
General Hospital, Verona, Italy
Duan C.
Cardiothoracic Surgical
Research, Department of Surgery, Allegheny University Hospitals,
Pittsburgh, Pennsylvania
Edström L.
Department of Neurology,
Karolinska Hospital and Institute, Stockholm, Sweden
El Messlemani A.
Department of Biomedical
Sciences, University of Padova, Italy
Eriksson L.I.
Anaesthesiology and Intensive
Care, Karolinska Hospital and Institute, Sweden
Faulkner
J.A.
Institute of Gerontology,
Departments of Physiology, and Biomedical Engineering, The
University of Michigan, Ann Arbor, MI, USA
Ferrari G.
The Telethon-HS Raffaele
Institute for Gene Therapy of Genetic Diseases, Milano, Italy
Ferrari R.
Chair of Cardiology, University
of Brescia, Italy
Fidzianska A.
Department of Neurology, Warsaw
Medical Academy, Warsaw, Poland.
Fiszman M.Y.
INSERM U153, Institut de
Myologie, Hopital Pitié-Salpêtrière, Paris, France
Fromes Y.
INSERM U153, Institut de
Myologie, Hopital Pitié-Salpêtrière, Paris, France
Gealow K.
Medtronic, Inc., Minneapolis,
USA
Gemelli M.
Division of Cardiology, Legnago
General Hospital, Verona, Italy
Giannini E.
Department of Neuroscience,
Section of Neurology, University of Pisa, Italy
Giardini E.
Institute of Plastic Surgery,
University of Padova, Italy
Giordano A.
Cardiology Division IRCCS
Fondazione Clinica del Lavoro, Gussago, Italy
Giurisato E.
C.R.I.B.I. Center, University
of Padova, Italy
Gosselin L.E.
Department of Physical Therapy
and Exercise Science, State University of New York, Buffalo USA
Hagerman F.C.
Department of Biological
Sciences and College of Osteopathic Medicine, Ohio University,
Athens, Ohio, USA
Hartigan-O'Connor D.J.
Dept. of Human Genetics
University of Michigan, Ann Arbor, Michigan, USA
Hauser M.A.
Dept. Human Genetics,
University of Michigan, Ann Arbor, Michigan, USA
Hausmanowa-Petrusewicz I.
Neuromuscular Unit, Medical
Research Center, Polish Academy of Sciences, Warsaw, Poland
Hikida R.S.
Department of Biological
Sciences and College of Osteopathic Medicine, Ohio University,
Athens, Ohio, USA
Hooper T.L.
Department of Human Anatomy and
Cell Biology, University of Liverpool & Department of
Cardiothoracic Surgery, Wythenshawe Hospital, Manchester, UK
Ikeda H.
First Department of Pathology,
Aichi Medical University, Aichi, Japan
Itoh G.
First Department of Pathology,
Aichi Medical University, Aichi, Japan
Jackson
M.J.
Muscle Research Centre,
Department of Medicine, University of Liverpool, UK.
Jakubiec-Puka
A.
Department of Cellular
Biochemistry, Nencki Institute of Experimental Biology, Warsaw,
Poland
Jarvis J.C.
Department of Human Anatomy and
Cell Biology, University of Liverpool, UK
Kaminska
A.M.
Neurological Department,
Medical Academy, and Neuromuscular Unit, Medical Research
Center, Polish Academy of Sciences, Warsaw, Poland
Kaulbach H.G.
Department of Cardiology,
Cardiovascular Research Institute CARIM, Maastrich, The
Netherlands
Koskinen S.A.O.
Biology of Physical Activity,
University of Jyvaskyla, Finland
Kovanen
V.
Departments of Health Sciences,
University of Jyvaskyla, Finland
Kurek J.
Melbourne Neuromuscular
Research Centre, St Vincent's Hospital, Fitzroy, Australia
La Rovere M.T.
Dept. of Cardiology, Centro
Medico di Montescano, "S. Maugeri" Foundation, IRCCS,
Montescano, Pavia, Italy
Larsson
L.
Noll Physiological Research
Center, The Pennsylvania State University, U.S.A., &
Department of Neurology, Karolinska Hospital and Institute,
Stockholm, Sweden
Leprotti C.
Internal Medicine I, Venice
City Hospital, Venice, Italy
Li X.
Department of Neurology,
Karolinska Hospital and Institute, Stockholm, Sweden
Lombardi P.
Cardiovascular Surgery,
University of Bologna, Italy
Lorusso R.
Department of Cardiothoracic
Surgery, Brescia, Italy
Loughna P.T.
Molecular Physiology Unit,
Department of Veterinary Basic Sciences, Royal Veterinary
College, London, UK.
Luecke T.
Department of Biological
Sciences and College of Osteopathic Medicine, Ohio University,
Athens, Ohio, USA
Lukoyanova N.
Institute of Theoretical and
Experimental Biophysics RAS, Pushchino, Russia
Lynch G.S.
Institute of Gerontology, The
University of Michigan, Ann Arbor, MI, USA
Maglara A.
Department of Medicine,
University of Liverpool, Liverpool L69 3GA
Magovern G.J.Sr.
Cardiothoracic Surgical
Research, Allegheny University of the Health Sciences:
Department of Surgery, Allegheny University Hospitals,
Pittsburgh, Pennsylvania
Magovern J.A.
Cardiothoracic Surgical
Research, Allegheny University of the Health Sciences &
Department of Surgery, Allegheny University Hospitals,
Pittsburgh, Pennsylvania
Malyshev S.
Institute of Theoretical and
Experimental Biophysics RAS, Pushchino, Russia
Marchetti C.
Department of Human Pathology,
University of Pavia, Italy
Marchini A.
Cardiology Department, Brescia,
Italy
Margreth
A.
C.N.R. Unit for Muscle Biology
and Physiopathology, Department of Biochemical Sciences,
University of Padova, Italy
Mason P.
Molecular Physiology Unit,
Department of Veterinary Basic Sciences, Royal Veterinary
College, London, UK.
Massimino M.L.
C.R.I.B.I. Center, University
of Padova, Italy
Mavilio F.
The Telethon-HS Raffaele
Institute for Gene Therapy of Genetic Diseases, Milano, Italy
Mazzoleni F.
Institute of Plastic Surgery,
University of Padova, Italy
McArdle
A.
Muscle Research Centre,
Department of Medicine, University of Liverpool, UK.
McArdle F.
Muscle Research Centre,
Department of Medicine, University of Liverpool, UK.
Menshova O.
Institute of Theoretical and
Experimental Biophysics RAS, Pushchino, Russia
Meola M.
Department of Medicine,
University of Pisa, Italy
Mikus P.M.
Cardiovascular Surgery,
University of Bologna, Italy
Minetti C.
Muscle Disease Service,
Department of Pediatrics, University of Genova, G. Gaslini
Institute, Italy.
Morelli E.
Department of Medicine,
University of Pisa, Italy
Mortara
A.
Department of Cardiology,
Centro Medico di Montescano, "S. Maugeri" Foundation, IRCCS,
Montescano, Pavia, Italy
Muneretto
C.
Cardiovascular Surgery,
University of Brescia, Italy
Ossowska K.
Department of
Neuropharmacology, Institute of Pharmacology of the Polish
Academy of Sciences, Cracow, Poland
Pette D.
University of Konstanz, Faculty
of Biology, D-78457 Konstanz, Germany
Podlubnaya
Z.
Institute of Theoretical and
Experimental Biophysics RAS, Pushchino, Russia
Poggi P.
Institute of Histology,
University of Pavia, Italy
Poole-Wilson
P.
A.
Cardiac Medicine, Imperial
College School of Medicine, National Heart and Lung Institute,
Dovehouse Street, London
Prenger K.
Departments of Cardiology,
Cardiac Surgery and Anaesthesiology; Cardiovascular Research
Institute Maastricht, The Netherlands
Pullan D.M.
Department of Human Anatomy and
Cell Biology, The University of Liverpool, UK
Rajnoch C.
Institute of Myology, La
Pitiè-Salpetrère Hospital. Paris, France
Ravara B.
CNR Unit for Muscle Biology and
Physiopathology and Department of Experimental Biomedical
Sciences, University of Padova, Padova, Italy
Rieder M.A.
Milwaukee Heart Project,
Milwaukee, WI, USA
Rizzi C.
Department of Biomedical
Sciences, University of Padova, Italy
Rossi B.
Department of Neuroscience,
Section Neurology, University of Pisa, Italy
Rossini K.
Department of Biomedical
Sciences, University of Padova, Italy
Rubin Y.
The Department of
Cardiothoracic Surgery, Carmel Medical Center, the Department of
Biological Engineering, I.I.T. Haifa, Israel
Salmons S.
Department of Human Anatomy and
Cell Biology, University of Liverpool, UK
Salvatori G.
Department of Human Genetics,
University of Michigan, Ann Arbor, Michigan, USA
Sandri C.
Department of Biomedical
Sciences, University of Padova, Italy
Sandri M.
Department of Biomedical
Sciences and The Institute of Experimental and Laboratory
Medicine, University of Padova, Italy
Sandri M
Department of Biomedical
Sciences and The Institute of Experimental and Laboratory
Medicine, University of Padova, Italy
Scalise D.
Cardiothoracic Surgical
Research, Allegheny University of the Health Sciences and
Department of Surgery, Allegheny University Hospitals,
Pittsburgh, Pennsylvania
Scelsi
R
Department of Human Pathology,
University of Pavia, Italy
Schiaffino S.
Department of Biomedical
Sciences and C.N.R. Unit for Muscle Biology and Physiopathology,
University of Padova, Italy
Schreuder J.J.
Departments of Cardiology,
Cardiac Surgery and Anaesthesiology; Cardiovascular Research
Institute Maastricht, The Netherlands
Schulze G.
Institute for
Neuropsychopharmacology, Free University, Berlin, Germany
Schwartz K.
INSERM U153, Institut de Myologie, Hopital Pitié-Salpêtrière, Paris, France
Shofti R.
The Department of Cardiothoracic Surgery, Carmel Medical Center, the Department of Biological Engineering, I.I.T. Haifa, Israel
Shortland A.P.
Department of Clinical
Engineering, The University of Liverpool
Shpagina M.
Institute of Theoretical and
Experimental Biophysics RAS, Pushchino, Russia
Smink M.
Departments of Cardiology,
Cardiac Surgery and Anaesthesiology; Cardiovascular Research
Institute Maastricht, The Netherlands
Staron R.S.
Department of Biological
Sciences and College of Osteopathic Medicine, Ohio University,
Athens, Ohio, USA
Suzuki Y.
First Department of Pathology,
Aichi Medical University, Aichi, Japan
Takala T.E.S.
Biology of Physical Activity,
University of Jyvaskyla, Finland
Tang A.T.M.
Department of Human Anatomy and
Cell Biology, University of Liverpool & Department of
Cardiothoracic Surgery, Wythenshawe Hospital, Manchester, UK
Tchekanov G.V.
Milwaukee Heart Project,
Milwaukee, WI, USA
ter Keurs H.
University Calgary, Calgary,
Canada
Testolin L.
Cardiovascular Surgery,
University of Padova, Italy
Tews D.S.
Department of Neuropathology,
Johannes-Gutenberg-University, Mainz, Germany
Tong
J.
First Department of Pathology,
Aichi Medical University, Aichi, Japan
Trainini J.C.
Hospital Presidente Peròn,
Buenos Aires, Argentina
Trumble D.R.
Cardiothoracic Surgical
Research, Allegheny University of the Health Sciences and
Department of Surgery, Allegheny University Hospitals,
Pittsburgh, Pennsylvania
Udaltsov S.
Institute of Theoretical and
Experimental Biophysics RAS, Pushchino, Russia
Uretzky G.
The Department of
Cardiothoracic Surgery, Carmel Medical Center, the Department of
Biological Engineering, I.I.T. Haifa
van der Nagel T.
Department of Cardiology,
Cardiovascular Research Institute Maastricht, The Netherlands;
van der
Veen F.H.
Departments of Cardiology,
Cardiac Surgery and Anaesthesiology, Cardiovascular Research
Institute Maastricht, The Netherlands
van der Veen F. H
Departments of Cardiology,
Cardiac Surgery and Anaesthesiology, Cardiovascular Research
Institute Maastricht, The Netherlands
Vescovo
G.
Internal Medicine I, Venice
City Hospital, Venice, Italy
Vescovo
G
Internal Medicine I, Venice
City Hospital, Venice, Italy
Volterrani M.
Cardiology Division IRCCS
Fondazione Clinica del Lavoro, Gussago, Italy
Walsh S.
Department of Biological
Sciences and College of Osteopathic Medicine, Ohio University,
Athens, Ohio, USA
Wendeln H.
Department of Biological
Sciences and College of Osteopathic Medicine, Ohio University,
Athens, Ohio, USA
White J.
Melbourne Neuromuscular
Research Centre, St Vincent's Hospital, Fitzroy, Australia
Wolf T.
The Department of
Cardiothoracic Surgery, Carmel Medical Center, the Department of
Biological Engineering, I.I.T. Haifa
Wolfarth S.
Department of
Neuropharmacology, Institute of Pharmacology of the Polish
Academy of Sciences, Cracow, Poland
Zackrisson H.
Department of Neurology and
Anaesthesiology and Intensive Care, Karolinska Hospital and
Institute, Sweden and
Zander G. L.
Milwaukee Heart Project,
Milwaukee, WI, USA
Zennaro R.
Internal Medicine I, Venice
City Hospital, Venice, Italy
Zogno M.
Cardiac Surgery Department,
Brescia, Italy
Zuliani F.
Institute of Plastic Surgery,
University of Padova, Italy
ABSTRACTS
SKELETAL MUSCLE RESPONSES TO
EXERCISE-INDUCED OXIDATIVE STRESS
M.J. Jackson, A. McArdle and F. McArdle
Muscle Research Centre,
Department of Medicine, University of Liverpool, UK.
It is now well established
that contractile activity leads to an increase in the production
of reactive oxygen species (free radicals and/or hydrogen
peroxide) within skeletal muscle. However the consequences of
this increase and the desirability of reducing their production
is the subject of debate. The superoxide radical is produced as
a consequence of the increased mitochondrial oxygen consumption
during aerobic exercise with the eventual production of hydrogen
peroxide following further reduction of superoxide (1). Many
investigators have examined the possibility that this increase
in free radical generation is the cause of exercise-induced
muscle damage resulting in loss of cell viability, force
generation and muscle pain, but the evidence that this is an
inevitable consequence is not compelling (2) There is increasing
evidence that oxidative stress may be used by cells as a signal
for an adaptive response and some evidence suggests this may be
true for exercising skeletal muscle. Muscle cells exposed to a
non-damaging contractile activity respond by increasing the
expression of a number of genes, including a those coding for
the antioxidant enzymes and for other cytoprotective proteins,
such as heat shock proteins. This has been reported to be
triggered by exercise-induced oxidative stress within the muscle
(3).
Financial support from the
Wellcome Trust and Ministry of Agriculture, Fisheries and Food
is gratefully acknowledged.
References
1. Loschen G, Azzi A, Richter C and Flohe L. FEBS Lett. 42: 68-72, 1974
2. Jackson MJ, McArdle A, and O'Farrell S. In: Immunopharmacology of Free Radical Species. Eds. D. R. Blake and P.G. Winyard. Pub. Academic Press, 1995: pp. 175-182
3. Salo DC, Donovan CM, Davies
KJA. Free Rad. Biol. Med. 11: 239-246, 1991.
FORCED LENGTHENING CONTRACTION
INDUCED MUSCLE DAMAGE IN RATS AT DIFFERENT AGES-ACTIVATION OF
COLLAGEN SYNTHESIS RELATED GENE EXPRESSION
V. Kovanen, A.M. Ahtikoski1, S.A.O. Koskinen1 T.E.S. Takala1 and L.E. Gosselin2
Departments of Health Sciences
and (1) Biology of Physical Activity, University of Jyvaskyla,
Finland; (2) Department of Physical Therapy and Exercise
Science, State University of New York, Buffalo USA
The expression of fibrillar
collagens themselves as well as prolyl 4-hydroxylase (P4H), the
regulatory posttranslational modificator enzyme in collagen
biosynthesis, increases coordinatively both at the mRNA and
protein level after eccentric exercise induced muscle damage
(1). This has been shown to occur in young adult animals.
Therefore, the present study investigated the effect of age (6-,
12-, and 26 mo) on collagen metabolism and its regulation in
male Fischer 344 rat tibialis anterior (TA) muscle 7 days after
eccentric contractions. TA muscle were subjected to eccentric
contractions by electrical stimulation of the common peroneal
nerve (120 Hz, 0.2 msec pulse duration, 20 contractions/min, 2x5
min bouts with 5 min rest between bouts). Contralateral TA was
used as a control muscle. Steady state mRNA levels were analyzed
using Northern and slot blot hybridization and densitometric
quantification of the autoradiographs. P4H activity was measured
according to Kivirikko and Myllylä (2). ß-glucuronidase (ß-GU)
activity was used as a biochemical marker of muscle damage.
There was an age-associated decline in the mRNA levels of type I
and III collagens, and a- subunit of P4H, as well as P4H enzyme
activity in both control and injured muscles. When compared to
the contralateral muscles, the relative responses to injury were
significantly higher in the 26-mo than in the 6-mo rats. The
mRNA levels of TGF-ß1, the important growth factor regulating
collagen biosynthesis, did not change with age and increased to
same extent in each age group after muscle damage. The activity
of ß-GU indicated a lower degree of damage in old rats compared
to the younger ones in spite of exposure to an indentical injury
protocol, which may reflect age-dependent differences in the
timetable of the responses related to muscle damage. The results
suggest that older rats have a high capacity to respond to
muscle damage and regeneration by activating collagen synthesis
related gene expression.
References
1. Han X, Wang W, Komulainen J, Koskinen SAO, Kovanen V, Vihko V, Takala TES: Increased expression of collagen and related genes in rat skeletal muscle after acute strenuous exercise. Am J Phjsiol, resubmitted.
2. Kivirikko KI, Myllylä R:
Posttranslationnal enzymes in biosynthesis of collagen:
Intracellular enzymes. Methods in Enzymology. 82:245-304,1982
The financial support of the
Ministry of Education, Finland, is gratefully acknowledged.
THE IMMUNOHISTOCHEMICAL ANALYSIS
OF DYSTROPHIN AND SOME MYOFIBRILLAR PROTEINS (DESMIN,
a-ACTININ AND TROPOMYOSIN) IN SKELETAL MUSCLE ELECTRICALLY
STIMULATED IN EXTENSION
A. Jakubiec-Puka, D. Biral1 and A. Fidzianska2
Dept. of Cellular Biochemistry, Nencki Inst. of Experimental Biology, Warsaw, Poland;
(1)Dept. of Biomedical
Sciences, The University of Padova, Italy; (2)Dept. of
Neurology,Warsaw Medical Academy and Neuromuscular Unit.,
Medical Research Center, Polisch Academy of Sciences, Warsaw,
Poland2
It is a well-know fact from the previous study, that the muscle that has been work-overloaded in extension gets easily demaged. Those findings make us examine some structural muscle proteins (desmin a-actinin and tropomyosin) by the immunohistochemical methods. In addition the dystrophin was examined, the loss of which was observed recently in the muscle stimulated in extension (Biral et al. BAM, in press)
Methods. The rat soleus and extensor digitorum longus muscles ware maintained in an extended position and the sciatic nerve was stimulated continuously for 4-24 h by low frequency. As controls served muscles stimulated in a neutral position and intact muscles. Immediatelly after decapitation each whole muscle was excised, maintaining its length carefully and frozen. Examination was performed of the longitudinal (desmin, a-actinin and tropomyosin) and transverse (dystrophin) cryostat-sections. Monoclonal antibodies against the above-mentioned proteins were used.
Results. In longitudinal section of the control muscles the transverse striation features were present in all investigated muscle fibres, stained by all the mentioned antibodies against myofibrillar proteins. In fibres of the muscles stimulated in extension the transverse striation features were sometimes preserved, but often the transverse striation was irregular and disturbed, or absent, at all. Some longitudinal structures were frequently observed within such fibres, shown by all antibodies applied. These fibres also contained structures resembling the contraction bands, or they were fragmented. Those changes were observed, too, occasionally in the muscle stimulated in a neutral position, but never in the control ones. Intriguing was that desmin seemed to be the most resistant among the myofibrillar proteins examined; desmin was seen even within the heavily demaged fibres.
Transverse sections of the same muscles were stained with anti-dystrophin antibody. Numerous fibres of the muscles stimulated in extension, which contain the fibres with the above-described abnormalities of the myofibrillar proteins, showed some lack of dystrophin in sarcolemma. This phenomenon was not observed either in the control muscles or in those stimulated in a neutral position. Coexistence the losed dystrophin and the changed organization of the myofibrillar proteins needs some detailed study.
ARE LIM-DOMAIN CONTAINING
PROTEINS INVOLVED IN THE REGULATION OF MUSCLE PHENOTYPE ?
P.T. Loughna and P. Mason
Molecular Physiology Unit,
Department of Veterinary Basic Sciences, Royal Veterinary
College, London, UK.
Lim domain proteins are a
family of proteins that are thought to regulate protein-protein
interactions or transcription. The term LIM derives from the
first letter of each of the three genes from which the LIM motif
was first discovered (1). The LIM protein MLP (Muscle LIM
Protein, which is expressed at high levels in both cardiac and
skeletal muscle, is suggested to be a regulator of myogenic
differentiation and the temporal expression of it's transcript
in cultures of C2 cells is similar to that of myogenin (2). We
have previously described a LIM domain containing protein,
expressed at high levels only in skeletal muscle (3), which we
termed SLIM (Skeletal muscle LIM-protein). The expression of
this LIM protein in C2 cells exhibits a different pattern to
that of MLP and it's ontogenic pattern of expression in vivo
exhibits a degree of muscle-specificity. During development
dramatic increases in the transcript level for this protein
coincide with periods of rapid muscle growth albeit in a
muscle-specific manner. We have examined, in the rat, the
effects of immobilisation of muscles in the shortened (disuse
atrophy) position and lengthened (passive stretch) upon SLIM
mRNA levels. Disuse atrophy was associated with a dramatic
decrease in the level of this transcript whereas passive stretch
was not. We have also examined the effect of short-term
denervation upon the expression of several different LIM protein
transcripts in muscle of differing fibre type compositions.
These data suggest that certain LIM proteins may play a role in
the regulation of skeletal muscle phenotype in the post-natal
animal.
References
1. Sanchez-Garcia I, Rabbitts TH: Trends in Genetics 1994; 10: 315-320.
2. Alber A, Halder G, Caroni P: Cell 1994; 79: 221-231.
3. Morgan MJ, Madgwick AJ,
Charleston B, Pell JM, Loughna PT: Biochem Biophys Res Commun
1995; 212: 840-846.
ACUTE QUADRIPLEGIA AND LOSS OF
MUSCLE MYOSIN IN PATIENTS TREATED WITH NON-DEPOLARIZING
NEUROMUSCULAR BLOCKING AGENTS AND CORTICOSTEROIDS. UNDERLYING
CELLULAR AND MOLECULAR MECHANISMS
L. Larsson1,2, X. Li2, L.
Edstrom2, L.I. Eriksson3, H. Zackrisson2, 3, C. Argentini4 and
S. Schiaffino4
(1) Noll Physiological
Research Center The Pennsylvania State University, U.S.A., (2)
Department of Neurology and(3) Anaesthesiology and Intensive
Care, Karolinska Hospital and Institute, Sweden and (4)
Department of Biomedical Sciences and C. N. R. Unit for Muscle
Biology and Physiopathology, University of Padova, Italy
Objectives: The purpose of this study was to investigate the mechanisms underlying the acute quadriplegic myopathy (AQM) after treatment with non-depolarizing neuromuscular blocking agents (NMBA) and corticosteroids (CS) in the intensive care unit (ICU) by using sensitive biochemical, molecular biological and cellphysiological techniques.
Background: Long-term treatment with NMBA and CS in the ICU is not benign and an increasing number of patients with the AQM have been reported in parallel with increased use of these drugs. AQM is an important cause of morbidity in the ICU, and hence also has a significant economic impact on ICU and hospital resources.
Design/Methods: Muscle biopsies were taken and electrophysiological examinations performed in the acute phase and during recovery in the AQM patients. Regulation of muscle contraction and myofibrillar of protein synthesis was studied using cellphysiological, ultrasensitive electrophoretic, in situ hybridization, and histopathological techniques critically ill patients who had been given massive doses of CS in combination with variable doses of NMBA. All patients developed paralysis of spinal nerve-innervated muscles. Cranial nerve-innervated muscle, sensory and cognitive functions were, on the other hand, well maintained after discontinuation of NMBA treatment.
Results: The major observations in the AQM patients were: 1) a general decrease in myofibrillar protein contents, 2) specific but highly variable partial or complete loss of myosin and myosin-associated proteins, 3) very low thick:thin filament protein ratios, 4) absence of myosin mRNA, and 5) a dramatically impaired muscle cell force generating capacity in the acute phase of the AQM. During clinical improvement, normal expression of myosin mRNAs, re-expression of thick filament proteins, and an increased specific tension were observed.
Conclusions: Acute
quadriplegic myopathy is associated with a specific decrease in
thick filament proteins related to an altered transcription
rate. Although the decreased content of thick filament proteins
is important for the prolonged muscle weakness, it is not the
primary cause of the muscle paralysis in the acute stage, in
which impaired muscle membrane excitability probably plays a
more significant role. Several factors contribute to this
condition, but the action of corticosteroids appears to be the
predominant one, with potentiation by neuromuscular blocking
agents, immobilization, and probably also concurrent sepsis.
CARDIAC ASSISTANCE FROM SKELETAL MUSCLE: AVOIDING
ISCHAEMIA IN THE LATISSIMUS DORSI MUSCLE GRAFT
S. Salmons1, A.T.M. Tang1,2,
J.C. Jarvis1, and T.L. Hooper1, 2
(1)Department of Human Anatomy and Cell Biology; University of Liverpool
(2)Department of
Cardiothoracic Surgery, Wythenshawe Hospital, Manchester , UK
Functional grafts of skeletal muscle can be used to provide cardiac assistance. The latissimus dorsi muscle (LDM) is particularly suitable, as it has a discrete blood supply from the thoracodorsal artery, entering close to its proximal insertion, and can therefore be raised as a pedicled flap and transferred into the chest. However the muscle cannot be raised without dividing the perforating arteries that enter its distal portion, and this results in ischaemic damage when the muscle is stimulated electrically.
To address this problem, we focused on communication between branches of the thoracodorsal artery proximally and the collateral vessels distally. Anastomotic connections have been reported in the literature. The importance of anastomoses in the LDM is that they provide, in principle, a route whereby blood can be supplied to the distal part of the muscle via an existing vascular network, without the delays required for neovascularization. Using a fluorescent microsphere technique to measure regional blood flow in the sheep LDM, and fluorescence microscopy to image microspheres in capillaries, we obtained the first definitive evidence that arterial anastomoses not only exist but are functional under physiological conditions. We went on to show that electrical stimulation of the muscle prior to mobilizing it as a graft abolished proximodistal gradients in flow, enhancing blood flow to the distal portion of the graft. This rendered the muscle resistant to the ischaemia associated with cooling, handling, electrocautery and loss of resting tension, which could all contribute to collapse of the anastomotic channels during a clinical procedure.
In procedures such as dynamic cardiomyoplasty, prestimulation of the LDM and the use of less demanding stimulation patterns for conditioning and activation would allow earlier introduction of effective cardiac assistance, and avoid the fibrofatty replacement that can otherwise prejudice the functionality of the graft in the long term.
The financial support of the British Heart Foundation is gratefully acknowledged.
References
1. Salmons S, Tang ATM, Jarvis JC, Degens H, Hastings M, Hooper TL. Morphological and functional evidence, and clinical importance, of vascular anastomoses in the latissimus dorsi muscle of the sheep. J Anat, in press.
2. Tang ATM, Jarvis JC, Hooper
TL, Salmons S. Observation and basis of improved blood flow to
the distal latissimus dorsi muscle: a case for electrical
stimulation prior to grafting. Cardiovasc Res, in press.
INCREASED SUSCEPTIBILITY TO
MUSCLE DAMAGE FOLLOWING EXERCISE IN NEPHROTIC PATIENTS WITHOUT
RENAL FAILURE
C. Chisari, A. Cupisti1, E.
Morelli1, M. Meola1, G. Barsotti1, B. Rossi
Dpt. of Neuroscience, Sect. Neurology, University of Pisa, Italy
(1) Dpt. of Medicine, University of Pisa, Italy
The nephrotic syndrome is a protein-wasting disorders affecting total body protein metabolism, often leading to reduction of lean body mass and changes of muscle cell composition.
The aim of this study was to investigate the susceptibility to muscle cell damage in nephrotic patients following submaximal physical exercise, by detection of the creatinkinase (CK) plasma level changes.
Fourteen patients effected by primary nephrotic syndrome, without chronic renal failure, underwent an exercise test on cycle ergometer for 20 minutes at constant speed (60 r.p.m.). In each subject, the work rate (watt-expressed) was established as 70% of maximum power theoretically calculated on the sex, age, weight and height basis. CK plasma levels (U/L) were determined before and 1, 3, 6, and 24 hours after the exercise.
Following exercise, CK plasma levels became higher in nephrotics than in normal control. That is, the amount of CK increments were greater in nephrotics than in controls since the first hour after the end of the exertion. These changes, both as absolute values and as percentage of the basal values, correlate positively to daily urinary protein losses and inverse linear relationship has been detected with albumin serum levels at 3 hours and at 6 hours time. Instead, no correlation has been observed between the amount of plasma CK increases and age, body weight, plasma creatinine, plasma cholesterol or haematocrit.
These result demonstrate that a greater than normal increase of CK plasma levels occurs in nephrotics following the physical exercise. The suggests an increased susceptibility to muscle injury in nephrotic patients probably related to the protein depletion and/or to modifications of muscle cell metabolism.
References
1. Maroni BJ, Staffeld C, Young VR, Manatunga A, Tom K: Mechanism permitting nephrotic patients to achieve nitrogen equilibrium with a protein-restricted diet. J Clin Invest 1997; 99: 2479-2487.
2. Armstrong RB: Mechanism of exercise-induced muscle fibre injury. Sports Med 1991; 12: 184-207.
3. Van der Muelen JH, Kuipers
H, Drukker J: Relationship between exercise-induced muscle
damage and enzyme release in rats. J Appl Physiol 1991; 71 (3):
999-1004.
FORCE AND POWER OUTPUT OF
SKELETAL MUSCLES FROM YOUNG, ADULT AND OLD MDX MICE
J.A. Faulkner1, 2, 4, G.S.
Lynch1 and J.S. Chamberlain3
(1) Institute of Gerontology,
and (2) Departments of Physiology, (3) Human Genetics, and (4)
Biomedical Engineering, The University of Michigan, Ann Arbor,
MI, USA
Duchenne muscular dystrophy
(DMD) is an X-linked recessive disorder resulting from a
mutation in the gene that encodes dystrophin. In human beings,
DMD is characterized by progressive skeletal muscle
degeneration. The mdx mouse, which has a dystrophin gene
mutation and lacks dystrophin expression in muscle tissues, has
been used in a wide range of investigations probing the
underlying mechanisms of DMD (1). Of the muscles studied in the
mdx mouse, only the diaphragm muscle exhibits extensive and
progressive degeneration, fibrosis and severe functional
deficits comparable to the clinical symptoms observed in limb
muscles of boys with DMD (3). Despite the numerous studies of
the morphology and capabilities for force development of the
limb muscles of the mdx mouse, considerable controversy exists
as to the muscle mass and maximum force development compared
with comparable data on age-matched control C57BL/10 mice (2).
We tested the hypotheses that extensor digitorum longus (EDL)
and soleus muscles from young (6 month) and adult (16-18 month)
mdx mice are larger but weaker than those from age- matched
control muscles, whereas for old (24 month) mdx compared with
old control mice, muscles are smaller and weaker. A corollary to
our primary hypothesis is that EDL and soleus muscles of old
compared with young mdx mice demonstrate decreases in muscle
mass and absolute and normalised force and power. The maximum
force production and power output of EDL and soleus muscles from
age-matched mdx and control mice were measured in vitro at 258C
and normalized by cross-sectional area and muscle mass
respectively. Compared with muscle masses of age-matched control
muscles, masses of EDL and soleus muscles of young and adult mdx
mice were ~120% to ~140% of control values, but those of old mdx
and control mice were not different. In the main, the maximum
forces developed by muscles from mdx and age-matched control
mice of all ages were not different, but the specific maximum
forces of muscles from mdx mice were generally 80% of the
age-matched control value. The EDL and soleus muscles of young
and adult mdx mice exhibited hypertrophy and weakness and in old
age muscles of mdx mice showed atrophy and losses in specific
force and normalized power. Losses in structure and function
were more severe in soleus than in EDL muscles. The functional
deficits observed in muscles of old mdx mice extend the
usefulness of this animal model for studying the mechanisms of
dystrophinopathies.
This work was supported by N.I.H grants AG-06157 (JAF) and AR-40864 (JSC), the Muscular Dystrophy Association (JSC), a C. J. Martin Research Fellowship (GSL). The mice were maintained by the Animal Core of the Nathan Shock Center AG-13283 (JAF).
References
1. Cox GA, Cole NM, Matsumura K, Phelps SF, Hauschka SD, Campbell KP, Faulkner JA, Chamberlain JS: Overexpression of dystrophin in transgenic mdx mice eliminates dystrophic symptoms without toxicity. Nature 1993; 364: 725-729.
2. Dupont-Versteegden EE, Mccarter RJ: Differential expression of muscular dystrophy in diaphragm versus hindlimb muscles of mdx mice. Muscle and Nerve 1992; 15: 1105-1110.
3. Lynch GS, Rafael JA, Hinkle
RT, Cole NM, Chamberlain JS, Faulkner JA: Contractile properties
of diaphragm muscle segments from old mdx and old transgenic mdx
mice. Am J Physiol 1997; 272 (Cell Physiology 41): C2063-C2068.
FUNCTIONAL BEHAVIOUR OF NATIVE
RYANODINE RECEPTOR/Ca2+-RELEASE CHANNEL IN SLOW SKELETAL
MUSCLE SR OF YOUNG AND OLD RATS
A. Margreth, E. Bortoloso and
E. Damiani
C.N.R. Unit for Muscle Biology
and Physiopathology, Department of Experimental Biochemical
Sciences, University of Padova, Italy
Prolongation of isometric
twitch contraction time, presumably as the result of altered
sarcoplasmic reticulum (SR)-mediated Ca2+-fluxes is observed in
aging skeletal muscle of human and animal species, such as the
rat, although with yet unclarified differences depending on the
strain of animals and the fiber type composition of the muscle
(1, 2, 3). An age-related decline of SR Ca2+-pumps, as
originally suggested from studies on skinned skeletal muscle
fibers (4), is not supported by direct biochemical studies on
isolated SR vesicles from fast skeletal muscle (2). Likewise, no
significant age-related differences in immunoreactive Ca2+-pump
protein content or in Ca2+-ATPase activity accounted for the
age-associated decrease of Ca2+-transport activity in
slow-muscle SR (3). An apparent uncoupling of ATP-hydrolysis
from Ca2+-transport might result from an age-related increase of
Ca2+-efflux through the Ca2+-release channel /Ryanodine receptor
(RyR1). A recent study (5), which assessed SR Ca2+-release
indirectly in chemically-skinned muscle fibers from rat soleus,
i.e. by following tension development, indicated an
age-associated decrease in the caffeine threshold of the SR. The
present [3H]-ryanodine binding study was designed to verify the
validity of this assumption, using SR-enriched membrane vesicles
from the soleus of young and old animals, and [3H]-ryanodine as
a specific probe of the functional state of native RyR1. Our
findings demonstrate no significant age-related difference in
caffeine sensitivity of the receptor, in the presence of
low-concentrations of Ca2+. Dose-dependent activation of RyR1 by
calmodulin was studied following preactivation by maximizing
concentrations of caffeine and also did not show any difference
between young and adult animals. Aging did not affect the
membrane density of RyRs and of the associated junctional SR
protein triadin, while appearing to be associated with a
relative increase in the slow cardiac isoform of calsequestrin.
Our results seem to exclude the possibility of an age-related
change in the intrinsic properties of RyR1 equivalent to isoform
transition (6).
References
1. Damiani E, Larsson L, Margreth A: Age-related abnormalities in regulation of the ryanodine receptor in rat fast-twitch muscle. Cell Calcium 19, 15-27, 1996.
2. Danieli-Betto D, Betto, Megighian A, Midrio M, Salviati G, Larsson L: Effects of age on sarcoplasmic reticulum properties and histochemical composition of fast- and slow-twitch rat muscles. Acta Physiol Scand 154, 59-64, 1995.
3. Larsson L, Ansved T: Effects of ageing on the motor unit. Prog. Neurobiol. 45, 397-458, 1995.
4. Larsson L, Salviati G: Effects of age on calcium transport activity of sarcoplasmic reticulum in fast- and slow-twitch rat muscle fiberes. J. Physiol. 419, 253-164, 1989.
5. Li X, Hughes S, Salviati G, Teresi A, Larsson L: Thyroid hormone effects on contractility and myosin composition of soleus muscle and single fibres from young and old rats. J. Physiol. 494, 555-567, 1996.
6. Narayanan N, Jones D, Xu A,
Yu J: Effects of aging on sarcoplasmic reticulum function and
contraction duration in skeletal muscles of the rat. Am. J.
Physiol. 271, C1032-C1040, 1996.
Work supported by EEC
(Bio-Med, project BMH4-CT 96-0174).
AGE-RELATED MUSCLE PLASTICITY AS
A RESPONSE TO LONG-TERM ELECTRICAL STIMULATION
V.S. Chekanov, M.A. Rieder,
G.L. Zander and Q. Cheng
Milwaukee Heart Project,
Milwaukee, WI, USA
In adult organisms, when electrical stimulation of skeletal muscle is stopped after 8 weeks of training, all acquired changes (physiological, morphological, and biochemical) revert to control after 10-14 days (delay period). this is the reason that training of the skeletal muscle before cardiomyoplasty is not done. We hypothesized that the muscle of the growing organism obtains more plasticity ( and "memory" at the gene level) and would not revert during the delay period.
Six adult sheep and 4 lambs received 8 weeks of electrical stimulation (ES) of the left latissimus dorsi muscle (LDM), with the right LDM serving as control. After 8 weeks of ES, the contractile force (CF) in the adult sheep decreased to 81±7% at 20g/kg preload; the CF in lambs decreased to 82±3% (p>0.05 vs. adult sheep). After 2 weeks delay, CF in adult sheep increased to 97±2%; CF in the lambs was only 93±1% (p>0.05)
During a 30 minute stress test, the untrained right LDM of adult sheep lost 43±5% CF; lamb right LDM lost 39±4% CF (p>0.05). Conditioned LDM of adult sheep lost only 8±3% CF during the same stress test; lamb CF lost 7±2% (p>0.05). The same test was repeated after 2 weeks delay. it was then evident that there are differences between adult and young muscle. After 30 minutes of testing, the LDM of adult sheep lost 33±6% CF. lamb CF lost only 12±2% (p>0.05). The LDM in the lamb continued to be fatigue resistant even after a 2 week delay period.
After 8 weeks of ES, the lactate dehydrogenase (LDH) fraction 5 in adult sheep decreased to 74±4% of total LDH (compared to 96±3% before ES). In lambs, the LDH-5 fraction decreased to 77±8% (compared to 91±5% before ES. p>0.05 vs. adult sheep). After 2 weeks delay, this LDH level increased to 83±3% in adult, yet continued to decrease to 68±3% in lambs (p<0.05 vs. adult sheep). LDH fractions 1 and 2 in adults were 2.5±09% of total LDH before ES, 8.4±0.4% after ES, and 4.6±0.3 after delay. In the lambs the levels were 2.5±0.9%, 6.7 ± 1.9%, and 7.2±1.5%, respectively.
Changes seen in the percent of mitochondrial area were similar: 5.2±2.0% before ES, 6.9±1.3% after ES, and 5.4 ± 1.3% after the delay period in adults; and 3.3 ± 0.5%, 6.5 ± 0.5%, and 5.1±0.4%, respectively in lambs. In the lambs there were also changes in the percent area occupied by muscle fibers: 81±2% before ES, 76±4% after ES, and 68±6% after the delay period. The number of nuclei per millimeter squared increased from 567±52 before ES TO 1012±76 after ES, and tented towards baseline after a 2 weeks delay (891±48, p<0.05 vs. baseline).
Conclusion: This data show
that young skeletal muscle obtains more plasticity than adult
muscle and "remembers" the obtained properties longer than the
adult muscle.
REGENERATIVE CAPABILITY OF SENILE
RAT SKELETAL MUSCLE
A.M. Kaminska(1, 2), A.
Fidzianska1, 2, G.Schulze3, H. Coper3, K. Ossowska4, S.
Wolfarth4, I. Hausmanowa-Petrusewicz2
(1) Neurological Department,
Medical Academy, (2) Neuromuscular Unit, Medical Research
Center, Polish Academy of Sciences, Warsaw, Poland; (3)
Institute for Neuropsychopharmacology, Free University, Berlin,
Germany. (4) Department of Neuropharmacology, Institute of
Pharmacology of the Polish Academy of Sciences, Cracow, Poland
A question frequently asked in the study of senile muscle is whether satellite cells that remain in muscle retain their myoblastic potential. Satellite cells in aged muscle appear dormant on the basic of their cytology and exhibit little or no mitotic activity. Experimental in vivo studies of senile muscle regeneration are sparse. Electronmicroscopic studies of the regenerating senile muscle have not- to our knowledge-yet been reported. Thus, it is the purpose of the present study to determine the ultrastructural features of bupivacaine (BPVC) induced muscle regeneration in senile rats.
To determine the regenerative capacity of senile muscle we injected anterior tibial (AT) muscle of rats aged 33-36 months with myotoxic agent (BPVC). The patterns of skeletal muscle regeneration were compared by the use of electron microscopy in young and old rats. Our results clearly showed that senile muscle was able to regenerate in response in senescence, however, was slower, distempered and markedly impaired as compared to young adul animals. Retardation of reparative response in old animals was already seen in early stages of regeneration, with less vigorous proliferation of myogenic cells and decreased activity of macrophages involved in phagocytosis of cellular debris. The phenomenon observed only in senile muscle was the presence of multiple single activated satellite cells non fusing with the parent fiber. Up to two weeks after injury, in contrast to young rats, regenerating senile muscle still showed prominent features of immaturity.
Decrease in satellite cell number, their diminished proliferative potential, delayed phagocytosis and insufficient innervation of the newly formed fibers are the main factor responsible for the impaired muscle regeneration in senescence. Thus it seems that age is the one of the principal determinants of the success of muscle regeneration.
This project was supported in
part by Deutscheforschungsgemeinschaft (grant DFG 436 Pol
113/63/09(S)) and Bundesministerium für Bildung und Forschung
(grant XO 8461).
THE ROLE OF HEAT SHOCK PROTEINS
IN CYTOPROTECTION OF ADULT AND AGED SKELETAL MUSCLE
Anne McArdle, Antonia Maglara
and Malcolm J. Jackson
Department of Medicine,
University of Liverpool, Liverpool L69 3GA
Molecular chaperones constitute an essential cellular system promoting correct cellular protein folding and translocation to a large variety of cellular compartments (Morimoto et al, 1994). These chaperone proteins belong to the family of proteins known as heat shock proteins (HSPs) or stress proteins, the synthesis of which is increased following a variety of stresses (for example, anoxia, hyperthermia or changes in intracellular pH). All cells respond to stress by a specific increase in the synthesis of HSPs and this increased content of HSPs then confers significant cytoprotection to the cell against subsequent stress. For example, hyperthermia induces HSPs in cardiac tissue and this confers resistance to the heart against subsequent, normally damaging, ischaemia (Plumier and Currie, 1996).
The possibility that HSPs provide protection to skeletal muscle has received less attention. HSPs are produced by skeletal muscle and increased content of HSPs in mouse muscle or myotubes in culture reaches a peak at 12 - 18 hours following hyperthermia (McArdle and Jackson, 1996). We have established a range of model systems of muscle damage by intracellular calcium overload, increased free radical activity, a failure of energy homeostasis or an induction of apoptotic cell death and have demonstrated that a prior induction of HSPs provides protection against some of these damaging processes.
The decline in skeletal muscle function with ageing has been recognised for a considerable time. Skeletal muscle from aged populations demonstrate a considerable force deficit, increased susceptibility to damage and poor recovery from that damage in comparison with muscle from young individuals (Zerba et al, 1990; Brooks and Faulkner, 1990; McBride et al, 1995). There is considerable evidence to suggest that tissues from ageing animals have a reduced ability to respond to stress by the production of HSPs (Liu et al, 1996) and it may be that this attenuated response to stress in muscles of aged animals may play a role in the functional deficit which occurs in skeletal muscle with ageing.
References
1. Brooks SV and Faulkner JA. 1990. Am. J. Physiol. 258:C436-C442.
2. Morimoto RI, Tissieres A and Georgopoulos C (Eds.).1994.The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbour Laboratory Press, Cold Spring Harbor.
3. Liu AYC, Lee YK, Manalo D and Huang LE. 1996. In: Stress-Inducible Cellular Responses (Feige U, Morimoto RI, Yahara I and Polla BS Eds). Birkhauser Verlag, Basel.
4. McArdle A and Jackson MJ. 1996. Biochem. Soc. Trans. 24:485S.
5. McBride TA, Gorin FA and Carlsen RC. 1995. Mech. Ageing Dev. 83:185-200
6. Plumier JCL and Currie W. 1996. Cell Stress and Chaperones, 1:13-17.
7. Zerba E, Komorowski TE and
Faulkner JA. 1990. Am. J. Physiol. 258:C429-C435.
The authors would like to
thank the Wellcome Trust and Research into Ageing for financial
support.
EFFECTS OF AGEING ON THE
MORPHOLOGY OF THE HUMAN SKELETAL MUSCLE
Roberto Scelsi, Carla
Marchetti and Paola Poggi
Dipartimento di Patologia
Umana ed Ereditaria, Istituto di Anatomia Umana ed Istituto
della Universita' di Pavia. Italy
Literature data on skeletal
muscle in the aging man are few and they concern qualitative
rather then quantitative analysis of muscle fibres. In
particular these studies used middle-aged subjects and their
scope is related to pathology. The vastus lateralis muscle was
studied in 40 male sedentary subjects, ranging in age from 30 to
89 years. A qualitative and quantitative analysis of muscle
fibres was carried out by light and electron microscopy and by
an automatic interactive image analyzer system. Biopsies were
taken from subjects subdivided into four age groups: 30-50,
60-70, 71-80, 81-90 years. attention was focused on the fibre
type distribution, the size and the amount of mitochondria. The
amount of sarcoplasmic lipid droplets and the intramuscular
capillarity. Main changes seen in the age groups are indicative
of a sequence of events within senescent skeletal muscle fibres.
The muscle displayed increasing type I fibres and consistent
type 2 fibre atrophy with advancing age. Mithocondral size and
percentage per fibre area decrease with age, while lipid droplet
percentage per fibre area increases. The capillary/fibre ratio
and the capillary density were correlated with muscle fibre size
and the morphometric results indicate an increase of capillarity
with age. The present results suggest a possible transformation
of fibre type population in aging men. The reduction of the
energy requirements with age may be the cause of the decrease of
mitochondria in muscle fibres.
IS
HYPERTROPHY LIMITED IN ELDERLY MUSCLE FIBERS? A COMPARISON OF
ELDERLY AND YOUNG STRENGTH-TRAINED MEN
R.S. Hikida, S. Walsh, H.
Wendeln, T. Luecke, N. Barylski, G. Campos, F.C. Hagerman and
R.S. Staron.
Department of Biological
Sciences and College of Osteopathic Medicine, Ohio University,
Athens, Ohio, USA
Nucleo-cytoplasmic relationships were compared in strength-trained young and elderly men to determine whether the ability of muscle fibers to hypertrophy were similar. Young (age 22.5+5.8 years) and elderly (65.0+6.0 years) men were divided into a training group to induce hypertrophy,and a control group. They performed strength training for the quadriceps femoris muscle group. Biopsies of the vastus lateralis muscle were taken before and after training and examined by electron microscopy, immunohistochemistry for nuclear analysis,and histochemistry (mATPase) to determine fiber type, satellite cell:myonucleus ratio, and myonuclei per cross-sectional profile. Strength training increased strength from 52 to 92% in the elderly, and 25 to 117% in the young,depending upon the exercise. Although the relative percentage increase of the elderly strength was equal to the young, the elderly began 40-85% weaker. Training increased the absolute strength of the elderly to that of untrained young men.
Muscle fibers of untrained elderly were significantly smaller than untrained young, bat with training, their fibers hypertrophied by 36% to the size of untrained young. Along with larger fibers, the cytoplasm to nucleus ratio increased by 24%, which was not significant (p=0.12). The number of myonuclei did not change with training.
In the young, the fibers hypertrophied by 25% with training. In the hypertrophied fibers, the cytoplasm to nucleus ratio did not change (-1%), suggesting an increased number of myonuclei. The myonuclear count increased by 15% but this was not significant (p=0.15).
In conclusion, elderly muscle
fibers are capable of considerable hypertrophy after strength
training, but this hypertrophy is limited ( as shown by their
hypertrophying only to the size of untrained young fibers), most
likely due to the reduced capacity to add nuclei to the fibers.
Since addition of nuclei is a function of satellite cells, this
suggests that satellite cell capabilities have declined in the
elderly. Although the cellular mechanisms show how fiber
hypertrophy is limited in the elderly, other factors, most
notably motor neuron degeneration and loss, have been shown to
account for the major portion of strength loss and atrophy of
muscle mass in the elderly.
MUSCLE FUNCTION DECLINE IN THE
ELDERLY: BIOCHEMICAL INDICES OF EVALUATION
C. D'Alessandro, C. Chisari,
E. Giannini, M. Bresci, B. Rossi
Department of Neuroscience,
Section of Neurology, University of Pisa , Italy
During ageing processes neuromuscular apparatus undergoes structural and functional modification involving both nervous system and muscular effector. The functional decline of the elderly is characterised by a reduced oxygen consumption (VO2), which fatigue resistance is based on. There is also a more or less important decline of cardiocirculatory and respiratory capacity. However, the more evident decay involves the muscle mass which is effected by quantitative and qualitative alterations: besides a reduction of the free fat mass, with a contemporary increase of the fat mass, a reduction of muscle fibres (in particular type II fibres) diameters and number can be observed.
With the aim of an in vivo evaluation of muscle function in the elderly, we study lactic acidemia in blood samples obtained from 32 elderly and 10 young subjects before, during and after an incremental exercise performed on a treadmill. Results obtained show normal lactate resting levels in both groups. At the end of the exercise and 5' and 10' during recovery the elderly show significantly higher values (p<0.05). At 30' after the end of the exercise lactate returns to the basal values in both groups.
These data show a precocious
resort to the glycolitic metabolism during a prolonged exercise
and confirm, in vivo, a reduced aerobic capacity in the elderly.
References
1. Antonutto G, Di Pranpero PE: The concept of lactate threshold. J Sports Med Phys Fitness 1995, 35: 6-12.
2. Essen-Gustavsson B, Borges O: Histochemical and metabolic characteristics of human skeletal muscle in relation to age . Acta Physiol Scand 1986, 126: 107-114.
3. Rogers MA, Hagberg JM,
Martin WH, Ehsani AA, Holloszy JO: Decline in VO2MAX in master
athletes and sedentary men. J Apply Physiol 1990, 68: 2195-2199.
CONTROL OF BLOOD FLOW TO SKELETAL
MUSCLE IN HEART FAILURE
P.A. Poole-Wilson
Cardiac Medicine, Imperial College School of Medicine, National Heart and Lung Institute,
Dovehouse Street, London
Since changes in central
haemodynamics do not easily explain the symptoms of patients
with chronic heart failure, there has been considerable interest
in the possibility that signals initiating the symptoms of heart
failure emanate from skeletal muscle. These signals might be
activated by the metabolic consequences of exercise in muscle.
The metabolic changes are consequent upon the workload
undertaken by the muscle, the nature of the muscle itself and
the blood flow. Many investigators have studied blood flow in
the periphery in heart failure. The control of flow may be
different in patients with treated as opposed to oedematous
heart failure and in the lower limbs with a large muscle mass
compared to the arms or hand with a small muscle mass. The
increased systemic vascular resistance is partly due to the
reduction in the muscle mass. The residual increase in
resistance must be attributed either to histological changes or
to functional changes within the vascular itself.The study of
blood flow is complex because comparisons with normal persons is
fraught by limitations of methods. Plethysmography measures
blood flow in a given volume of muscle whereas other techniques
such as the measurement of blood velocity in arteries by the
Doppler technique measures total flow to a limb. Results are
often expressed as percentages or increments. The major
difficulty is that in patients with severe heart failure the
total blood flow to the lower limbs is reduced. The
interpretation of any change in that blood flow compared to the
change in normal persons in difficult if not impossible because
of the alteration in the base-line value. A popular belief is
that endothelial function is diminished in heart failure. The
evidence for that assertion is somewhat limited. Another
possibility is that the base-line flow is modified by the
anatomy of peripheral vessels and the response to exercise is
limited due to a reduction in the response to an accumulation of
metabolites.
ARTERIAL BARORECPTOR FUNCTION IN
CHRONIC HEART FAILURE: CLINICAL AND PHYSIOLOGICAL IMPLICATIONS
A. Mortara and M.T. La Rovere
Dept. of Cardiology, Centro
Medico di Montescano, "S. Maugeri" Foundation, IRCCS,
Montescano, Pavia, Italy
Sinoaortic and cardiopulmonary baroreceptors normally exercise a restraining influence on resting central sympathetic activity. IN CHF patients these normal tonic inhibitory reflexes are depressed contributing to sympathetic excitation. Baroreflex abnormalities in CHF have been reported both in experimental and human studies in association with a prolonged exposure to low cardiac output and reduced blood pressure. The exact mechanism of this baroreceptor dysfunction is still uncertain (1, 2), but independent of the causal mechanisms, it has been shown to be reversible after optimization of medical treatment or after cardiac transplantation (3,4).
Baroreflex sensitivity (BRS) may be safely quantified by the phenylephrine method in normal subjects and in patients with cardiac diseases (5), and it has been proposed as a valid index of the capability to reflexly increase parasympathetic activity. By this technique, the vasoactive drug phenylephrine is given to raise the systolic arterial pressure by 15-30 mm Hg thus provoking a reflexly increase in mean RR interval. The slope of the linear regression between changes in systolic blood pressure and RR interval is considered as representing BRS (ms of RR change/1 mm Hg of SAP increase) (5).
In patients with a recent myocardial infarction (MI), a decreased BRS has been regarded as a powerful marker of poor prognosis (6). In CHF, BRS resulted markedly depressed and it was found to clearly parallel the deterioration of clinical and hemodynamic status (7). The mechanism of arterial baroreflex dysfunction in CHF is probably multifactorial and may be located in all components of the reflex arc (1). Moreover, activation of the renin-angiotensin system in heart failure by increased plasma levels of angiotensin II may act on baroreflex control of sympathetic activity and heart rate both directly in the vasomotor and cardiac centers in the brain and in the peripheral nerve terminals, facilitating norephinephrine release and inhibiting acetylcholine release. CHF patients may also have abnormalities of sinus node responsiveness to changes in efferent traffic to the heart (1). As in post-Mi patients, it was observed that a depressed BRS, assessed by the phenylephrine technique, was significantly associated with poor survival (R.R. 2.7, 95%C.I. 1.6-4.7) and that its quantification added prognostic information to the predictive accuracy of the other established risk factors such as age, LVEF and maximum O2 consumption during exercise. The association with mortality was particularly evident in those patients with ischemic heart disease and in those with depressed ventricular function and severe mitral regurgitation (7).
in conclusion, it is
demonstrated that arterial baroreflex modulation of heart rate
may be safely quantified by the phenylephrine technique even in
patients with mild to severe heart failure. In, this method may
be considered as an indicator of both neural and hemodynamic
capability to reflexly increase parasympathetic activity.
Baroreflex modulation of heart rate was clearly more depressed
in the advanced stages of the disease and this depression was
significantly associated with higher incidence of cardiac death.
References
1. Eckberg DL, Sleight P. Congestive heart failure. In: Eckberg DL, Sleight P eds. Human baroreflexes in health and disease, Clarendon Press, Oxford. 1992: 399-436.
2. Thames MD. Kinugawa T, Smith ML, Dibner-Dunlap ME. Abnormalities of baroreflex control in heart failure. J Am Col Cardiol 1993; 22A: 56A-60A.
3. Ellebogen KA, Mohanty PK, Szentpetery S, Thames MD. Arterial baroreflex abnormalities in heart failure: reversal after orthotopic cardiac transplantation. Circulation 1989; 79: 51-58.
4. Marin-Neto JA, Pintya A.O., Gallo L Jr, Marciel BC. Abnormal baroreflex control of heart rate in decompensated congestive heart failure and reversal after compensation. Am J Cardiol 1991; 67: 604-610.
5. La Rovere MT, Mortara A, Schwartz PJ. Baroreflex sensitivity. J Cardiovasc Electrophysiol 1995; 6: 761-774.
6. La Rovere MT, Bigger JT Jr, Marcus FI, Mortara A, Schwartz PJ for the ATRAMI Investigators. Baroreflex sensitivity and heart rate variability in prediction of total cardiac mortality after myocardial infarction. Lancet 1998; 351: 478-484.
7. Mortara A, La Rovere MT,
Pinna DG, Prpa A, Maestri R, Febo O, Pozzoli M, Opasich C,
Tavazzi L. Arterial baroreflex modulation of heart rate in
chronic heart failure. Clinical and hemodynamic correlates and
prognostic implications. Circulation 1997; 96: 3450-3458.
A. Coats and S. Anker
Cardiac Medicine NHLI London,
Dovehouse Street, London, UK
Muscle wasting is known to occur in many patients with chronic heart failure (CHF).
Recent research indicates that body weight is kept stable due to the replacement with fat tissue. Body weight remains stable as well as skeletal muscle strength. In cachectis patients with CHF muscle loss progresses, but also fat tissue loss is observed, and body weight declines. The presence of body wasting is strong indicator of impaired prognosis independent of other established prognostic markers (peak VO2 , NYHA class, LVEF, plasma sodium), possibly because it is sensitive indicator that neurohormonal and immunological alterations have reached a clinically relevant level.
The specific cause of muscle
wasting remains unknown, but early reports suggest that the
interaction of increasing catabolic factors (stress hormones,
cytokines), a failing anabolic response (anabolic steroid
hormones), and the development of hormone resistance syndromes
(growth hormone, insulin) may contribute to the development of
muscle wasting in CHF patient. First trials to study the
muscular effects of altering the catabolic/anabolic balance or
underway.
SKELETAL MUSCLE MICROBIOPSIES FOR
ASSESSING MHC COMPOSITION:
A MARKER OF SEVERITY IN CHF
G. Vescovo, L. Dalla Libera1,
C. Catani1, 2, R. Zennaro, G.B. Ambrosio and U. Carraro1, 2)
Internal Medicine I, Venice
City Hospital, Venice; 1 CNR Unit for Muscle Biology and
Physiopathology , Padova; 2 Department of Biomedical Sciences,
University of Padova,Italy
Congestive Heart Failure (CHF) is characterized by the occurrence of a specific myopathy with preferential synthesis of fast more fatigable myosins (MHC) and a decreased expression of the slow fatigue resistant aerobic MHC. By means of a new electrophoretic method developed in our laboratories we have been able to study the MHCs composition of the gastrocnemius in patients with CHF by taking 100-150 µg microbiopsies with a Menghini soft-tissue needle. We have found a strong correlation between indexes of severity of cardiac failure, such as NYHA class, diuretic consumption, ejection fraction and cardiopulmonary indexes of exercise tolerance (peak VO2, anaerobic threshold) and the MHCs composition suggesting that the impaired exercise capacity in patients with CHF is at least in part due to the shift of the skeletal muscle pattern toward fast fibres. We have also correlated the magnitude of the improvement in exercise tolerance in patients with CHF after 6 months treatment either with ACE inhibitors or with AII antagonist with the net changes in skeletal muscle MHCs. We found that the improvement in peak V02 correlates significantly with the change in MHCs.
The same modifications in MHCs
composition have been observed in an animal model of
monocrotaline induced CHF.
IMMUNOLOGICAL DISORDERS,
APOPTOSIS AND CARDIOVASCULAR INJURIES
S. Curello, C. Ceconi and R.
Ferrari
Chair of Cardiology,
University of Brescia, Italy
There is increasing evidence of immunologic disorders in the progression of congestive heart failure (CHF). Such an assumption was postulated due to a number of facts: 1) plasma circulating levels of the cytokine tumor necrosis factor alpha (TNFa), interleukin (IL)-2, and IL-6 are elevated in severe CHF and associated with a worse prognosis; 2)inducible nitric oxide synthase (iNOS) is detected in the failing human heart and in circulating monocytes; in both cases, iNOS expression is correlated with TNFa, and associated with increased short term mortality; 3) sera from CHF patients (NYHA class IV) induce a significant increase in the rate of apoptosis on human endothelial cells. This effect is partially inhibited by the presence of an anti-human TNF-a antibody; 4) alterations in the lynphocyte subtypes: reduction of CD4, increased CD4/CD8 ratio and augmented FAS antigen expression.
In conclusion, all this
evidence suggest that immunomodulation could represent an
important skill for feature CHF therapy.
DISRUPTION OF PLASMA MEMBRANE IN
RAT MYOCARDIAL INFARCTED CELLS AS MONITORED BY USE OF
LANTHANUM IONS
J. Tong, H. Ikeda, Y. Suzuki,
G. Itoh
First Department of Pathology,
Aichi Medical University, Aichi, Japan
Previous investigations have shown that DNA fragmentation in infarcted myocardial cells of both human autopsy cases and rat experimental models occurs. In the present study, we are examining whether the integrity of rat infarcted myocardial cell membrane, focusing on the early lesions of acute myocardial infarction (AMI) at 2-6 hours is disrupted or not. Rat AMI (Wistar strain, 6 weeks old, male) was induced by permanent occlusion of the left coronary artery without reperfusion. The development of DNA fragmentation in myocardial cells of AMI was determined by the TdT-mediated dUTP-biotin nick end labeling (TUNEL). Then, the integrity of the plasma menbrane of AMI foci which had revealed TUNEL positive areas was exsamined electron microscopically using lanthanum ions (La(NO3)3).
Two hours after the induction of AMI, TUNEL positive nuclei were found in a limited number of myocardial cells in subendocardial region. TUNEL positive nuclei increased both in number and in area, thereafter.
Intracellular lanthanum was not detected in the myocardial cells at 2 hours after occlusion. At 3 hours, intracellular depositions of lanthanum were detected in some myocardial cells, Furthermore, most of the myocardial cells showed intracellular depositions of lanthanum at 4-6 hours after ischemia.
The evidence obtained here way
suggest that the internucleosomal cleavage of DNA occurs in the
nuclei of infarcted myocardial cells at 2 hours after coronary
occlusion and that the process of impaired plasma membrane
permeability occurs at 3 hours after occlusion. The frequency of
myocardial cells having the intracellular deposition of
lanthanum increased thereafter.
References
1. Tanaka M, Ito H, Adachi S, Akimoto H, Nishikawa T, Marurno F, et al. Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes. Circ Res 1994; 75: 426-33
2. Gottlieb RA, Burieson KO, Kloner RA. Babior BM,Engier RL. Reperfusion injury induces apoptosis in rabbit cardiomyocyties. J Clin Invest 1994; 94: 1621-8
3. Itoh G, Tamura J, Suzuki M, Suzuki Y, Ikeda H, Koike M, et al. DNA fragmentation of human infarcted myocardial cells demonstrated by nick end labeling method and DNA agarose gel electrophoresis. Am J Pathol 1995; 146: 1325-31.
4. Reimer KA, Ideker RE. Myocardial ischemia and infarction: anatomic and biochemical substrates for ischemic cell death and ventricular arrhythmia. Hum Pathol 1987; 18: 462-75.
5. Gavrieli Y. Sherman Y, Benson SA. Identification of programmed cell death in situ via specific labeling of nuclesr DNA fragmetation. J Cell Biol 1992; 119: 493-501.
6. Gold R, Schmied M, Giegerich G, Breitschopf H, Hartung HP, Toyka KV, et al. Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation technique. Lab Invest 1994; 71: 219-25.
7. Facchinetti A, Tessarollo M, Mazzocchi R, Kingston DC, Biasi G. An improved method for the detection of DNA fragmentation. J Immunol Methods 1991; 136/ 125-31.
8. Selye H, Bajusz E, Grasso S, Mendell P. Simple techniques for the surgical occlusion of coronary vessels in the rat. Angiology 1960; 11: 398-(é).
9. Batistatou A, Greene LA. Internucleosomal DNAcleavage and neuronal cell survival/death. J Cell Biol 1993; 122: 523-32.10) Kerr JFR, Harmon BV. Definition and incidentce of apoptosis: an historical perspective. In: Tomei LD, Cope FO, editors. Apoptosis: the molecular basis of cell death. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1991: 5-29.
11. Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 1980; 68: 251-306.
12. Searle J, Kerr JFR, Bishop CJ. Necrosis and apoptosis. distinct model of cell death with fundamentally different significance. Pathol Annu 1982; 17: 229-59.
13. Fukuda K, Kojiro M, Chiu JF. Demonstration of exstensive chromatin cleavage in tranaplanted Morris hepatoma 7777 tissue: apoptosis or necrosis ? Am J Pathol 1993; 142: 935-46.
14. Tpminaga T. Kure S, Narisawa K, Yoshimoto T. Endonuclease activation following focal ischemic injury in the rat brain. Brain Res 1993; 608: 21-6.
15. Kajstura J, Cheng W, Reiss K, Clark WA, Sonnenblich EH, Krajewski S, et al. Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size iri rats. Lab Invest 1995; 74: 86-107.
16. Contran RS, Kumar V.
Robbins S. The heart. In: Contran RS, Kuman V. Robbins S.
editors. Robbins pathologic basis of disease. 4th ed.
Philadelphia: Saunders, 1989; 597-656.
HEMODYNAMIC RESULT AFTER
CARDIOMYOPLASTY IN A CHRONIC HEART FAILURE MODEL CESSATION OF
ELECTRICAL STIMULATION FOR 12 HOURS DAILY
V.S. Chekanov, M.A. Rieder,
G.V. Tchekanov
Milwaukee, WI, USA
A major concern in determining the efficacy of cardiomyoplasty (CMP) has been whether synchronous stimulation of the latissimus dorsi muscle (LDM) is essential for maximum benefit, or if the heart wrap itself is enough to stop or minimize the progressive dilation and remodelling of cardiac mass. With the cardiomyostimulator turned on, cardiomyoplasty patients adapt well to daily stresses. However, when a maximal assist effort is not needed, perhaps patients would benefit if their stimulator were switched off (e.g. at night while sleeping). This would allow the LDM an opportunity to rest for several hours daily,perhaps preventing muscle deterioration from continuous contraction. The main aim of our investigation was to evaluate whether hemodynamic results after cardiomyoplasty were impaired when continuous stimulation for 12 hours daily.
A model of chronic heart
failure was created in 4 sheep by performing an arterio-venous
anastomosis.Two weeks later, cardiomyoplasty (CMP) was
performed; electrical stimulation (ES) training in a continuous
mode was begun after a two week delay period. Doxorubicin was
administered intravenously once a weeks to enhance heart
failure. Hemodynamic parameters were investigated 2.5 months
post-CMP by echocardiography with the stimulator on and turned
off momentarily. The stimulator was then turned off 12 hours
daily for two weeks. LV parameters were retested.
Continuous ES Stim. off 12hours daily
-------------------------------------------------------------------------------------
LV
Parameters
Stim.
On Stim.Off Stim.
On Stim. Off
Syst. Area (cm2 5.9 6.8 5.8 6.9
Syst. Volume (ml) 10.8 13.9 10.9 14.9
Syst. Long Axis (mm) 31. 31.6 31.2 31.3
Diast. Area (cm2) 11.1 14.3 15.0 14.5
Diast. Volume (ml) 27.1 35.4 27.7 39.4
Diast. Long Axis (mm) 43.5 48.8 43.2 48.5
EF 66 52 64 52
Syst, systolic; Diast,
diastolic; Stim, stimulator. Values are means.
Conclusions: After 2 weeks
with the stimulator turned off for 12 hours daily, LV paremeters
did not differ from data during continuos ES. Damage to muscle
after CMP due to continuous ES without rest may be prevented by
periodic cessation of stimulation. Hemodynamic results do not
seem to be impaired by short episodes of rest daily.
EXERCISE-INDUCED MUSCLE DAMAGE IN
MUSCULAR DYSTROPHIES: EVALUATION BY SERUM CPK DETERMINATION
C. Chisari, E. Giannini, M.
Bresci, C. D'Alessandro and B. Rossi
Department of Neuroscience,
Section of Neurology, University of Pisa, Italy
Creatinphosphokinase (CPK) is a dimeric protein which catalyzes the reversible reaction ADP+ phosphocreatine <---> ATP+ creatine. It's a key enzyme of muscle fibers metabolism and the increment of CPK serum levels is identified both in healthy subjects performed a strenuous exercise, and in patients affected by primitive or secondary myopathies.
This parameter is identified as index of increased membrane permeability and muscular damage.
We studied 27 patients affected by primitive myopathy: 8 affected by Becker Muscular Dystrophy (BDM), 10 affected by Myotonic Dystrophy (MD), 8 affected by Fascioscapulohumeral Muscular Dystrophy (FSH), and 6 subjects as control.
Each subject underwent to an exercise of same intensity and duration, which consisted of walking on a treadmill during 15 minutes, at constant speed, chosen as the highest but still comfortable for each subject. Haematic CPK activity is determined by venous sampling before and after 1, 3, 6, 24h after the exercise.
The analysis of curves obtained from these data allowed to evidence a significative increment of CPK hematic levels after exercise only in BDM where it's known that a quantitative and/or qualitative deficit of dystrophin exists; instead in DM and FSH myopathies the CPK hematic levels results similar to controls. In these last myopathies, at present, any alteration of structural protein is not noticed, and probably muscular damage pathogenesis is not exclusively mechanical.
So, it's possible to study
functionally the dystrophic muscle by a test of simple
execution, not invasive and reproducible. Therefore, this
approach results useful in the dystrophic patient's follow up,
to planning individual rehabilitation programs and, if these
data will be confirmed by a larger casuistry, in the diagnostic
iter of different muscular dystrophies.
References
1. Jackson MJ, Round DIM, Newham DJ, Edwards RHT. An examination of some factors influencing CPK in the blood of patients with muscular dystrophy. Muscle & Nerve 1987; 10: 15-21.
2. Kuipers H. Exercise-induced muscle damage. Int J Sports Med 1994; 15: 132-135.
3. van der Meulen, JK, Kuipers
H, Drukker J. Relationship between exercise-induced muscle
damage and enzyme release in rats. J Appl Physiol 1991; 3:
999-1004.
THE ROLE OF THE PLASTICITY OF
SKELETAL AND CARDIAC MUSCLES AND POLYMORPHISM OF MYOSIN ON
SURVIVING MAMMALS IN HIBERNATION
Z. Podlubnaya, N. Lukoyanova,
I. Akopova, M. Shpagina, S. Malyshev, S. Udaltsov and O.
Menshova
Institute of Theoretical and
Experimental Biophysics RAS, Pushchino, Russia
Among the physiological states with temporal reduced vital activity the hibernation is the most pronounced one. At this period all physiological systems of hibernators are switched to minimal regime of functioning. Upon arousing the animals revert to the original active state without pathological consequences. The capacity to endure the great fluctuations of the activity of all physiological systems on retention of their coordinated action makes the hibernators an unique object for studying the mechanisms of the mobilization of reserve possibilities of organism for survival in extreme and pathological situations including those incompatible with life of man and animal. Unfortunately, in study of hibernation mechanisms the proper attention was not paid to executive apparatus of skeletal and cardiac muscles and to its individual proteins. The purpose of this study was to determine how plasticity properties of skeletal and cardiac muscles of ground squirrels and polymorphism of their myosins are used by mammalian organism in hibernation. It has been revealed that actin-activated ATPase activities of the skeletal muscles myosins of hibernating (Mhib) and arousing (Mar) ground squirrels are less by 60 and 20%, correspondingly, than that of the myosin of active animals (Mact). Ca2+-sensitivity of Mhib and Mar in the presence of rabbit skeletal muscle non-regulated actin (AMhib and AMar) are less by 60 and 55%, correspondingly, in comparison with that of AMact. SDS-gel electrophoresis analysis of the isoform composition of myosin light and heavy chains at the different stages of hibernation has revealed that the content of essential LC3 in preparations of skeletal muscle Mhib decreases up to 35% as compared with that of Mact. However, the amount of LC3 in Mar increases almost by 2 times within 1.5-2 hours of arousal. The fast isoform 2B of myosin heavy chains undergoes similar changes at the corresponding stages of hibernation. It is most likely that such changes in myosin isoform composition underlie the above changes in functional properties of skeletal muscle myosin contributing to the inhibition of the moving ability of skeletal muscle upon hibernation.
In contrast to situation with
skeletal muscle myosin we have not revealed any changes in
isoform composition of cardiac myosin light chains. However,
actin-activated ATPase activity of cardiac Mhib and its
Ca2+-sensitivity are decreased by 20% in comparison with those
of cardiac Mact. This lowering are probably resulted from the
changes in isoform composition of cardiac myosin heavy chains.
The qualitative and quantitative dissimilarities in adaptive
changes of composition and properties of skeletal and cardiac
muscles myosins upon hibernation and arousal are likely
determined by the differencies in the regime of functioning
these muscles. Whereas for skeletal muscles it is necessary to
inhibit completely the moving capacity upon hibernation the
heart of hibernating animal maintains some level of contractile
activity (for 4-20 beats/min) which recovers on arousal (280-310
beats/min) more quickly than that in skeletal muscles. There are
reasons to suppose that similar adaptive mechanisms are also
switched at the first stages of some cardiomyopathies that makes
hibernation an unique model for studying these mechanisms and
for elucidating the criteria of the reversibility of contractile
dysfunctions.
References
1. Akopova I, Shpagina M, Malyshev S, Meníshova O, Podlubnaya Z: Light chains of myosin in dilated cardiomyopathy: markers of adaptive and pathological stages. Abstr. 6th World Congr. Heart Failure - Mechanisms and Management, (Geneva, Switzerland, May 17-20), 1998.
2. Morano I, Adler K, Agostini B, Hasselbach W: Expression of myosin heavy and light chains and phosphorylation of the phosphorylatable myosin light chain in the heart ventricle of the European hamster during hibernation and in summer. J Muscle Res Cell Motil 1992; 13: 64-70.
3. Podlubnaya Z, Lukoyanova N,
Udaltsov S, Shpagina M: The role of the myosin of ground
squirrels skeletal muscles in inhibition of their activity upon
hibernation. J Muscle Res Cell Motil 1997; 18: 252-253.
Supported by RFBR grants n.
97-04-48346 and n. 96-04-48756.
MONITORING MUSCLE TROPHISM AND
DAMAGE/REGENERATION IN EXPERIMENTAL AND CLINICAL MUSCLE
TRANSPLANTS
C. Rizzi, K. Rossini1, U.
Carraro1, E. Giardini2, F. Zuliani2 and F. Mazzoleni2
(1) Department of Biomedical
Sciences; (2) Institute of Plastic Surgery, University of
Padova, Italy.
Motor units of skeletal
muscles differ in their ability to withstand fatigue at
different sustainable power outputs and therefore they are
differentially damaged by different level of exercise and/or
distrophic events (ischaemia/reperfusion damage and/or
denervation). Several independent parameters can be evaluated to
type skeletal myofibers and muscle damage/regeneration. Ease,
reliability, sensitivity and discriminating power make SDS
PAGE-immunoblotting of: 1) myosin heavy chains and myoglobin
(markers of myofiber transformation/regeneration), ubiquitin and
CD11 (markers of actual damage/inflammation) and of
extracellular matrix proteins (markers of cumulative damage),
the first choice in experimental and clinical applications,
because only a-few-milligram muscle biopsy is needed to perform
undisputable fiber typing and histopathology. Indeed all these
markers of myofiber transformation and/or damage can be
quantified in a few muscle cryostat sections making the
follow-up of skeletal muscle adaptation to
transposition/transplant feasible at both experimental and
clinical settings. Combining myoglobin and CPK serum leakage
follow-up and needle muscle biopsies muscle trophism can be
semi-continuosly monitored. Results illuminating the power and
limits of these approaches in clinical muscle transplants will
be reported.
TIME-COURSE OF EXERCISE AND APOPTOSIS IN
DYSTROPHIN-DEFICIENT MUSCLE OF MICE
K. Rossini1, M. Sandri1, 2, A.
El Messlemani1 and U. Carraro1
(1) Department of Biomedical
Sciences; and (2) The Institute of Experimental and Laboratory
Medicine, University of Padova, Italy
Apoptosis is a process of cell
death occuring 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 spontaneus
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 16 hours) and, the morning after, Tibialis
Anterior of both hindlimbs removed. We checked the activity of
mice by monitoring the covered distance, the time when the wheel
was moving, the maximum and the average speed. While 2-hours
runner mice, sacrificed after additional 14 hours of housing in
a normal cage, covered 1.76 ± 0.2 (mean ± SE) Km with a average
speed of 1.9 ± 0.01 Km/h and with activity time of 36%, 16-hours
runner mice covered 5.3 ± 0.39 Km with a average speed of 1.8 ±
0.1 Km/h and actual time of activity was of 18%. Apoptosis was
assessed by the terminal deoxynucleotidyl transferase assay and
expressed as number of apoptotic nuclei for mm3 of muscle
tissue. Control non-runner mice present 40 ± 11 (mean ± SE)
apoptotic myonuclei/mm3, 2-hours runner mice 84 ± 13 (p = 0.04
against control) and 16-hours mice 159 ± 46 (p = 0.04 against
control; p = 0.23 against 2-hours). Interstitial nuclei/mm3 were
74 ± 6 in mdx control group, 349 ± 76 in 2-hours group (p =
0.001 against control) and 188 ± 49 in 16-hours one (p = 0.06
against control; p = 0.06 against 2-hours). Beside confirming
that apoptosis present in mdx mice at rest drammaticaly
increases after exercise, results suggest that inflammation
decreases with exercise while the apoptotic process becomes more
manifested after a bout of heavy exercise followed by 14 hours
rest. This is exactly the needed time to activate the gene
program for apoptosis in any known cell.
The financial support of
TELETHON - ITALY to the project "Role of apoptosis of myofibers,
satellite cells and endothelia in exercise-induced muscle damage
and in progression of muscular dystrophies (n. 968)" is
gratefully acknowledged. Supported in part by funds from the
Italian C. N. R. to the Unit for Muscle Biology and
Physiopathology, and by the Italian M.U.R.S.T. 60% (recipient
U.C.).
EXPRESSION OF FasL IN SKELETAL MUSCLE CELLS
M. Sandri1, 2, C. Sandri2, K.
Rossini2, C. Rizzi2, U. Carraro2, E. Giurisato3, M. Cantini2, 3,
M.L. Massimino3 and P. Arslan1
(1) Institute of Experimental
and Laboratory Medicine; (2) Department of Biomedical Sciences;
(3) C.R.I.B.I. Center, University of Padova, Italy.
Acute muscle injury result in rapid inflammation of the injured site, which is accompanied by regional necrosis of the injured muscle. Substantial elevations of inflammatory cells occur within 1-24h of injury, persist for several days and return to control concentrations over a period of several days to weeks. This implies that strong regulatory mechanism may be in place to control these reactions, on the contrary a lack of this control induce a persistent presence of inflammatory cells. We tested the hypothesis that myoblast contribute to the regulation of immuno response producing FasL, a well known inducer of apoptotic process in the immuno system. The presence of FasL in C2C12 cell culture were detected by RT-PCR, in situ hybridization, RNAse protection, immunoprecipitation and immunocytochemistry. The results showed that FasL expression occurs in myoblasts but not in myotubes and is cell cycle related. The presence of macrophages in myoblast cell culture increased the expression of FasL and macrophages underwent to apoptosis. The apoptosis of macrophages were blocked by inhibition of FasL system. The same data were obtained in primary muscle cell culture.
In vivo study during
regeneration of a marcaine treated muscles confirm the
expression of FasL in myobast and the presence of apoptosis in
inflammatory cells. Our observations suggest an important role
of myoblasts in the control of imflammation during muscle
regeneration, moreover open new perspectives in the pathogenesis
of myositis and in the field of FasL engineered myoblats.
VENTRICULAR REDUCTION SURGERY IN
GOATS WITH DILATED HEARTS
M. Smink, F.H. van der Veen,
K. Prenger and J.J. Schreuder
Departments of Cardiology, Cardiac Surgery and Anaesthesiology;
Cardiovascular Research
Institute Maastricht, The Netherlands
Introduction: It was in the beginning of 1996 that Dr. Randas Batista presented his results of ventricular reduction surgery (VRS) to the world. This therapy is developed to treat patients with dilated cardiomyopathy. His results were impressing but needed more evidence to see that cardiac function improved from ventricular reduction. We,at that time, had studied the effect of cardiomyoplasty (CMP) in patients with dilated cardiomyopathy and could show that gradual reduction of dilation occurred within 6 to 12 month after surgery. It was clear that acute surgical reduction of left ventricular (LV) volume might be of potential interest to NYHA class IV patients who did not tolerate CMP to well. At that time we had developed an animal model of cardiac dilation using the AV-shunt goat. In the present study, this model was selected to examine the effect of VRS on cardiac function and dimensions, using the conductance catheter.
Material and Methods: In eight female goats (40-83 kg) we performed an AV-shunt between the carotid artery and the left jugular vein. In a pervious study from this laboratory we showed that a period of about 12 weeks is required to obtain an enlarged heart. This model is based on a
decrease in arterial pressure
and a doubling of the cardiac output. This model of
heart-enlargement is also characterized by low mortality and
shows no signs of heartfailure. After the period of 12 weeks the
goats were anaesthesized, the AV-shunt was closed and the
extracorporeal circulation was initiated. Subsequently, part of
the left ventricular wall was surgically excised. During the
operation the heart dimensions and contractility of the left
ventricle were observed with the conductance catheter.
|
CO
l/min |
EF % |
EDV ml |
dPdt mmHg/s |
dPdt+ mmHg/s |
PER ml/s |
PER ml/s |
||
| before VRS |
2.61 |
19 |
117 33 |
|||||
All goats who were admitted for VRS, following chronic overload, had dilated hearts. Earlier experiments had shown an EDV in normal goats of 70±13 ml, vs 117 ±33 ml in the present study. The direct effect of the shunt can be shown by the increase of the cardiac output from 4.7±0.6 l/min to 9.6±2.6 l/min. VRS reduced the LV size considerably in the present study. The EDV reduced to 38% of the initial EDV, the ESV to 37% of the initial ESV, and as a result, SV reduced from 86 ml to 32 ml. The PER and PFR, however, decreased about 100%. The high EF of 69% before surgery and 67% after surgery ( with a closed AV-shunt ) confirm the adequate contractility of the left ventricle in our present study.
Conclusion: The surgical
technique of left ventricular reduction according to Batista is
feasible in goats with a chronic AV-shunt, but the dilated
hearts of these goats are not suitable to demonstrate the
potential positive effect of ventricular reduction surgery on
LV-function
APOPTOTIC MUSCLE FIBER LOSS IN SPINAL
MUSCULAR ATROPHIES
D.S. Tews
Department of Neuropathology,
Johannes-Gutenberg-University, Mainz, Germany
There is evidence that in neurodegenerative diseases motor neurons may undergo apoptotic cell death. Likewise, apoptosis as one mechanism of muscle fiber loss was found in denervated muscle fibers in spinal muscular atrophy (1-3) and in amyotrophic lateral sclerosis (4).
DNA-fragmentation, a hallmark of apoptosis, was identified by in situ-labeling in muscle fibers of patients with infantile and adult forms of spinal muscular atrophies (SMA), and to a lesser extent, in peripheral neuropathies indicating that DNA-fragmentation is not specific to spinal muscular atrophies but a common feature in defect innervation. Studying the expression of the apoptosis-promoting protein bax constant expression was found in muscle fibers. The expression of bax correlated with defective innervation of muscle fibers indicated by upregulation of N-CAM. While in SMA with early onset atrophic as well as normo-and hypertrophic muscle fibers displayed expression of bax, muscle fibers in late-onset SMA and peripheral neuropathies showed bax-expression only in atrophic fibers. The same expression pattern was seen for ICE, mediating cell death by cleavage of actin filament. Bcl-2 and bcl-x, both inhibiting apoptosis by acting in an anti-oxidant manner, were only expressed in atrophic muscle fiber, predominantly in late-onset SMA and peripheral neuropathies. The lack of expression of these apoptosis-inhibitory proteins in early infantile SMA was considered to be associated with muscle fiber immaturity due to defective innervation. Less or lacked expression of apoptosis-protecting factors such as bcl-2 cause inability to neutralize high bax-levels and will secondarily promote muscle fiber death.
These findings indicate that defective innervation may prompt muscle fibers to activate an intrinsic 'suicide' programme which is promoted by the pro-apoptotic factors bax and ICE. Nevertheless, these are also anti-apoptotic strategles in muscle fibers by expression of the bax-antagonist bcl-2 being able to neutralize high bax levels. Although the role of apoptosis in denervating disorders still remains far from clear, more knowledge of this emerging mechanisms may give relevant information for possible therapeutic interventions to modify the rate of muscle fiber loss in lack of effective primary therapy.
References
1. Fidzianska A, Goebel HH, Warlo I: Acute Infantile spinal muscolar atrophy. Muscle apoptosis as a proposed pathogenetic mechanism. Brain 1990: 113: 433-45
2. Tews DS, Goebel H: DNA-fragmentation and bcl-2 expression in infantile spinal muscular atrophy. Neuromusc Disord 1996; 6: 265-273
3. Tews DS, Goebel HH: Apoptosis-related proteins in skeletal muscle fibers of spinal muscular atrophy. J Neuropat Exp Neurol 1997: 56: 150-156
4. Tews DS, Goebel HH, Meinch
HM: DNA-fragmentation and apoptosis-related proteins of muscle
cell in motor neuron disorders. Acta Neurol Scand 1997; 96:
380-386
APOPTOSIS OF MYOFIBERS IN
DUCHENNE MUSCULAR DYSTROPHY
M. Sandri1, 2, C. Minetti3, K.
Rossini1 and U. Carraro1
(1) C.N.R. Unit for Muscle
Biology and Physiopathology, Department of Biomedical Sciences,
University of Padova; (2) Institute of Experimental and
Laboratory Medicine, University of Padova; (3) Muscle Disease
Service, Department of Pediatrics, University of Genova, G.
Gaslini Institute, Italy
The current view that
apoptosis precedes necrosis in death of dystrophin-deficient
muscle fibers of mdx mouse is well advanced (Sandri et al.,
1995; Tidball et al., 1995; Smith et al., 1995). Moreover we
described an increase of apoptotic myonuclei in
dystrophin-deficient mice two days after spontaneous exercise
(Sandri et al., 1997). To investigate the role of apoptosis in
human muscular dystrophy, muscles from 11 patients of different
ages affected by Duchenne muscular dystrophy were analyzed for
apoptosis. Apoptosis was assessed by terminal deoxynucleotidyl
transferase assay, furthermore expression of bcl-2 and bax was
examined by immunohistochemistry. While being very rare in
normal muscles (less than 0.1%), apoptotic nuclei were detected
in dystrophic muscles especially at interstitial level.
Nevertheless few dystrophin deficient myofibers with centrally
located nuclei showed a positive reaction for DNA fragmentation.
A mosaic pattern of bcl-2/bax positive myofibers characterized
dystrophic muscles so the relative proportion of pro- and
anti-apoptotic proteins differs among muscle fibers in
correlation with the presence of apoptotic myonuclei. In the
interstitium apoptotic cells were identified as macrophages and
as activated satellite cells. These are the first findings that
show an apoptotic process in adult muscle fibers of human
Duchenne dystrophy shedding new light on muscle damage and on
its progression in dystrophinopathies.
References
Sandri A, Minetti C, Pedemonte
M, Carraro U: Apoptotic Myonuclei in human Duchenne Muscular
Dystrophy. Laboratory Investigation 1998; in press.
The financial support of
TELETHON - ITALY to the project "Role of apoptosis of myofibers,
satellite cells and endothelia in exercise-induced muscle damage
and in progression of muscular dystrophies (n. 968)" is
gratefully acknowledged. Supported in part by funds from the
Italian C. N. R. to the Unit for Muscle Biology and
Physiopathology, and by the Italian M.U.R.S.T. 60% (recipient
U.C.).
SKELETAL MUSCLE MYOPATHY IN CHF:
THE ROLE OF APOPTOSIS IN DETERMINING ATROPHY AND CHANGES IN
MHCS
G. Vescovo, L. Dalla Libera1,
G.B. Ambrosio, R. Zennaro, C. Leprotti and M. Sandri2
Internal Medicine I, Venice
City Hospital, Venice; (1) CNR Unit for Muscle Biology and
Physiopathology , Padova; (2) Department of Experimental
Biomedical Sciences, University of Padova, Italy
CHF is characterised by a limb
skeletal muscle myopathy with shift from the slow aerobic,
fatigue resistant, to the fast, anaerobic fibres, and by the
occurrence of muscle bulk loss.We have investigated the role of
Apoptosis (A) in the skeletal muscle hindlimbs myopathy in an
experimental model of CHF. CHF was induced in 7 males
Sprague-Dawley rats with 30mg/kg monocrotaline. 5 age and
diet-matched control were also studied. They were killer after
27 days. The time course of A was also studied in additional
animals at days 0,17,24, and 30. At day 27 the electrophoretic
analysis of Myosin Heavy Chains (MHC) demonstrated in the
Tibialis Anterior of the CHF rats the occurence of a myopathy
with disapperance of the slow MHC1 and relative increase of
MHC2a (p=.02) and MHC2b (p=.001). With in situ nick-end
labelling (TUNEL)(T) we found in CHF rats a significantly higher
number of T positive endothelial (E) nuclei (0.43%±0.24 vs
controls 0.08%±0.02, p<.02) and T positive Myofiber (M)
nuclei (0.031%±0.012 vs controls 0.0025%±0.005, p<.02)
distinguished on the basis of double laminin staining. At 30
days A showed a further rise in E (0.7%±0.31) and M
(0.15%±0.001) A, accompanied by a drop in M cross sectional area
(1427+491 vs 1215+290, p<.03) and muscle weight/body weigh
(1.78 ± .02 vs 1.63 ± 0.02, p<.001) and by an 800% increase
in plasma TNFa. Western blot showed a decreased expression of
Bcl-2 at day 27 (1924±65, arbitrary units) and a further drop at
day 30 (1475±392, p<.01 vs controls) in CHF. Double staining
for T and antibodies anti MHC2b+2x showed that A occurs in all
the M type. ßANP and right ventricle mass/volume correlated
significantly with the number of A nuclei (p=.015, p=.0001). In
CHF M A lead to muscle atrophy. E A may produce an imbalance in
M nutrition with relative ischemia that trigger the preferential
synthesis of fast anaerobic MHC as an adaptive mechanism or
alternatively induce M death.
Dirk Pette
University of Konstanz,
Faculty of Biology, D-78457
Konstanz, Germany
Skeletal muscles are composed of a wide variety of fiber types, each with a specific protein isoform pattern and metabolic enzyme activity profile. Increasing evidence suggests that fiber types are not fixed structures but represent versatile entities capable of changing their phenotypes in a flexible way (9). This plasticity relates to the potential of alternatively expressing different sets of myofibrillar protein isoforms adjusted to specific functional demands. In addition to qualitative changes in protein isoform patterns, changes in the expression levels of specific proteins, e. g. enzymes of anaerobic and aerobic energy metabolism or protein involved in Ca2+ -release and sequestration also lead to alteration in muscle fiber phenotypes.
Among the factors determining muscle fiber phenotypes, neuromuscular activity play an important role. Alterations in fiber type composition may thus be regarded as responses to specific functional demand. However, intrinsic programs and species-specific properties also play a role in determining the phenotypic patterns of muscle fibers and their malleability.
In general, elevated and reduced neuromuscular activity exert apposite effects (9). Increased activity induces fast-to-slow transitions in myofibrillar protein isoform, whereas decreased activity elicits slow-to-fast transitions. Enhanced neuromuscular activity also induces adaptive changes in energy metabolism, e.g., improved fuel and oxygen supply, enhanced fuel uptake by elevations in specific carrier proteins, and increases in mitochondrial content and enzyme levels of terminal substrate oxidation. Opposite metabolic changes occur in response to reduced neuromuscular activity. The adaptive changes in myofibrillar protein isoform expression and metabolic exhibit dose-response relationships and are reversible upon cessation of the inducing signal.
Chronic low-frequency stimulation (CLFS) has been used during the past decades to systematically investigate the effects of increased neuromuscular activity on skeletal muscles of small mammals (8,10). CLFS represent a highly reproducible experimental protocol of enhanced contractile activity. Contrary to exercise training were motor units are activated in a graded and hierarchical manner, CLFS simultaneously activates all motor units of the target muscle and thus elicits the full range of adaptive responses. Moreover, the standardized experimental protocol of CLFC makers it possible time course studies and to relate stimulation-induced changes at the molecular levels of organization to altered functional properties.
CLFS of fast-twitch muscles elicits a series of orchestrated changes affecting all functional elements of the muscle fiber. As a result, a fast-twitch, fast-fatigable muscle is gradually transformed into a slower contracting, less fatigable muscle. This transformation is the result of changes in the expression of numerous genes. Briefly, CLFS modifies both the isoform pattern of contractile and regulatory myofibrillar proteins, the proteins composition of the Ca2+ - regulatory system, and the isozyme profile and enzyme activity pattern of energy metabolism. At the cellular level, these changes correspond to sequential fast-to-slow fiber type transition. However, depending on the species and other factors (e.g., thyroid hormone level), the range of inducible fiber type transitions and their starting points are found to vary. For example, CLFS of mouse tibialis (TA) muscle causes negligible changes in myosin isoform and fiber type composition. In rat TA muscle, CLFS leads to fiber type transitions which occur mainly within the fast phenotypes and, as documented by studies at the mRNA and protein level, restricted to transition from type IIB-- type IID/X--type IIA (5). Fast to slow transitions (type IIA--type I) are difficult to archive by CLFS in rat fast-twitch muscle, but may be attained if CLFS is combined with hypothyroidism. In contrast, fast-to-slow conversions are easily elicited in rabbit TA muscle where CLFS-induced fast-to-slow fiber type transitions start at the level of type IID/X fibers. Time course studies and single fiber analyses at both the mRNA and protein level have shown that these transitions proceed in the order of type IID/X -- type IIA --type Iß (4?11). The transformation process is mirrored by a pronounced increase in the population of hybrid fibers characterized at both mRNA and protein levels by the coexistence of two or more isoforms of contractile, regulatory, and Ca2+ -handling proteins. The coexistence of several isoforms of a given protein in transforming fibers is partly due to their co-expression, but also results from their relatively long half-lives (12). Moreover, isoform transitions of different proteins do not necessarily follow identical time courses. Different turnover rates of the subunits of heteromeric proteins, e.g., myosin light and heavy chains, troponin subunits TnT, TnI, TnC, explain why hybrid proteins composed of fast and slow subunits (hybrid isomyosins and troponins) appear transiently during the transformation process (6,7).
The sequence of fiber type
transitions and related changes in myosin heavy chain (MHC)
isoforms during fast-to-slow conversion obviously follows a
scale of gradated differences in ATPase activity and tension
cost (1). Microbiochemical analyses of single rabbit muscle
fibers classified according to their MHC isoform complements,
yielded a similar order of fiber types with regard to
differences in their [ATP]/[ADPfree] ratios (2). These
observations and our previous finding that CLFS leads to an
almost immediate and persistent drop in the [ATP]/[ADPfree]
ratio of low- frequency stimulated muscles (3), is in line with
the suggestion that long-term changes in the energy state of the
muscle fiber, namely the phosphorylation potential of the
adenylic acid system,might act as an important signal triggering
changes in gene expression that ultimately lead to fiber type
transitions.
References
1. Bottinelli R, Canepari M, Reggiani C, Stienen GJM: Myofibrillar ATPase activity during isometric contraction and isomyosin composition in rat single skinned muscle fibers. J. Physiol. Lond. 1994; 481: 663-675.
2. Conjard A, Peuker H, Pette D: Energy state and myosin isoforms in single fibers of normal and transforming rabbit muscle, 1998; submitted.
3. Green HJ, Düsterhöft S? Dux L, Pette D: Metabolite patters related to exhaustion, recovery, and transformation of chronically stimulated rabbit fast-twitch muscle. Pflügers Arch. Eur. J. Physiol. 1992; 420: 359-366.
4. Hämäläinen N, Pette D: Expression of an a-cardiac like myosin heavy chain in diaphragm, chronically stimulated, and denervated fast-twitch muscles of rabbit. J. Muscle Res. Cell Motil. 1997; 18: 401-411.
5. Jaschinski F, Schuler M, Peuker H, Pette D: Transitions in myosin heavy chain mRNA and protein isoforms of rat muscle during forced contractile activity. Am. J. Physiol. 1998; 274: C365-C371.
6. Leeuw T, Pette D: Coordinate changes in the expression of troponin subunit and myosin heavy chain isoforms during fast-to-slow transition of low-frequency stimulated rabbit muscle . Eur. J; Biochem. 1993; 213: 1039-1046.
7. Leeuw T, Pette D:
Coordinate changes of myosin light and heavy chain isoforms
during forced fiber type transitions in rabbit muscle.
MUSCLE REGENERATION BY BONE MARROW-DERIVED
MYOGENIC PROGENITORS
G. Ferrari, G. Cossu1 and F.
Mavilio
The Telethon-HS Raffaele
Institute for Gene Therapy of Genetic Diseases, Milano, and (1)
Department of Medical Embryology and Histology, University of
Rome, Italy.
In post-natal life, growth and
repair of skeletal muscle fibers is carried out by a population
of resident, mononuclear myogenic precursors, the satellite
cells. Upon muscle injury, or in chronic degenerative
myopathies, satellite cells divide and fuse to repair or replace
the damage/degenerated fibers. The number of resident satellite
cells in the adult muscle is relatively small, far smaller that
the number of committed myogenic precursors which populate the
muscle tissue shortly after an injury. Several explanations to
this apparent paradox have been proposed, from migration of
satellite cells from adjacent fibers, or even neighboring
muscles, to recruitment to myogenesis of resident, non-myogenic
cells such as fibroblasts or mosonchymal progenitors. To
investigate the presence of mesoderm-derived myogenic
progenitors, we first injected into the regenerating tibialis
anterior (TA) of scid/bg mice genetically-marked bone marrow
(BM) cells, obtained from a transgenic line in which a nlslacZ
gene is under the control of the muscle-specific MLC3P promoter
(C57/MlacZ). After injection of total BM we found regenerating
ß-gal+ nuclei. Since ß-gal expression, in our system, is not
only a donor cell genetic marker, but also a muscle
differentiation marker, these results indicate that a population
of cells within the BM actively participated in the formation of
new muscle fibers. To test whether these myogenic progenitors
could be physiologically recruited from the BM, and access a
site of muscle regeneration via peripheral circulation, we
transplanted into sublethally irradiated scid/bg mice allogeneic
C57/MlacZ BM cells. When muscle regeneration is induced in the
transplanted animals, lacZ-positive nuclei are found
incorporated into the regenerated fibers. BM-derived myogenic
progenitors could be serially transplanted into second recipient
immunodeficient mice. These data indicate that circulating
BM-derived cells can migrate into areas of muscle degeneration,
undergo myogenic differentiation, and participate to
regeneration of the damaged fibers. These data may have
potential implications for cell and gene therapy of severe
muscle-wasting disorders, such as Duchenne muscular dystrophy.
ADENOVIRAL MEDIATED DELIVERY OF
FULL-LENGTH DYSTROPHIN TO IMMUNOCOMPETENT ADULT MDX MUSCLE
G. Salvatori1, M.A. Hauser1,
C. Barjot1, D.J. Hartigan-O'Connor1, D. Calnek1, C. DelloRusso1,
2, A. Amalfitano1, JF Faulkner2, and J.S. Chamberlain1
Depts. of (1) Human Genetics
and (2) Physiology, University of Michigan, Ann Arbor, Michigan,
USA
Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, which normally expresses a 14 kb mRNA in muscle. One approach to treating DMD is by viral delivery of full-length or truncated dystrophin cDNAs. We have created a variety of full-length and truncated dystrophin expression vectors and examined their utility for gene therapy. Generation of transgenic mice on the mdx (dystrophin null) background enabled functional analyses of dystrophin clones, and these transgenic mouse studies indicated that although truncated dystrophins are highly functional, delivery of a full-length clone would probably be needed to eliminate all symptoms of DMD.
To deliver full-length
dystrophin to muscle, a gutted adenoviral vector system has been
developed that uses a replication defective helper virus for
growth. This system enables high titer growth (>1011
transducing units/ml) of gutted virus with a cloning capacity
>30 kb. The helper virus lacks Ad-Polymerase rendering it
defective for replication and late gene expression.
Incorporation of LoxP sites into the helper enables purification
of gutted vector with minimal contamination by the DeltaPOL
helper using 293 cells that express Ad-POL and Cre recombinase.
These gutted viruses efficiently transduce adult mdx muscle and
express dystrophin for >60 days in immunocompetent mice. The
results suggest that gutted adenoviral vectors could be viable
system for gene therapy of DMD.
The financial support of the
Muscular Dystrophy Association (USA) is gratefully acknowledged.
References
1. Phelps SF, Hauser MA, Cole NM, Rafael JA, Hinkle RT, Faulkner JA, Chamberlain JS: Expression of full-length and truncated dystrophin mini-genes in transgenic mdx mice. Hum Mol Genet 1995; 4:1251-1258.
2. Kumar-Singh R, and Chamberlain JS: Encapsidated adenovirus mini-chromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells. Hum Mol Genet 1996; 5:913-921.
3. Hauser MA, Kumar-Singh R, Amalfitano A, Hauschka SD, and Chamberlain JS: Improved adenoviral vectors for gene therapy of Duchenne muscular dystrophy. Neuromusc Disord 1997; 7:277-283.
4. Amalfitano A, Hauser MA,
Serra D, Begy C, and Chamberlain JS: Isolation and
characterization of improved adenoviral vectors deleted for the
E1, E2b, and E3 genes. J Virol 72:926-33, 1998.
THE INFLUENCE OF LEUKAEMIA
INHIBITORY FACTOR (LIF) ON THE DYSTROPHIC PROCESS IN MUSCLE
AND ITS POTENTIAL IN MYOBLAST TRANSFER THERAPY.
L. Austin, J. Bower, J.White,
J. Kurek, and T. Bennett1
Melbourne Neuromuscular
Research Centre, St Vincent's Hospital, Fitzroy; (1) Bernard
O'Brien Microsurgery Research Institute, St Vincent's Hospital,
Fitzroy, Australia
Leukaemia inhibitory factor (LIF) has been shown to be very effective in the proliferation of myoblasts in culture, it has no effect on fibroblasts and does not influence fusion of myoblasts to form myotubes. When perfused at a low dose (2ug/day) into crushed mouse muscle, the muscle repair is accelerated leading to larger fibres than found in controls. When LIF is perfused into the non-injured vastus lateralis muscle of the mdx mouse, again larger, but not more fibres result. Thus LIF has been shown to have positive effects in both damaged, normal muscle in vivo
and mdx muscle in vitro.
Primary myoblasts have been grow from C57 BL 10 mouse muscle and
used, under a variety of conditions to study the development of
dystrophin positive fibres when injected into mdx mouse muscle.
It was found that treatment of myoblasts in culture with LIF,
had no advantage over controls. When LIF was injected together
with the myoblasts, the effect was approximately doubled. When
the myoblasts were injected and a controlled release alginate
rod releasing LIF at about 10 ug/day was placed adjacent to the
muscle, the effect was increased fivefold. All assessments were
carried out one month after injection and no immunosuppressant
drugs were used. The diaphragm of the mdx mouse is known to
undergo muscle degeneration in a manner similar to Duchenne
muscular dystrophy. LIF charged alginate roads, releasing LIF at
about 10 ug/day were attached to the undersurface of a
hemidiaphragm of mdx mice. These were attached at 3 months of
age and the diaphragm assessed at 6 months. It was found that
the continual perfusion with LIF lead to very significant
improvements in the muscle structure of the hemidiaphragm and to
a lesser extent, that of the contralateral hemidiaphragm. In
addition, there was a significant reduction in fat deposits in
the diaphragm. No other growth factor has been shown to be
beneficial in damaged or dystrophic muscle and these results
strongly suggest that LIF is the growth factor most likely to
the beneficial in the treatment of dystrophic muscle.
References
1. Austin L, Bower J, Kurek J, and Vakakis N.: Effect of LIF and other cytokines on marine and human myoblast proliferation, 1992. J.Neurol Sci 112: 185-191.
2. Kurek J, Bower J, Romanella M, and Austin L.: LIF treatmrnt stimulates muscle regeneration in the mdx mouse, 1996. Neurosci Lett 212: 167-170.
This project was supported by
National Health and Medical Research Council and AMRAD Corp.
HUMAN MACROPHAGES RELEASE A
MYOBLAST-SELECTIVE MYTOGEN
E. Giurisato, L. Dalla Libera
(1), B. Ravara (1), M.L. Massimino and M. Cantini
CRIBI Biotechnology Center and
Department of Experimental Biomedical Sciences; (1)CNR Unit for
Muscle Biology and Physiopathology c/o Department of
Experimental Biomedical Sciences, University of Padova, Padova,
Italy
In response to a variety of
injury such as chemical, mechanical or biological agents adult
skeletal muscle is able to regenerate by the activation of a
special type of cells, named satellite cells, which in few days
strongly proliferate and fuse to form new myofibres. The muscle
destruction and successive regeneration is accompanied in vivo,
during the early stages, by an inflammatory process in which
macrophages hold a key role in muscle integrity reconstitution,
at first by removing the cellular debris and later by producing
a variety of cytokines responsible for mitosis activation and
chemotaxis of satellite cells. Recently we demonstrated that in
an in vivo culture in which both myoblasts and macrophages have
grown, the myotube formation is strongly increased by the
presence of acid stable, heat-labile, soluble growth factor(s)
secreted by macrophages. We present here new data concerning the
medium of culture and the role of macrophages on myoblast
proliferation, and in particular we demonstrate that: i)
macrophages release the factor also in a totally serum-free
medium; ii) muscle growth induced by macrophage is mainly the
consequence of an increased myoblast proliferation as revealed
by the presence of increased number of MyoD-positive myonuclei,
while fibroblasts show no increase; iii) the muscle specific
cytokine(s) released by activated macrophage has A molecular
weight in the range 50-10 kDa as determined by both
microfiltration and chromatographic experiments.
The financial support of
Telethon-Italy to the project "Myoblast-selective mitogen(s)
released by macrophages as a tool muscle regeneration and gene
therapy" (Grant n. A.101 to L.D.L.) is gratefully acknowledged.
MILIEU-DEPENDENT DIFFERENTIATION
OF SKELETAL MYOBLAST IMPLANTED INTO THE MYOCARDIUM: CURRENT
EVIDENCE
R. C.-J. Chiu
The Montreal General Hospital,
Montreal, Quebec, Canada
This presentation reviews the evidence available so far in support of the hypothesis that skeletal myoblast from adult animals, when implanted in the myocardium, may differentiate into musclefibers expressing the phenotype of cardiac myocytes.
The plasticity of myoblast differentiation depending on the milieu they are placed have been illustrated by Nathanson MA et al in 1978 when they demonstrated that cloned skeletal myoblasts grafted onto the demineralized bone could trans-differentiate into cartilage. More recently, Robinson et al showed that C-2, C-12 cells, a cell line derived from skeletal myoblasts, when implanted into the myocardium, will differentiate into cells with a number of phenotypic characteristics of a cardiac myocyte. Monoclonal antibody labelling revealed expression of connexin-43 on the cell wall at the junction between the implanted cell and the native cardiac myocyte, which developed into intercalated disks as can be seen under electron microscopy.
Kao R et al were the first to
implant primary skeletal myoblasts (satellite cells) into a
cryo-lesion created in a canine myocardium. four to eight weeks
later, they were able to observe fully differentiated striated
muscle at the implant site, surrounded by the scar tissue
developed as a result of cryo-injury. These muscle fibers showed
a number of morphological characteristics of cardiac fibers,
including the presence of intercalated disks and a centrally
located nucleus, which is distinct from the peripherally located
skeletal muscle fiber nuclei. Using lao-z reporter gene as a
cell marker, we were able to demonstrate the survival of the
implanted satellite cells in the myocardium, and more recently,
using a fluorescent compound dapi (4',6-diamidino-2-phenyl
indole) as the cell marker, we observed clearly labelled, fully
differentiated muscle fibers derived from the implanted
satellite cells. additional preliminary evidence suggest that
the expression of cardiac phenotype in these myocytes may be
facilitated by a culture condition which prevents high cell
density and confluence in vitro, as well as by the contact of
the implanted myoblasts with native cardiac myocytes. Further
characterization of optimal culture and implant conditions may
advance this approach to become useful in the clinical
management of patients with heart failure.
HOW TO IMPROVE IN VIVO CARDIAC GENE TRANSFER
Y. Fromes, H. ter Keurs1, K.
Schwartz and M.Y. Fiszman
INSERM U153, Institut de
Myologie, Hopital Pitié-Salpêtrière, Paris, France(1) University
Calgary, Calgary, Canada
In vivo gene transfer to the
heart muscle may represent, for the future, a major therapeutic
goal in human pathology. Single shot injection, even in the
coronary arteries, induces little gene transfer to the
myocardium. Perfusion rises the efficiency in a significant way,
as demonstrated by experiments using perfused heart muscle.
Considering that, during the cardiac cycle, myocardial perfusion
occurs only during the diastole, we have designed a
microinjection system that allows sequential gene delivery,
paced by the ECG. This system allows to synchronize the
injections with the diastole which should improve the gene
delivery to the myocardium. Using a Wistar rat model and
adenoviral vectors with a reporter gene, we show that it is
indeed the case since transfected cardiomyocytes can be
detected. The transfected cells are still found close to the
major coronary branches and ways to improve the method will be
further discussed. Interestingly, it is noteworthy to mention
that this method of delivery has excellent haemodynamic
tolerance.
NEW DEVELOPMENTS IN
CARDIOMYOPLASTY
J.C. Chachques, A. Berrebi, Ch
Rajnoch1, M Fiszman1, K Schwartz1 and A. Carpentier.
Department of Cardiac Surgery,
Broussais Hospital, and (1) Institute of Myology, La
Pitiè-Salpetrère Hospital. Paris, France.
A large proportion of the end-stage heart failure patients need a therapeutic approach other than the current standard modalities, due to the restricted number of heart donors and the high cost and drawbacks of mechanical assist devices.
Following the first clinical case of cardiomyoplasty, performed at the Broussais Hospital, more than 1000 patients have undergone this procedure worldwide. At our institution, 104 patients have since been operated upon.
Functional improvements and survival benefit have been observed by the majority of surgical and cardiologic groups working in cardiomyoplasty. To clearly investigate the ventricular function following cardiomyoplasty, new investigations have been performed using left ventricular pressure/volume (PV) loop analysis. These studies demonstrated the complementary roles of the muscle wrapping itself (girdling effect) and the dynamic assist (systolic squeeze).
The current research programs on cardiomyoplasty are very encouraging, they include the use of minimally invasive video-assisted surgical techniques (the last 6 cases at Broussais Hospital have been performed using this approach), the evaluation of a vascular delay (2 weeks) between muscle dissection and cardiac wrapping, and the modification of the postoperative electrostimulation protocol (using an intermittent latissimus dorsi pacing: "demand cardiomyoplasty").
Our group is also working on "cellular cardiomyoplasty", an approach to implant autologous primary skeletal muscle cells in the damaged myocardium, that potentially may contribute to the contractile performance of the heart.
It is clear that there is an
international need for autologous biological heart support
system. For this reason, over the last 10 years, cardiomyoplasty
has generated great enthusiasm for scientific studies, perhaps
more than other surgical techniques. The impressive number of
international publications confirms this interest: from 1985 to
1998 there are nearly 1400 publications (1100 experimental and
300 on clinical cardiomyoplasty).
DYNAMIC CARDIOMYOPLASTY AND AORTOMYOPLASTY:
THE BUENOS AIRES EXPERIENCE
J.C. Trainini
Hospital Presidente Peròn,
Buenos Aires, Argentina
The aim of the present study is to evaluate results obtained applying dynamic cardiomyoplasty to patients with dilated cardiomyopathy and severe ventricular dysfunction (Functional Class III-IV, New York Heart Association).
A dynamic cardiomyoplasty procedure was performed in 15 patients with a mean age of 59.2 ± 6.4 years old. Despite the medical treatment with inhibitors of the converted enzyme or vasodilators, these patients required 2.2 ± 0.7 hospitalisation/patient/year owing to congestive heart failure in the year before dynamic cardiomyoplasty was applied. In 8 patients the aetiology of the cardiomyopathy was idiopathic, ischemic-necrotic in 6 and Chagas' disease in the other.
Hemodynamic studies were done preoperatively in all patients and every six months postoperatively.
Twelve patients had a follow-up for two years.. The following values related to two-years evaluation improved significantly in comparison with baseline: functional class (1.7 ± 0.6 versus 3.06 ± 0.2); radionuclide left ventricular ejection fraction ( 29.7 ± 5% versus 23.6 ± 3%). Walking test values increased from 332 ± 127 meters to 421 ± 102 meters. Left ventricular diastolic diameter remained unchanged (72.7 ± 7 mm versus 72.3 ± 8mm).
Improvement was observed in functional capacity and left ventricle systolic function parameters two years after cardiomyoplasty was applied.
AORTOMYOPLASTY. Six patients
were included with mean age of 52 years and a Functional Class
(FC) III-IV. One patient died two months later (ventricular
arrhythmia) before completing the stimulation protocol. The
other 5 patients improve the FC with a value of I-II at 12
months postoperative average.
INTERMITTENT STIMULATION AND VASCULAR DELAY
ENHANCE FUNCTION OF CONDITIONED MUSCLE
C. Duan, D.R. Trumble, J.A.
Magovern, I.Y. Christlieb, D. Scalise and G.J. Magovern Sr.
Cardiothoracic Surgical
Research, Allegheny University of the Health Sciences:
Department of Surgery, Allegheny University Hospitals,
Pittsburgh, Pennsylvania
Normal latissimus dorsi (LD)
muscle is powerful but prone to fatigue. Continuously stimulated
LD muscle is fatigue-resistant, but generates significantly less
power. In cardiomyoplasty (CMP), ischemia after muscle isolation
has been reported. For clinical applications, conditioned muscle
needs to be powerful, fatigue-resistant and well-vascularized.
In this study, rabbit LD muscle were used to test the hypothesis
that periodic intervals of rest (12 hr/day) and vascular delay
can improve the functional capacity of trained skeletal muscle.
Animal were assigned to five groups: 6-wk continuous
stimulation; 12-wk intermittent stimulation without division of
collateral vessels (preserved); 12-wk intermittent stimulation
with division of collateral vessels after training (isolated);
12-wk interval stimulation with a 3-wk vascular delay between
isolation and training (vascular delay); and control. LD muscle
were stimulated using burst stimuli (25 Hz, 250 msec burst
duration, 53 contractions/min). Muscle strength and endurance
were tested using a custom muscle ergometer. Results indicate
that intermittent burst stimulation prevents power loss in
fatigue-resistant muscle and increases contractile function
beyond baseline values. Intermittent stimulation without
division of collateral vessels increased maximum isometric force
generation compared with both the continuous stimulation group
(391%) and control muscles (175%). intermittent stimulation also
improved endurance capacity relative to continuous burst
stimulation. At the conclusion of the 40-minute fatigue test,
control LD muscles retained less than 1% of their initial work
capacity. Stimulated muscles in the 6wk-continuous group
fatigued from 205,0 ± 22,4 g-cm to 124,3 ± 8,2 g-cm after the
first ten minutes of testing and retained 71,5 ± 19,5 g-cm of
their initial stroke work at 40 minutes. Muscles in 12-wk
interval stimulation (preserved) group had the highest initial
stroke work (449,7 ± 92,4 g-cm) and the highers remaining stroke
work (234,7 ± 50,1 g-cm) at 40 minutes. Fatigue tests for the
12wk-interval (preserved) group were continued to 3 or 8 hours
and yielded the following temporal stroke-work profile: 239,3 ±
54,7 g-cm at 1 hr; 213,0 ± 61,0 g-cm at 3 hr, 258,0 g-cm at 5
hr, 181 g-cm at 7 hr, and 165,0 g-cm at 8 hr. The effect of
vascular delay was estimated by comparing the functional changes
among the three intermittent stimulation groups. Maximum
isometric forces were decreased in isolated (19%) and vascular
delay muscles (28%) compared with the preserved group. The
endurance capacity of isolated LD were significantly lower than
the preserved and vascular delay groups. At the conclusion of
the 8 hr fatigue test, isolated LD muscles retained 7% of their
initial work capacity (from 526 g-cm to 36 g-cm) at 3 hr, and
generated less than 9 g-cm stroke work after 4 hr. LD muscles in
the vascular delay group retained 43% of their initial work
capacity (from 268 g-cm to 114 g-cm) after 8 hr. these findings
suggest that the use of intermittent rest and vascular delay
periods may enhance the efficacy of CMP.
References
1. Arpesella G, Milkus P, Lombardi P, Pierangeli A, Giannoni A, Zampieri S, Catani C, and Carraro U: Activity-rest regimen of latissimus dorsi stimulation for cardiomyoplasty: anatomy, isomyosins and sustained power of sheep LD up to one year. Basic Appl Myol 7: 45-53; 1997
2. Overgoor MLE, Carroll SM, Carroll CM, et al: Does pacing induced angiogenesis enhance latissimus dorsi muscle flap survival? Eur Surg Res 27: 99-100; 1995.
3. Duan C, Trumble DR,
Christlieb IY, Magovern JA, and Magovern GJ Sr: Improved
function in muscles trained via interval stimulation. Basic Appl
Myol 8: 35-40; 1998.
DEMAND DYNAMIC CARDIOMYOPLASTY: 18-MONTH
FOLLOW-UP
C. Muneretto (1,4), M.
Barbiero (2), G. Docali (2), C. Brunazzi (2), M. Gemelli (2), K.
Gealow (3), L. Testolin (1,4), D. Casarotto (4) and U. Carraro
(5)
(1) Cardiovascular Surgery,
University of Brescia, Italy; (2) Division of Cardiology,
Legnago General Hospital (Verona); (3) Medtronic, Inc.,
Minneapolis, USA; (4) Cardiovascular Surgery, University of
Padova; (5) C.N.R. Unit for Muscle Biology and Physiopathology
and Department of Biomedical Sciences, University of Padova,
Italy;
Full transformation of
latissimus dorsi muscle from a fast-fatigable to a slow
fatigue-resistant muscle has been considered until recently as
the key step in Dynamic Cardiomyoplasty. Unfortunately muscle
power has been showed to decrease with extent of fast to slow
transformation. On the basis of Arpesella's experimental data,
we have developed a new clinical protocol based on activity-rest
stimulation which provides resistance to fatigue at higher
muscle power. The extent of transformation of contractile
characteristics of the LD flap can be related to the stimulation
protocols used, i.e. the amount of impulses delivered per day.
Beside the sheep experiments of Arpesella team, corroborating
results have been recently presented in a rabbit model
confirming that long-term daily stimulation increases blood flow
but decreases muscle mass, while "interval stimulation", that is
an activity-rest regime of stimulation, preserves muscle mass
and force. Indeed, transformation is reversed by a "demand"
stimulation, i.e., with an activity-rest regime which rests LD
several hours per day. The result is achieved with an
activity-rest LD stimulation obtained by a heart frequency
dependent stimulation cut-off. Thus, in patients LD muscle is
rested during low activity periods. Preliminary study included
four patients with cardiac heart failure due to dilatative
cardiomyopathy in NYHA class III with a mean preoperative peak
VO2 of 12 ml/kg/min. There are no deaths and the patients are in
NYHA class I after a follow up of 18 months. One year after
Demand Dynamic Cardiomyoplasty peak VO2 increased up to 35% when
compared with preoperative values. If these preliminary data
will be confirmed in a larger cohort, Demand Dynamic
Cardiomyoplasty could offer long-standing benefits to manage
pharmacologically-intractable heart failure.
References
1. Carraro U, Barbiero M, Docali G, Brunazzi C, Lorusso R, Rinaldi M, Gazzoli F, Viganò M, Gerometta PS , Barbier P, Biglioli P, Casarotto D, Muneretto C: Dynamic Cardiomyoplasty: Long-term viability demonstrated by non-invasive on-line analysis of dynamic contractile characteristics of human LD flap in Italian subjects. J Cardiovasc Diagn P, in press.
2. Carraro U, Docali G, Barbiero M, Brunazzi C, Lorusso R, Muneretto C: Dynamic Cardiomyoplasty: Implement a basic concept to allow non invasive monitoring of dynamic characteristics of the LD flap, in D. Popovic (ed): Proceedings of the IFESS97 Conference, (Burnaby, British Columbia, Canada, August 16-21) 1997, 51-52.
3. Duan C , Trumble DR , Christlieb IY, Magovern JA, and Magovern GJ Sr: Improved function in muscles trained via interval stimulation. Basic Appl Myol 1998, in press
The financial support of MURST
is gratefully acknowledged.
TUTORIAL ON LATISSIMUS DORSI WRAP MECHANOGRAM
AT BEDSIDE
U. Carraro (1), M. Barbiero
(2), G. Docali (2), A. Cotogni (2) and C. Brunazzi (3)
(1) C.N.R. Unit for Muscle
Biology and Physiopathology, Department of Biomedical Sciences,
University of Padova; (2) Division of Cardiology, Legnago
General Hospital (Verona); (3) Cardiology, Pieve di Coriano
Hospital (Mantua); Italy
In the cohort of patients operated in Padova, Dynamic Cardiomyoplasty was performed according to the Carpentier and Chachques procedures, but the LD wrap conditioning period was shortened to one month. Beginning one-two week after surgery with one impulse, an impulse was added each week for a total of four impulses per burst. The pulse interval was 23 msec (43 Hz), and the LD was stimulated every third cardiac cycle. Each subject's LD flap was monitored bed-side using a standard polygraph (Siemens MegaCart or Mingophon). Originally developed for monitoring cardiac apical motion (apicocardiogram) and heart sounds, we have used this technology to provide a simple, non-invasive way to monitor LD contraction. A signal registering the contraction and relaxation of the LD can be measured by placing the transducer normally used for recording the apicocardiogram over the rib window through which the LD enters the thoracic cavity. ECG and heart sounds are recorded simultaneously with the pressure changes due to LD flap contraction-relaxation. From this LD "mechanogram" the following parameters relating to LD contraction are determined: 1) the activation threshold, 2) the clinically acceptable LD activation amplitude, 3) the tetanic fusion frequency (TFF) of the LD, 4) the duration of the complete mechanical event (contraction and relaxation), and 5) the optimal synchronization timing between cardiac events and LD contraction.
The activation threshold is easily determined by measuring the peak contraction at different amplitudes of stimulating voltage (from 1 to 8 Volts). The point at which the muscle is first activated is easily recognized, even in patients whose LD contraction is not readily identified through standard palpation of the axillary region. We chose a clinical stimulation amplitude at half the difference between the threshold and the maximal contraction accepted without discomfort for the patient. This is not the maximal activation of the LD, but in such a way the non-activated portion of the flap is "spare LD," available in case of long-term-activity muscle damage.
The dynamic characteristics of the LD flap are also determined from the LD response to stimuli delivered at increasing frequency rate. The LD tetanic fusion frequency can be identified by delivering triplets (up to 70 msec interpulse intervals) or doublets at intervals ranging from 16 to 200 msec (63 to 5 Hz, respectively). The percent of relaxation between two stimuli delivered at a 100 to 200 msec interval could also be measured. Of course, the faster the LD flap the greater the extent of muscle relaxation between the two impulses. The duration of the complete mechanical event in response to four impulses delivered at 23 msec intervals could be also measured.
Finally, the optimal synchronization between the cardiac cycle and contraction of the LD flap is determined. This can be done using the mitral and aortic valve tones as measured on the phonocardiogram or, preferably, by connecting the mechanogram signal directly to echocardiography equipment. In this way, the LD mechanogram events can be directly and simultaneously compared to the high-resolution images of cardiac events, either M-mode imaging of valve motion or Doppler imaging of the left ventricle outflow. Precise tuning based on the actual mechanical events of LD contraction and relaxation can be achieved rather than tuning based only on the electrical impulses delivered to the LD as observed on the ECG. The onset of LD contraction can be programmed to occur at the start of the isovolumic contraction phase of cardiac systole or just at the start of ejection.
Patients were submitted to an activity-rest stimulation regime by special programming of the cardiomyostimulator (Transform(r), Model 4710, Medtronic, Inc., Minneapolis, MN, USA). These patients were operated in June 1996 (a 48-year-old man and a 46-year-old woman). They were submitted to an activity-rest regime after six to nine months of continuous daily stimulation. The patients' average heart rates during the day and night were first determined by 24-hour Holter analysis. For both patients, the average heart rate at night was less than 80 bpm and greater than 80 bpm during the day. The lower rate on the pacing channel of the cardiomyostimulator was then programmed to 80 but with minimum values for pacing amplitude and pulse width. In this way, the device will be pacing most of the time during night or resting hours but at a very low, sub-capture level. By programming the muscle channel output to "Sense," rather than "Sense + Pace," muscle stimulation will occur only when the heart rate goes above 80 bpm. When the heart rate decreases, indicating a period of low activity, the device begins to pace and muscle stimulation is inhibited most of the time, allowing the muscle to rest. With the device programmed accordingly, repeat Holter studies have shown approximately 8 to 10 hours per day of reduced muscle stimulation; 7 to 8 hours at night plus 1 to 2 more hours during the day. This intermittent stimulation has been well tolerated by the patients with no sleeping disturbances.
References
1. Carraro U, Barbiero M, Docali G, Brunazzi C, Lorusso R, Rinaldi M, Gazzoli F, Viganò M, Gerometta PS , Barbier P, Biglioli P, Casarotto D, Muneretto C: Dynamic Cardiomyoplasty: Long-term viability demonstrated by non-invasive on-line analysis of dynamic contractile characteristics of human LD flap in Italian subjects. J Cardiovasc Diagn P, in press.
2. Carraro U, Docali G, Barbiero M, Brunazzi C, Lorusso R, Muneretto C: Dynamic Cardiomyoplasty: Implement a basic concept to allow non invasive monitoring of dynamic characteristics of the LD flap, in D. Popovic (ed): Proceedings of the IFESS97 Conference, (Burnaby, British Columbia, Canada, August 16-21) 1997, 51-52.
3. Carraro U, Muneretto C,
Barbiero M, Brunazzi C, Docali G, Barbaresi F, Testolin L,
Marcassa A, Casarotto D, Picotti P, Bandello A, Marchi C:
Cardiomioplastica Dinamica: come scegliere e preparare i
pazienti. Medicus news 1997; 4: 22-25.
VALIDATION OF LATISSIMUS DORSI
WRAP MECHANOGRAMWITH FLUOROSCOPIC CONTRACTION ANALYSISIN THREE
PATIENT GROUPS
F.H. van der Veen, M.
Barbiero1, M. Smink, R. Lorusso2, M. Volterrani3, T. van der
Nagel, and U. Carraro4
Department of Cardiology,
Cardiovascular Research Institute Maastricht, The Netherlands;
(1) Division of Cardiology, Legnago General Hospital, Legnago
(Verona); (2) Department of Cardiothoracic Surgery, Brescia; (3)
Department of Cardiology, Gussago (Brescia); (4) CNR Unit for
Muscle Biology and Physiopathology, University of Padua, Italy
Selection of stimulator settings in patients after cardiomyoplasty (CMP) should be performed with non-invasive evaluation of latissimus dorsi muscle (LDM) contraction. Relevant settings concern the delay after the QRS-complex, the stimulus amplitude and the burst duration. Two methods have been reported which characterize mechanical properties of the LDM. Firstly, fluoroscopic evaluation of LDM contraction by measuring the distance of the stimulation electrodes within the LDM (ref1). Secondly, mechanographic evaluation of LDM contraction by extra-thoracic recording of the LDM sound (ref2). In the present study both techniques were applied in three groups of patients at least 3 months after CMP surgery. In the first group (n = 4), patients were included who had followed the general stimulation protocol (LDM heart ratio 1:2). In the second group (n = 4), patients had been on a 1:4 LDM heart ratio, and in the third (n = 4), patients had been stimulated according to the so-called "demand" protocol. The following stimulator settings were applied: amplitude 5 or 10 Volts, delay 25-125 msec, a burst with 1, 3 or 6 pulses.
Results: comparison of the contraction/relaxation curves with both methods revealed a significant identity. Both the curve duration and the relative amplitude showed a difference of less than 5%. Surprisingly, the delay after the QRS complex was 30± 20 msec longer with the mechanogram technique, which might point to a rather slow stimulus conduction in the distal direction. The three different patient groups had significant slower muscles if the stimulation protocol was more intensive (1:2). The cycle length at 6 pulse bursts increased from 440 ± 105 msec to 650 ± 180 msec.
Conclusions: The mechanogram offers a reliable, fast, cheap and non-ivasive method to study stimulator setting in patients after cardiomyoplasty. LDM stimulation after CMP transforms the muscle in a slow-muscle, unless "on demand" stimulation is applied.
References:
1. van der Veen FH, Lucas CMHB, Lorusso R, van der Nagel T, Penn OCKM, Wellens HJJ: A new method to select stimulus strengh after cardiomyoplasty. J Cardiac Surg 1991; Vol 6, No 1, Suppl 259-264,.
2. Carraro U, Docali G,
Barbiero M, Brunazzi C, GealowK, Casarotto D, Munaretto C: Deman
dynamic cardiomyoplasty. Improved clinical benefits by
non-invasive monitoring of LD flap and long-term tuning of its
dynamic contractile characteristics by activity-rest regime.
Basic and Applied Myology 1998; 8: 11-15.
HOW TO EVALUATE LONG-TERM HEMODYNAMIC RESULTS IN
CARDIOMYOPLASTY
V.S. Chekanov
Milwaukee, WI, USA
Correct evaluation of the hemodynamic long-term results is one of the difficult problems regarding cardiomyoplasty, especially if trying to compare results from different institutions. Below is a proposed new method to simplify the evaluation of hemodynamic results following cardiac surgery.
The main focus is the
calculation of a hemodynamic coefficient (graded from 1 to 10,
with 10 being the best) as an average of available hemodynamic
parameters. Seven such parameters, all important for patients
with pre end stage congestive heart disease and possible
candidates for cardiomyoplasty, are presented in the table
below.
1
2
3
4
5
6
7
8
9
10
LVEDV
(ml)
>550
500-549
450-499
400-449
350-399
300-349
250-299
200-249
150-199
<150
LVESV
(ml)
>450
400-499
350-399
300-349
250-299
200-249
150-199
100-149
50-99
<50
LVEF
(%)
<10
11-17
18-24
25-31
32-38
39-45
46-52
53-59
60-66
>67
RVEF
(%)
<10
11-17
18-24
25-31
32-38
39-45
46-52
53-59
60-66
>67
PCWP
mmHg
>27
25-26
23-24
21-22
19-20
17-18
15-16
13-14
11-12
<10
MPAP
mmHg
>82
75-81
68-74
61-67
54-60
47-53
40-46
37-39
26-32
<25
Peak
VO2
ml/min
<10
10.1-11.5
11.6-13
13.1-14.5
14.6-16
16.1-17.5
17.6-19
19.1-21.5
21.6-23
>23
The number of parameters may increase or decrease according to the studies performed.
Below are examples of patients from the Milwaukee Heart Institute which will show how the table can be used to generate the hemodynamic coefficient.
Example 1. Before
cardiomyoplasty: LVEDV -310 ml (category 6); LVESV -257 ml (5);
MPAP -52mmHg (6); PCWP -31 mmHg (1); Peak VO2 -16.9 ml/min (6);
LVEF -20% (3); RVEF -16% (2).Hemodynamic coefficient is:
6+5+6+1+6+3+2=29/7=4.14. One year after cardiomyoplasty: LVEDV
-274ml (7); LVESV -190ml (7); Peak VO2 -16.7 ml/min (6); LVEF
-26% (4); RVEF -32% (5). Hemodynamic coefficient is:
7+7+6+4+5=29/5=5.8.
Example 2. Before cardiomyoplasty: LVEDV -273 ml (7); LVESV -213 ml (8); LVEF -25% (4); RVEF -46% (7); MPAP -80 mmHg (1); PWCP -25 mmHg (2); Peak VO2 -16 ml/min (5). Hemodynamic coefficient is 7+8+4+7+1+2+5=34/7=4.86. Six months after cardiomyoplasty: LVEDV -320 ml (6); LVESV -260 ml (5); LVEF -22% (3); RVEF -42% (6); Peak VO2 -13.8 ml/min (4). Hemodynamic coefficient is 6 + 5 + 3 + 6 + 4 = 24 : 5 = 4.8.
Conclusion: Utilizing this new
method for evaluating hemodynamic results, it is possible to
compare: the status of patients before and after surgery,
different surgical techniques, different patient populations,
and data from different institutions.
FATAL RHYTHM DISTURBANCES AFTER
CARDIOMYOPLASTY PROCEDURE: IS COMBINED AICD IMPLANTATION THE
OPTIMAL SOLUTION?
R. Lorusso, A. Marchini1, F.
Bianchetti1, A. Curnis1, M. Volterrani2, A. Giordano2, G.
Coletti, M. Zogno
Cardiac Surgery and (1)
Cardiology Departments, Brescia, (2) Cardiology Division IRCCS
Fondazione Clinica del Lavoro, Gussago, Italy
Background. The incidence of sudden death in chronic heart failure patients ranges between 10% and 40%. Ventricular arrhythmia represent the major cause of such episodes. Cardiomyoplasty does not seem to modify the patient outcome in this respect. The experience of our Centre is reviewed addressing the arrhythmic events and subsequent management.
Methods. Twenty-six patients underwent cardiomyoplasty procedure at the Brescia Hospital since 1991. Patient age ranged from 45 years to 73 (mean 62 years). Fourteen patients had ischemic cardiomyopathy, whereas 12 patients had idiopathic cardiomyopathy. Every patient underwent preoperative basal and exercise ECG, as well as Holter monitoring, which were repeated every six months postoperatively. One patient had preoperative EPS (electrophysiologic study) for an episode of sustained VT, showing inducible VT, and combined cardiomyoplasty and AICD implantation was deemed appropriate and performed. All patients are on Amiodarone after cardiomyoplasty procedure as a part of a postoperative protocol.
Results. Follow-up ranges from 18 months to 77 months (mean 42 months). Ten patients experienced sudden death (4 witnessed). Sudden death occurred in 7 ischemic patients and in 3 idiopathic patients. No arrhythmic episodes was present in their preoperative history. Preoperative basal or exercise ECG, and Holter recordings were devoid of malignant ventricular arrhythmia and no change in number and quality of supraventricular or ventricular arrhythmia were observed postoperatively. Two patients had sustained VT episodes with hemodynamic compromise at 3 and 5 years after surgery, respectively, and were successfully treated (external defibrillation). AICD was subsequently implanted becasue of evidence of inducible VT during EPS. Subsequent episodes of malignant ventricular arrhythmia occurred in 2 patients with AICD implanted, and terminated by the device. One patient had AICD intervention because of high ventricular rate during paroxysmal atrial fibrillation. No episode of sudden death or documented malignant ventricular arrhythmia was correlated to the clinical status or induced by hemodynamic impairment.
Conclusions. Our study clearly
underlines the importance of combined AICD implantation in
cardiomyoplasty patients. Despite favorable changes in
ventricular geometry, ventricular arrhythmia seem to occur
anyway, and not correlated with the patient hemodynamic state.
Patients affected by ischemic cardiomyopathy are at higher risk
for developing unfavorable events. Amiodarone therapy may
provide additional rescue, but does not appear to significantly
modify the risk from postoperative sudden death.
NUMERICAL PREDICTION AND EXPERIMENTAL MEASUREMENT
OF HYDRAULIC WORK DURING CARDIAC ASSISTANCE WITH SKELETAL
MUSCLE VENTRICLES
D.M. Pullan1, 3, A.P.
Shortland2, S. Salmons1 and J.C. Jarvis1
(1)Department of Human Anatomy and Cell Biology, The University of Liverpool
(2) Department of Clinical Engineering, The University of Liverpool
(3) The Cardiothoracic Centre,
Liverpool NHS Trust
Skeletal muscle ventricles (SMVs) are hydraulic pumps formed from autografts of skeletal muscle arranged to provide extra hydraulic work to the cardiovascular system in heart failure. We have investigated one configuration of such a system in which the SMV acts as an aortic counterpulsator. The SMV is connected by a single conduit to the descending aorta and, by analogy with an intra-aortic balloon pump for example, contracts during diastole to enhance coronary perfusion and relaxes during systole to reduce the work of the left ventricle. We have investigated both by numerical modelling and now by experimental measurement how the degree of SMV assistance depends on the placement of the assist phase within the cardiac cycle. Cylindrical SMVs were made in pigs by wrapping the latissimus dorsi muscle around a PTFE former. The SMVs were electrically stimulated at 1 Hz for 4 weeks to render them resistant to fatigue. In a terminal procedure the SMVs were connected to the descending aorta via a GoreTex conduit. We recorded left ventricular volume via a conductance catheter and SMV volume via a sonomicrometer system. Pressures were measured within the left ventricle, aortic root and SMV. Flow was recorded from the aortic root, and proximal and distal to the site of anastomosis of the SMV conduit with the aorta.
The timing of SMV activation was controlled relative to the prevailing systolic and diastolic durations of the left ventricle. The delay between the QRS complex and the start of SMV activation was varied between 20% and 140% of the systolic duration and the duration of SMV activation was varied between 60% and 140% of the diastolic duration. Various timing combinations were delivered in a pseudo-random order, after a short period with the SMV OFF. The complete matrix was achieved in 6 experiments. In two of the experiments it was also possible to evaluate the isolated pressure-volume characteristic of the SMV.
Results and conclusions:
The pressure-volume loops for
the LV and SMV show that the timing of SMV action has a profound
effect on its influence on the LV. SMV action can reduce the
work done by the heart, but can also increase it if the timing
is inappropriate. The experimental results contain trends
similar to those predicted by our numerical model of the
assisted circulation.
The support of the British
Heart Foundation is gratefully acknowledged.
References
1. Jarvis JC, Kwende MMN,
Shortland AP, Eloakley, RM , Gilroy, SJ, Black RA and Salmons S
(1997) Relation between muscle contraction speed and hydraulic
performance in skeletal muscle ventricles. Circulation 96:
2368-2375.
CORONARY BLOOD FLOW AUGMENTATION AND AFTER LOAD
REDUCTION: A COMPARISON BETWEEN INTRA-AORTIC BALLOON AND
DESCENDING AORTOMYOPLASTY COUNTERPULSATION
G. Bolotin, T. Wolf, R.
Shofti, F. van der Veen, M. Smink, J. Schreuder, R. Lorusso, Y.
Rubin, G. Uretzky
The Department of
Cardiothoracic Surgery, Carmel Medical Center, the Department of
Biological Engineering, I.I.T. Haifa, Israel and the Department
of Cardiology, Maastricht,The Netherlands
Objective: Aortomyoplasty is a surgical procedure whose aim is to induce the hemodynamic benefits of the intra-aortic balloon pump (IABS). The objective of this study was to compare coronary flow augmentation and afterload reduction as are induced by either IABP or descending aortomyoplasty counterpulsation.
Methods: 15 mongrel dogs (18-35 kg) underwent descending aortomyoplasty (n = 8) and IABP application (= 7). Left anterior descending (LAD) coronary artery blood flow was measured using a Transonic Doppler flow probe. Left ventricular pressure and aortic pressures, proximally to either aortomyoplasty site or intra-aortic balloon position,were monitored continuously.
Results: Descending aortomyoplasty induced a 21% increase in the LAD blood flow integral in assisted beats (14 ± 6 ml/min) as compared to unassisted beats (11 ± 4 ml/min) (p < 0.001). This was comparable with the 24% rise in the LAD blood flow integral during IABP counterpulsation (from 9 ± 3 ml/min up to 12 ± 3 ml/min (p < 0.001)). On the other hand, wile IABP counterpulsation induced afterload reduction (14% reduction in the end diastolic proximal aortic pressures, from 106 ± 7 mmHG to 94 ± 9 mmHg (p < 0.001)7, descending aortomyoplasty did not induce afterload reduction.
Conclusions: Descending
aortomyoplasty is capable of reaching coronary blood flow
augmentation as is produced by the IABP. This may be important
for end-stage ischemic patient. However, the essential afterload
reduction induced by the IABP was not reproducible during
descending aortomyoplasty counterpulsation. Improving the
surgical technique of descending aortomyoplasty and attaining
afterload reduction is of additional value for congestive heart
failure patients.
ACTIVITY-REST STIMULATION REGIME FOR SKELETAL
MUSCLE CARDIAC ASSIST
G. Arpesella, P.M. Mikus, P.
Lombardi
Cardiovascular Surgery,
University of Bologna, Italy
In Dynamic Cardiomyoplasty chronic stimulation of the Latissimus Dorsi achieves full transformation of its myofibers, so that slowness and consequent delimited power limits its systolic support. A daily activity-rest regimen of stimulation could maintain a partial transformation of LD and its power output. After surgical shortening in sheep, LD were burst-stimulated either 10 or 24 hr per day. Two weeks after surgery and two, four, six and twelve months after stimulation, fusion frequency of tetanus, power output, and fatigue resistance of LD were assessed. LDs were biopsied at six months of stimulation, and sheep sacrificed at twelve months. After one year of 10 hr/day stimulation LD was substantially conserved and contained large amounts of fast type myosin. From two-month up to one-year of stimulation the power per muscle of the daily rested LD was three to four times higher than in 24 hr/day stimulated LD and its sustained power was bigger than that of the left ventricle.
Corroborating results were recently presented. In a rabbit model, long-term daily stimulation increases blood flow but decreases muscle mass, while "interval stimulation", that is an activity-rest regime of stimulation, preserve muscle mass and force. Also in a dog model activity-rest protocols are superior in increasing blood pressure and stroke work.
If these results will be
confirmed and extended to human muscle, we are confident that
they could be the experimental basis for a demand
cardiomyostimulation, whose discontinuous activity could offer
to cardiomyoplasty patients the long-standing advantage of a
faster and powerful muscle contraction.
ALTERNATIVE STIMULATION AND CONDITIONING
PROTOCOL TO TRANSFORM SKELETAL MUSCLE FOR CARDIOMYOPLASTY
F.H. van der Veen, M. Smink,
H.G. Kaulbach
Department of Cardiology,
Cardiovascular Research Institute CARIM, Maastrich, The
Netherlands
The present study examined whether partial fibre type transformation can be obtained with an intensity reduced training protocol and can serve as an alternative possibility to maintain force and fatigue resistance, when stimulating the muscle in cardiomyoplasty. Goats (n = 7) had two intramuscular electrodes in the proximal part of the latissimus dorsi (LD) muscle and a pulse generator implanted (Telectronics Pacing Systems, MYOSTIM 7220). A stimulation protocol with an average of 6 contractions per minute was used during a 3 months training period. Stimulation started at 1 pulse, while every 2 weeks 1 pulse was added to finally 6 pulses per burst. Also, a wide range of stimulation options was programmed at the start and at the end of the training period with measurements of the LD muscle force. Mean peak force did not change significantly after the training period (3.5 ± 17 NVS 5.8 ± 3.4 N) while type I fibre showed a large variation (40-90%) and were only marginally increased (from 24 ± 5% to 43 ± 26%). The amount of fatty tissue and connective tissue was slightly increased to 3.0 ± 1.4% and 3.0 ± 1.4%, respectively, while contraction was not increased significantly. Stimulation of the muscle at variable pulse width settings (range 150 to 650 usec) showed that contractile force was affected by pulse width variation at single pulse stimulation. However, at burst stimulation (6 pulses) an increase of pulse width was accompanied by a gradual increase in force. On the other hand, an increased from 1 to 6 pulses at constant amplitude and pulse width provoked also a gradual increase in contractile force.
Conclusion: 1) An intensity
reduced conditioning protocol for cardiomyoplasty maintains a
forceful muscle, which is accompanied by a large variation in
muscle fibre type distribution. 2) A gradual increase in the
number of pulses per burst, rather than an increase in pulse
width should be used to select the appropriate force of the LD
muscle in cardiomyoplasty.
Basic App Myol 8 (3), 1998
Abstracts of the International
Scientific Committee
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Akopova I., 254
Amalfitano A., 259
Ambrosio G.B., 252, 257
Anker S., 252
Argentini C., 246
Arpesella G., 266
Arslan P., 256
Austin L., 260
Barbiero M., 262, 263, 264
Barjot C., 259
Barsotti G., 247
Barylski N., 250
Bennet T., 260
Berrebi A., 261
Bianchetti F., 265
Biral D., 245
Bolotin G., 266
Bortoloso E., 248
Bower J., 260
Bresci M., 250, 254
Brunazzi C., 262, 263
Calnek D., 259
Campos G., 250
Cantini M., 256, 260
Carpentier A., 261
CarraroU., 237, 252, 255, 256, 257, 262, 263, 264
Casarotto D., 262
Catani C., 252
Ceconi C., 252
Chachques J.C., 261
Chamberlain J. S., 247, 259
Chekanov V. S., 248, 253, 264
Cheng Q., 248
Chisari C., 247, 250, 254
Chiu R. C-J., 260
Christlieb I. Y., 262
Coats A., 252
Coletti G., 265
Coper H., 249
Cossu G., 259
Cotogni A., 263
Cupisti A., 247
Curello S., 252
Curnis A., 265
D'Alessandro C., 250, 254
Dalla Libera L., 252, 257, 260
Damiani E., 248
DelloRusso C., 259
Docali G., 262, 263
Duan C., 262
Edström L., 246
El Messlemani A., 255
Eriksson L. I., 246
Faulkner J. A., 247, 259
Ferrari G., 259
Ferrari R., 252
Fidzianska A., 245, 249
Fiszman M.Y., 261
Fromes Y., 261
Gealow K., 262
Gemelli M., 262
Giannini E., 250, 254
Giardini E., 255
Giordano A., 265
Giurisato E., 256, 260
Gosselin L.E., 244
Hagerman F.C., 250
Hartigan-O'Connor D. J., 259
Hauser M. A., 259
Hausmanowa-Petrusewicz I., 249
Hikida R.S., 250
Hooper T.L., 246
Ikeda H., 252
Itoh G., 252
Jackson M. J., 244, 249
Jakubiec-Puka A., 245
Jarvis J.C., 246, 265
Kaminska A. M., 249
Kaulbach H.G., 266
Koskinen S.A.O., 244
Kovanen V., 244
Kurek J., 260
La Rovere M.T., 251
Larsson L., 246
Leprotti C., 257
Lombardi P., 266
Lorusso R., 264, 265, 266
Loughna P.T., 245
Luecke T., 250
Lukoyanova N., 254
Lynch G. S., 247
Maglara A., 249
Magovern G. J. Sr., 262
Magovern J. A., 262
Malyshev S., 254
Marchetti C., 250
Marchini A., 265
Margreth A., 248
Mason P., 245
Massimino M.L. 256, 260
Mavilio F., 259
Mazzoleni F., 255
McArdle A., 244, 249
McArdle F., 244
Menshova O., 254
Meola M., 247
Mikus P.M., 266
Minetti C., 257
Morelli E., 247
Mortara A., 251
Muneretto C., 262
Ossowska K., 249
Pette D., 258
Podlubnaya Z., 254
Poggi P., 250
Poole-Wilson P. A., 251
Prenger K., 256
Pullan D.M., 265
Rajnoch C., 261
Ravara B., 260
Rieder M. A., 248, 253
Rizzi C., 255, 256
Rossi B., 247, 250, 254
Rossini K., 255, 256, 257
Rubin Y., 266
Salmons S., 246, 265
Salvatori G., 259
Sandri C., 256
Sandri M., 255, 256, 257
Scalise D., 262
Scelsi R., 250
Schiaffino S., 246
Schreuder J.J., 256, 266
Schulze G., 249
Schwartz K., 261
Shofti R, 266
Shortland A.P., 265
Shpagina M., 254
Smink M., 256, 264, 266
Staron R.S., 250
Suzuki Y., 252
Takala T.E.S., 244
Tang A.T.M., 246
Tchekanov G. V., 253
ter Keurs H., 261
Testolin L., 262
Tews D.S., 256
Tong J., 252
Trainini J.C., 261
Trumble D. R., 262
Udaltsov S., 254
Uretzky G., 266
van der Nagel T., 264
van der Veen F.H., 256, 264, 266
Vescovo G., 252, 257
Volterrani M., 264, 265
Walsh S., 250
Wendeln H., 250
White J., 260
Wolf T., 266
Wolfarth S., 249
Zackrisson H., 246
Zander G. L., 248
Zennaro R., 252, 257
Zogno M., 265
Zuliani F., 255- 249 -
