Protective effect of hypothermia on contractile force in skeletal muscle

The clinical success of limb replantation and tissue transfer is partly dependent on the duration of ischemia experienced by the amputated part. This study focused primarily on the damage that occurs during this ischemic period. An experimental system was implemented that allowed the observation of contractile function in totally isolated skeletal muscle after ischemia. Contractile function was selected as an indicator of ischemic damage because normal function is the ultimate goal of replantation. All experiments were performed on the rat extensor digitorum longus. The muscles were subjected to ischemic periods of 1.5, 3.0, and 5.0 hours and were stored in either a hypothermic (4 degrees C) or a room-temperature (23 degrees C) environment during the ischemic interval. After the ischemic period, all muscles were transferred to a tissue bath and were subjected to contractility testing, followed by fatigue testing. In both groups, muscle function decreased as the ischemic interval was increased. A significant difference in function between the normal control and the muscles of both ischemic groups implied that ischemic injury had occurred in the hypothermic and room-temperature muscles, even with the relatively short 1.5-hour ischemic interval. After each ischemic interval however, the hypothermic muscles produced significantly greater contractile force than the room-temperature muscles in both the contractility and the fatigue tests. After 1.5 hours of ischemia, the contractile force in the hypothermic group was about three times as great as that observed in the room-temperature group. These results indicated that muscle function after a period of totally isolated ischemia is protected by hypothermic preservation. They also support the advisability of storage of amputated parts and free muscle flaps in hypothermic environments before replantation even after relatively brief intervals of ischemia.     

Immobilization effects on contractile properties of aging rat skeletal muscle

Recent advances in miniaturization have provided clinicians with hearing aids that can be comfortably inserted as far as the bony portion of the ear canal. It is possible to take advantage of these deeply inserted hearing aids in new ways. For example, the physical vibrations of microphone and receiver components may be used to improve hearing aid gain through bone conduction. Three cases are presented that will introduce this phenomenon for two transcranial CROS fittings and for one unilateral otosclerosis fitting. In each case, functional gain measurements under headphones were obtained with the hearing aid receivers acoustically plugged. Considerable gain was still present. Potential benefits, ramifications, and side effects are discussed.     

Structure of skeletal muscle in heart transplant recipients

OBJECTIVES: This study sought to define the ultrastructural characteristics of skeletal muscle in heart transplant recipients (HTRs) in relation to exercise capacity compared with that in age-matched control subjects. BACKGROUND: Muscle structural features seem to play an important role in the limitation of exercise capacity of HTRs long after transplantation. METHODS: The structure of the vastus lateralis muscle was analyzed by ultrastructural morphometry in 16 HTRs and 20 healthy control subjects. Maximal oxygen consumption (peak Vo2) was determined by an incremental exercise test. RESULTS: Peak Vo2 was significantly lower (by 35%) in HTRs. Fiber size, volume density of mitochondria and intramyocellular lipid deposits were not significantly different between HTRs and control subjects. In contrast, the capillary density and the capillary/fiber ratio were both significantly reduced in HTRs (by 24% and 27%, respectively). CONCLUSIONS: A normal volume density of mitochondria and a reduced capillary network are the main characteristics of muscle ultrastructure in HTRs by 10 months after transplantation. The muscle structural abnormalities and reduced exercise capacity might be related to immunosuppressive therapy with cyclosporine and corticosteroids as well as deconditioning.     

Developmental expression of dystrophin, dystrophin-associated glycoproteins and other membrane cytoskeletal proteins in human skeletal and heart muscle

Dystrophin, utrophin and the dystrophin-associated glycoproteins, beta-dystroglycan and adhalin, were analyzed, together with the membrane cytoskeletal proteins beta-spectrin, vinculin and talin, and adult and fetal myosin heavy chains, in 25 normal human fetuses from 8 to 24 weeks of gestation. Dystrophin was present in heart and skeletal muscle from 8 weeks although in the latter was mainly in the cytoplasm at this stage. Utrophin expression increased until around gestational weeks 19/21, but by 24 weeks immunostaining and immunoblot band intensities had reduced. Beta-dystroglycan was scarce in skeletal muscle at 8 weeks, increased with maturation and was more abundant in heart of the same age. Adhalin appeared later than beta-dystroglycan on skeletal muscle fiber surfaces, positivity became more intense as the fibers matured. In heart adhalin was detectable only in groups of cells at 12-16 weeks. From 8 weeks all fetal myotubes expressed beta-spectrin on their surfaces, while vinculin and talin positivity was mainly at the periphery of the fascicles, increasing with age. Adult slow myosin was seen in most myotubes at 10 weeks. Secondary myotubes then formed which increasingly expressed adult fast myosin, while still retaining fetal myosin. By 24 weeks most fibers expressing adult slow myosin had lost fetal myosin and were more mature in the expression of most membrane proteins. Muscle membrane organization during human fetal development is a complex process and takes place earlier in heart than skeletal muscle.     

Correlation between contractile strength and myosin heavy chain isoform composition in human skeletal muscle

PURPOSE: The purpose of this study was to investigate the relationship between myosin heavy chain (MHC) composition and maximal contraction strength of the human quadriceps femoris muscle. METHODS: Muscle biopsies were obtained from m. vastus lateralis in your highly physical active males (N = 7). The MHC composition of muscle homogenates was determined by electrophoresis techniques (SDS-PAGE). Isokinetic peak torque and constant-angle torque (50 degrees knee flexion) were obtained during slow (30 degrees.s-1), medium (120 degrees.s-1), and fast (240 degrees.s-1) maximal concentric and eccentric quadriceps contractions and expressed relative to muscle volume. RESULTS: The percentage of MHC II in the quadriceps muscle was positively correlated (rs = 0.61-0.93; P < 0.05-0.01) to maximal concentric quadriceps strength obtained at medium to high knee angular velocity. In contrast, no consistent pattern of correlation was observed for maximal eccentric quadriceps strength. CONCLUSIONS: The relationship observed between muscular MHC composition and maximal contractile strength is suggested to appear as a consequence of MHC -related differences in contractile force-velocity characteristics and/or contractile Rate of Force Development (RFD).     

A reperfusion interval reduces the contractile deficit in skeletal muscle following tourniquet ischemia

Bloodless surgical procedures on the extremities are achieved by application of a pneumatic tourniquet. The ischemia produced has deleterious effects on nerve and muscle function. It has been suggested that temporary interruption of ischemia by a reperfusion interval can prevent muscle and nerve injury. We investigated the muscle and nerve response to 3 hours of tourniquet ischemia, with and without a reperfusion interval after the first 2 hours of application, in a rodent model. Morphometric, contractile, and histologic parameters were measured. Tourniquet ischemia, with and without a reperfusion interval, results in muscle injury and a transient depression of muscle function. Introduction of a reperfusion interval reduces the severity of injury and increases the early rate of recovery. However, the later stages of recovery appear to be unaffected by reperfusion.     

Myosin heavy chain isoforms and dynamic contractile properties: skeletal versus smooth muscle

Torsades de pointes is a potentially life-threatening form of polymorphic ventricular tachyarrhythmia typically seen in the presence of repolarization-prolonging agents. We investigated this particular form of tachyarrhythmia in the isolated, perfused rabbit heart. The experimental model was designed to reproduce conditions that are clinically known to be associated with an increased propensity to the development of torsades de pointes. The class III agent clofilium (1 microM) and d,l-sotalol (10 microM), as well as the antibiotic erythromycin (30-150 microM) were infused in the presence of either normal (5.88 mM) or low (1.5 mM) potassium concentration in sinus-driven or atrioventricular (AV)-blocked hearts. Ventricular tachyarrhythmias spontaneously emerged in the clofilium-, d,l-sotalol-, and erythromycin-treated AV-blocked hearts. The episodes showed typical features of torsades de pointes found in humans. They developed within 4-12 min after the onset of infusion, were normally nonsustained, and only rarely degenerated into ventricular fibrillation. Electrical stimulation at cycle lengths <600 ms and perfusion with MgSO4 suppressed arrhythmic activity. In the d,l-sotalol- and erythromycin-treated hearts, torsades de pointes occurred only in the presence of hypokalemia and bradycardia, whereas, in the presence of clofilium, bradycardia alone caused torsades de pointes. Monophasic action-potential recordings demonstrated early afterdepolarizations in endocardial and epicardial recordings. Thus the isolated AV-blocked rabbit heart represents a model for studying drug-related torsades de pointes and its mechanism.     

Effects of beta 2-agonist administration and exercise on contractile activation of skeletal muscle fibers

Clenbuterol, a beta 2-adrenoceptor agonist, has therapeutic potential for the treatment of muscle-wasting diseases, yet its effects, especially at the single-fiber level, have not been fully characterized. Male C57BL/10 mice were allocated to three groups: Control-Treated mice were administered clenbuterol (2 mg.kg-1. day-1) via their drinking water for 15 wk; Trained-Treated mice underwent low-intensity training (unweighted swimming, 5 days/wk, 1 h/day) in addition to receiving clenbuterol; and Control mice were sedentary and untreated. Contractile characteristics were determined on membrane-permeabilized fibers from the extensor digitorum longus (EDL) and soleus muscles. Fast fibers from the EDL and soleus muscles of Treated mice exhibited decreases in Ca2+ sensitivity. Endurance exercise offset clenbuterol's effects, demonstrated by similar Ca2+ sensitivities in the Trained-Treated and Control groups. Long-term clenbuterol treatment did not affect the normalized maximal tension of fast or slow fibers but increased the proportion of fast fibers in the soleus muscle. Training increased the proportion of fibers with high and intermediate succinate dehydrogenase activity in the EDL and soleus muscles, respectively. If clenbuterol is to be used for treating muscle-wasting disorders, some form of low-intensity exercise might be encouraged such that potentially deleterious slow-to-fast fiber type transformations are minimized. Indeed, in the mouse, low-intensity exercise appears to prevent these effects.     

Phosphorylation of myosin regulatory light chain eliminates force-dependent changes in relaxation rates in skeletal muscle

The rate of relaxation from steady-state force in rabbit psoas fiber bundles was examined before and after phosphorylation of myosin regulatory light chain (RLC). Relaxation was initiated using diazo-2, a photolabile Ca2+ chelator that has low Ca2+ binding affinity (K(Ca) = 4.5 x 10(5) M(-1)) before photolysis and high affinity (K(Ca) = 1.3 x 10(7) M(-1)) after photolysis. Before phosphorylating RLC, the half-times for relaxation initiated from 0.27 +/- 0.02, 0.51 +/- 0.03, and 0.61 +/- 0.03 Po were 90 +/- 6, 140 +/- 6, and 182 +/- 9 ms, respectively. After phosphorylation of RLC, the half-times for relaxation from 0.36 +/- 0.03 Po, 0.59 +/- 0.03 Po, and 0.65 +/- 0.02 Po were 197 +/- 35 ms, 184 +/- 35 ms, and 179 +/- 22 ms. This slowing of relaxation rates from steady-state forces less than 0.50 Po was also observed when bundles of fibers were bathed with N-ethylmaleimide-modified myosin S-1, a strongly binding cross-bridge derivative of S1. These results suggest that phosphorylation of RLC slows relaxation, most likely by slowing the apparent rate of transition of cross-bridges from strongly bound (force-generating) to weakly bound (non-force-generating) states, and reduces or eliminates Ca2+ and cross-bridge activation-dependent changes in relaxation rates.     

Aorta and skeletal muscle NO synthase expression in experimental heart failure

Nitric oxide (NO), the free radical that accounts for the biological activity of endothelium-derived relaxing factor, is synthesized from L-arginine by NO synthase (NOS). There is evidence that NO availability is reduced in the peripheral vasculature of patients with congestive heart failure (CHF). The aim of this study was to investigate the expression of NOS in the descending aorta and in the skeletal muscles of rats subjected to heart failure. The alkaloid, monocrotaline, was used to induce pulmonary hypertension and cardiac failure in rats. The expression of both the constitutive (ecNOS) and the inducible (iNOS) isoforms of the enzyme was assessed by Western blot analysis. In CHF animals, the ecNOS location in the aorta is altered: the endothelial protein expression is substantially reduced (from 0.083 +/- 0.012 to 0.003 +/- 0.004 OD/microgram total proteins, P < 0.001) whereas the expression of ecNOS in the smooth muscle is increased (from 0.024 +/- 0.004 to 0.059 +/- 0.009 OD/ microgram total proteins, P < 0.01). The total aortic ecNOS is diminished in CHF respect to control animals (0.062 +/- 0.009 v 0.107 +/- 0.013 OD/microgram total proteins, P < 0.01). On the contrary, no difference in ecNOS protein expression was observed in the extensor digitorum longus and soleus muscles. Furthermore, iNOS was not detected in any of the tissues considered. In conclusion, experimental CHF causes a re-setting of the ecNOS protein expression in the descending aorta but not in skeletal muscles. The reduced abundance of ecNOS in the aortic endothelium is consistent with the impairment of the vasodilating function reported in patients with CHF.     

Apoptosis of skeletal muscle myofibers and interstitial cells in experimental heart failure

Congestive heart failure (CHF) is characterized by a limb skeletal muscle myopathy with shift from the slow aerobic, fatigue resistant fibers, to the fast, anaerobic ones, and muscle bulk loss. Apoptosis (A) has been recently demonstrated to play a role in several cardiovascular diseases. AIM OF THE STUDY: we have investigated the role of A in the skeletal muscle of the hindlimbs in an experimental model of CHF. ANIMALS AND METHODS: CHF was induced in 7 males 80-100 g Sprague-Dawley rats with 30 mg/kg monocrotaline. Five age and diet matched controls were also studied. The time course of A was also studied in additional animals at day 0, 17, 24 and 30 days. RESULTS: At day 27 the electrophoretic analysis of myosin heavy chains (MHCs) demonstrated in the CHF rats the occurrence of a myopathy, with disappearance of slow MHC1 in the Tibialis Anterior (TA), and a significant shift from the slow to the fast isoforms in the soleus and EDL. With in situ DNA nick-end labelling (TUNEL) we found in the TA of CHF animals a significantly higher number of TUNEL positive nuclei (0.43 +/- 0.24 v 0.08 +/- 0.02, P<0.02 and TUNEL positive myonuclei (0.031 +/- 0.012 v 0.0025 +/- 0.005, P<0.02). The time course of A showed a progressive rise in interstitial and myocyte A, accompanied by a drop in fibers cross-sectional area and muscle weight/body weight, that came out to be significant at 30 days. Western blot showed a lower expression of Bcl-2 at 27 days and a further drop at 30 days in the CHF rats. Double staining for TUNEL and antibody against anti-MHC2a and anti MHC2b + 2x showed that A occurs non-selectively in all the myofiber types. BetaANP and Right Ventricle Mass/Volume (RVM/V) correlated significantly with total apoptotic nuclei. CONCLUSIONS: In CHF myofibers A can lead to muscle atrophy. Endothelial cells A may produce an imbalance in myofibres nutrition with relative ischemia that triggers the preferential synthesis of fast anaerobic myosin as an adaptive mechanism or alternatively induce myofibres death.     

Chronic administration of taurine to aged rats improves the electrical and contractile properties of skeletal muscle fibers

Fimbriae or pili are essential adherence factors usually found in pathogenic bacteria to aid colonization of host cells. Three major structural pilin genes, fimA, sfaA, and papA, from Escherichia coli natural isolates were examined and nucleotide sequence data revealed elevated levels of both synonymous and nonsynonymous site variation at these loci. Examination of synonymous site variation shows a fivefold increase in fimA sites, relative to the housekeeping gene mdh; and similarly the sfaA and papA genes have increased synonymous sites variation relative to fimA. Nonsynonymous site variation is also elevated at all three loci but, in particular, at the papA locus (kN = 0.44). The kN/kS ratio for the three genes are among the highest yet reported for E. coli genes. Regional variation in nucleotide polymorphism within each of the genes reveal hypervariable segments where nonsynonymous substitutions exceed synonymous substitutions. We propose that at the fimA, papA, and sfaA genes, diversifying selection has brought about the increase levels of polymorphism.     

Effects of early severe malnutrition on heart and skeletal muscle lactate dehydrogenase

Following weaning, rats from litter sizes of 15 to 20 were subjected to severe food restriction for 10 weeks, and compared to rats from litter size 5 fed ad libitum. Percent M (muscle type) lactate dehydrogenase, M and H (heart type) subunit activity and lactate dehydrogenase activity (per g wet weight) determined both electrophoretically and spectrophotometrically were all significantly lower in separated left and right ventricles of the malnourished rats. There were no differences in skeletal muscle lactate dehydrogenase activity. Following 5 weeks of ad libitum feeding, the previously malnourished rats showed large increases in body weight. Now only the right ventricle showed slight decreases in H subunit and lactate dehydrogenase activity; other measurements in the right ventricle and all in the left ventricle had returned to control levels. Skeletal muscle lactate dehydrogenase activity was not different from controls.     

Direct and indirect assessment of skeletal muscle blood flow in patients with congestive heart failure

Techniques which are currently used to measure skeletal muscle blood flow (SMBF) in patients with congestive heart failure (CHF) are neither convenient nor accurate. They have led to discrepant results in patients with congestive heart failure and are, in part, responsible for the ongoing debate regarding the factors which limit the rise in body oxygen consumption during exercise in these patients. However, direct measurement of SMBF may not be needed during exercise in patients with severe CHF. Their skeletal muscles maximally extract oxygen. Consequently, increase in oxygen consumption by the skeletal muscles is only mediated by a concomitant increase in SMBF. In patients with severe CHF, peak body oxygen consumption attained during maximal exercise closely depends on the rise in SMBF, and thus provides an indirect measurement of SMBF.     

Altered expression of myosin heavy chain in human skeletal muscle in chronic heart failure

To explore further alterations in skeletal muscle in chronic heart failure (CHF), we examined myosin heavy chain (MHC) isoforms from biopsies of the vastus lateralis in nine male patients with class II-III (CHF) (left ventricular ejection fraction (LVEF) 26 +/- 11%, peak oxygen consumption (peak VO2) 12.6 +/- 2 mL.kg-1.min-1) and nine age-matched sedentary normal males (NL). The relative content of MHC isoforms I, IIa, and IIx was determined by gel electrophoresis as follows: The normal sedentary group (NL) had a higher percent of MHC type I when compared with the patients (NL 48.4 +/- 7% vs CHF patients 24 +/- 21.6%, P < 0.05, no difference between MCH IIa (NL 45.1 +/- 10.5% vs CHF 56.0 +/- 12.5%), and CHF patients had a higher relative content of MHC type IIx than did the normal group (NL 6.5 +/- 9.6% vs CHF 20.0 +/- 12.9%, P < 0.05. Three of nine patients had no detectable MHC type I. In patients relative expression of MHC type I (%) was related to peak VO2 (r = 0.70, P < 0.05). Our results indicate that major alterations in MHC isoform expression are present in skeletal muscle in CHF. These alterations parallel previously reported changes in fiber typing that may affect contractile function i skeletal muscle and possibly exercise performance. The absence of MHC type I in some CHF patients suggests that skeletal muscle changes in this disorder are not solely a result of deconditioning, buy may reflect a specific skeletal muscle myopathy in this disorder.     

Phosphorylation and IGF-1-mediated dephosphorylation pathways control the activity and the pharmacological properties of skeletal muscle chloride channels

1. In the present study we tested the hypothesis that insulin-like growth factor-1 (IGF-1) modulates resting chloride conductance (G(Cl)) of rat skeletal muscle by activating a phosphatase and that the chloride channel, based on the activity of phosphorylating-dephosphorylating pathways, has different sensitivity to specific ligands, such as the enantiomers of 2-(p-chlorophenoxy) propionic acid (CPP). 2. For this purpose G(Cl) in EDL muscle isolated from adult rat was first lowered by treatment with 5 nM 4-beta-phorbol 12,13 dibutyrate (4-beta-PDB), presumably activating protein kinase C (PKC). The effects of IGF-1 and of the enantiomers of CPP on G(Cl) were then tested. 3. IGF-1 (3.3 nM) had no effect of G(Cl) on EDL muscle fibres in normal physiological solution, whereas it completely counteracted the 30% decrease of G(Cl) induced by 4-beta-PDB. No effects of IGF-1 were observed on G(Cl) lowered by the phosphatase inhibitor okadaic acid (0.25 microM). 4. Ceramide, reported to activate on okadaic acid-sensitive phosphatase, mimicked the effects of IGF-1. In fact, N-acetyl-sphingosine (2.5-5 microM), not very effective in control conditions, increased the G(Cl) lowered by the phorbol ester, but not the G(Cl) lowered by okadaic acid. 5. In the presence of 4-beta-PDB, G(Cl) was differently affected by the enantiomers of CPP. The S(-)-CPP was remarkably less potent in producing the concentration-dependent reduction of G(Cl), whereas the R(+)-CPP caused an increase of G(Cl) at all the concentrations tested. 6. In conclusion, the PKC-induced lowering of G(Cl) is counteracted by IGF-1 through an okadaic acid sensitive phosphatase, and this effect can have therapeutic relevance in situations characterized by excessive channel phosphorylation. In turn the phosphorylation state of the channel can modulate the effects and the therapeutic potential of direct channel ligands.     

Clotrimazole, an antimycotic drug, inhibits the sarcoplasmic reticulum calcium pump and contractile function in heart muscle

Clotrimazole (CLT), an antimycotic drug, has been shown to inhibit proliferation of normal and cancer cell lines and its systemic use as a new tool in the treatment of proliferative disorders is presently under scrutiny (Benzaquen, L. R., Brugnara, C., Byers, H. R., Gattoni-Celli, S., and Halperin, J. A. (1995) Nature Med. 1, 534-540). The action of CLT is thought to involve depletion of intracellular Ca2+ stores but the underlying mechanism has not been defined. The present study utilized membrane vesicles of rabbit cardiac sarcoplasmic reticulum (SR) to determine the mechanism by which CLT depletes intracellular Ca2+ stores. The results revealed a strong, concentration-dependent inhibitory action of CLT on the ATP-energized Ca2+ uptake activity of SR (50% inhibition with approximately 35 microM CLT). The inhibition was of rapid onset (manifested in <15 s), and was accompanied by a 7-fold decrease in the apparent affinity of the SR Ca2+-ATPase for Ca2+ and a minor decrement in the enzyme's apparent affinity toward ATP. Exposure of SR to CLT in the absence or presence of Ca2+ resulted in irreversible inhibition of Ca2+ uptake demonstrating that the Ca2+-bound and Ca2+-free conformations of the Ca2+-ATPase are CLT-sensitive. Introduction of CLT to the reaction medium subsequent to induction of enzyme turnover with Ca2+ and ATP resulted in instantaneous cessation of Ca2+ transport indicating that an intermediate enzyme species generated during turnover undergoes rapid inactivation by CLT. The inhibition of Ca2+ uptake by CLT was accompanied by inhibition of Ca2+-stimulated ATP hydrolysis and Ca2+-induced phosphoenzyme intermediate formation from ATP in the ATPase catalytic cycle. Phosphorylation of the Ca2+-deprived enzyme with Pi in the reverse direction of catalytic cycle and Ca2+ release from Ca2+-preloaded SR vesicles were unaffected by CLT. It is concluded that CLT depletes intracellular Ca2+ stores by inhibiting Ca2+ sequestration by the Ca2+-ATPase. The mechanism of ATPase inhibition involves a drug-induced alteration in the Ca2+-binding site(s) resulting in paralysis of the enzyme's catalytic and ion transport cycle. CLT (50 microM) caused marked depression of contractile function in isolated perfused, electrically paced rabbit heart preparations. The contractile function recovered gradually following withdrawal of CLT from the perfusate indicating the existence of mechanisms in the intact cell to inactivate, metabolize, or clear CLT from its target site.