Depolarization accelerates the decay of K+ contractures in rat skeletal muscle fibers

The experiments examine the effects of membrane potential on the time course of K⁺ contractures in small bundles of rat soleus and extensor digitorum longus (EDL) muscle fibers. Control K⁺ contractures were induced by exposure to a 200 mmol/L K⁺ solution in polarized fibers with a resting membrane potential of −83 mV (3.5 mmol/L K⁺), while test contractures were evoked with 200 mmol/L K⁺ from −46 mV, after 5, 10, and 30 min in a 30 mmol/L K⁺ conditioning solution. The decay times of the test K⁺ contracture in depolarized fibers were faster than those of the control K⁺ contracture in both soleus and EDL. A maximum reduction of 60% in the time for the contracture to decay from 90% to 10% was seen in soleus fibers after depolarizations lasting 10 min, while a reduction of 45% was seen in the decay time of EDL fibers after a 5-min depolarizations. The amplitudes of the test contractures were 30% less than control after 5-min and 10-min depolarization and 50% less than control after 30 min. Analysis of the results suggests that the kinetics of excitation-contraction coupling may be altered in damaged muscle fibers. © 1996 John Wiley & Sons, Inc.     

Peripheral mechanisms of fatigue in muscles of normal and dystrophic mice.

We evaluated the contribution of different processes to fatigue of normal and dystrophic mouse muscles using an in vitro electromyography chamber. Fatigue was induced by repetitive nerve stimulation at 30 Hz for 0.5 s, every 2.5 s until tension decreased by about 50%. We monitored the compound nerve action potential (AP), compound muscle AP, and isometric tension responses to nerve stimulation, and compound muscle AP and tension responses to direct muscle stimulation. In normal mice, about 50% reduction in nerve-evoked tension occurred by 2.4 min in extensor digitorum longus (EDL), 4.8 min in diaphragm, and 9 min in soleus. Analysis of the responses revealed that the fatigue was caused by failure of more than one process in all muscles, and failure of nerve conduction did not contribute to fatigue in any muscle. Failure of neuromuscular transmission, muscle membrane excitation, and excitation-contraction (E-C) coupling and contractility accounted for 55, 45, and 0%, respectively, of the fatigue in EDL, for 21, 74, and 5% of the fatigue in diaphragm, and for 2, 54, and 44% of the fatigue in soleus. In dystrophic mice, while about 50% reduction in nerve-evoked tension occurred by 8.1 min in EDL and 5.6 min in diaphragm, only 29% reduction in tension occurred by 80 min in soleus. Failure of neuromuscular transmission, muscle membrane excitation, E-C coupling and contractility accounted for 22, 63 and 15% of the fatigue in EDL, for 21, 79, and 0% of the fatigue in diaphragm, and for 15, 59, and 26% of the fatigue in soleus. The proportion of slow-twitch oxidative fibers was more than normal in dystrophic EDL, but the same as normal in dystrophic diaphragm and soleus. The slower onset of fatigue was attributable to lesser failure of neuromuscular transmission in dystrophic EDL, and to lesser failure of E-C coupling and contractility in dystrophic soleus.     

Responses of intercostal muscle biopsies from normal subjects and patients with myasthenia gravis

In order to evaluate the mechanisms of weakness in muscles of patients with myasthenia gravis (MG), intercostal muscle biopsies were obtained from 9 normal subjects and 6 MG patients, and the compound muscle action potential (AP) and tension responses to nerve and muscle stimulation, and contracture responses on exposure to caffeine, were monitored in vitro. In normal muscle, on stimulation of the nerve or muscle at 30 to 100 Hz, the AP responses showed decrement in amplitude, one-third of which was attributable to failure of neuromuscular transmission and two-thirds to failure of muscle membrane excitation. On stimulation at 1 to 5 Hz, the AP responses showed very little decrement, while the contractile responses showed significant fade in tension, due to failure of E-C coupling or contractility. In muscle from patients with generalized MG, stimulation of the nerve at all frequencies (1 to 100 Hz) caused much greater decrement in APs and fade in tension responses than in normal muscle, due mainly to failure of neuromuscular transmission. However, at 100 Hz, 40% of the decrement in APs was due to failure of muscle membrane excitation, and at 1 to 5 Hz, 40% of the fade in tension was due to failure of E-C coupling or contractility, as in normal muscle. On direct stimulation the contraction and half-relaxation times were slower and the tetanic tension was smaller than in normal muscle, especially in the MG patient with thymoma. Caffeine-induced contractures were smaller in MG muscle than in normal muscle. These results indicate that while the weakness of MG muscle is due mainly to failure of neuromuscular transmission, it is also partly due to reduced E-C coupling or contractility.     

Mechanisms of fatigue in normal intercostal muscle and muscle from patients with myasthenia gravis

Fatigue mechanisms in normal intercostal muscle and muscle from patients with myasthenia gravis (MG) were evaluated by monitoring the compound muscle action potential (CMAP) and tetanic tension responses to repetitive nerve or muscle stimulation in vitro. When fatigue was induced by nerve stimulation at 30 Hz for 0.5 s every 2.5 s, about half of the original tension decreased after 30 min in normal muscle and 5 min in MG muscle. Analysis of the changes in area of CMAPs and tension indicated that impairment of neuromuscular transmission, muscle membrane excitation, and excitation-contraction (E-C) coupling and contractility accounted for 40%, 29%, and 31% of fatigue in normal muscle, and 83%, 0%, and 17% of fatigue in MG muscle. When fatigue was induced by muscle stimulation at 30 Hz, tension declined by a quarter after 30 min in normal muscle, but by a half after 17 min in MG muscle. Impairment of muscle membrane excitation and E-C coupling and contractility accounted for 58% and 42% of fatigue in normal muscle, and 22% and 78% of fatigue in MG muscle. Thus, fatigue of normal muscle is caused by impairment of at least four processes, and enhanced fatigue of MG muscle is caused by greater impairment of neuromuscular transmission, E-C coupling, and contractility. © 1993 John Wiley & Sons, Inc.     

Failure of neuromuscular transmission and contractility during muscle fatigue

In previous studies of muscle fatigue, tension was monitored from whole muscle, while action potentials were recorded from a few muscle fibers. To compare more accurately changes in these responses, an in vitro fluid electrode technique was employed to record the action potential of whole muscle simultaneously with tension during fatigue induced by nerve stimulation in the rat extensor digitorum longus (EDL), soleus, and diaphragm muscles. In each muscle, tension declined from the start of stimulation, while action potential amplitude initially increased slightly and then declined most rapidly in EDL, more slowly in diaphragm, and most slowly in soleus. Direct stimulation of the fatigued muscle produced the greatest increase in tension in EDL, next in diaphragm, and least in soleus. These results indicate that while failure of excitation-contraction coupling or of the contractile mechanism is the initial cause of fatigue in all the muscles studied, and remains the predominant cause throughout in the soleus muscle, failure of neuromuscular transmission plays an important role in fatigue after the first 15 seconds in EDL, and to a lesser extent, after the first 90 seconds in diaphragm.     

High-frequency fatigue in rat skeletal muscle: Role of extracellular ion concentrations

High-frequency fatigue (HFF), the decline of force during continuous tetanic stimulation (lasting 4–40 s), was studied in isolated bundles of rat skeletal muscle fibers. HFF was slower in slow-twitch soleus fibers than in fast-twitch red or white sternomastoid fibers; denervation accelerated fatigue in soleus. Maximal 200-mmol/L potassium contractures of normal amplitude were induced in fatigued fibers, suggesting that crossbridge cycling and the voltage activation of excitation–contraction coupling could still occur maximally, but that activation by action potentials was impaired. An increase in [Na⁺]_o slowed HFF, while a small increase in [K⁺]_o or reduction in [Cl^?]_o accelerated HFF. Increasing the tetanic stimulation frequency exacerbated fatigue. Recovery from HFF proceeded rapidly since force increased markedly within a few seconds when stimulation ceased. These results support the hypothesis that a redistribution of Na⁺, K⁺, and Cl^? across the transverse tubular membranes during repeated action potential activity induces fatigue by reducing the amplitude and conduction of action potentials. © 1995 John Wiley & Sons, Inc.     

β-Adrenergic potentiation of E–C coupling increases force in rat skeletal muscle

We examined the mechanism(s) which allow terbutaline, a β₂-adrenergic agonist, to increase isometric force in bundles of normal and denervated rat soleus fibers. Terbutaline (10 μmol/L) potentiated tetanic contractions during exposure to 1 mmol/L ouabain, 10 μmol/L nifedipine, or 0.5 mmol/L iodoacetate. Terbutafine induced equivalent increases in submaximal potassium (K⁺) contracture and tetanic force: these effects were mimicked by 2 mmol/L dibutyrl-cyclic AMP. Therefore, terbutaline increased force by a cyclic AMP-dependent mechanism other than enhancement of sodium-pump activity, dihydropyridine sensitive Ca²⁺ currents, glycolysis, or action potentials. Pretreatment with 1 mmol/L caffeine induced submaximal potentiation of peak tetanic force but prevented further potentiation by terbutaline. This suggested that terbutaline did not influence the myofilaments, but acted on the sarcoplasmic reticulum (SR) to increase the myoplasmic Ca²⁺ concentration and hence force production. We speculate that force is potentiated following β-adrenoceptor activation by a cyclic AMP-dependent phosphorylation of Ca²⁺ release channels to facilitate SR calcium release during tetanic stimulation. © 1993 John Wiley & Sons, Inc.     

The in vitro determination of susceptibility to malignant hyperthermia

To evaluate the reliability of the in vitro contracture test for susceptibility to malignant hyperthermia, we studied muscles from normal pigs and those susceptible to malignant hyperthermia. We performed the contracture test with various muscles from the same animal. Trapezius and intercostal muscles gave similar results, whereas the extensor digiti II muscle had lower sensitivities to both caffeine and halothane. Thus, the muscle chosen to determine susceptibility to malignant hyperthermia is important. In several animals, a false negative diagnosis would have resulted if only the distal muscle had been studied, and this was true even if weak contractures (less than 200 mg) were considered significant. In addition, we compared the response to caffeine or halothane of cut and intact muscle fibers. Although the cut fibers were depolarized, the sensitivity to these drugs was unchanged. Hence, results of the in vitro contracture test are independent of the resting membrane potential.     

Metabolic and nonmetabolic components of fatigue monitored with 31P-NMR

The goal of this study was to determine the roles of metabolic and nonmetabolic factors in muscle fatigue. Rat gastrocnemius muscles were fatigued by stimulation of the nerve (n = 6) or muscle (n = 4, after 2 days of denervation). ³¹Phosphorus nuclear magnetic resonance spectroscopy was used to measure levels of intracellular inorganic phosphate (Pi) and hydrogen ions (H⁺) (which are thought to inhibit contraction) and the high-energy phosphates, phosphocreatine (PCr), and ATP. For both indirect and direct stimulation, with fatigue to ≈60% initial tetanic force, [Pi] increased from ≈3.5 mmol/L to ≈20 mmol/L and [PCr] decreased from ≈27 mmol/L to ≈9 mmol/L. However, with continued fatigue to 25–35% initial tetanic force, neither [Pi] or [PCr] changed further. [ATP] and pH changed only slightly during fatigue. The results are consistent with early fatigue arising from metabolic inhibition of contraction; but later fatigue arising independent of metabolites, due to impaired activation beyond the neuromuscular junction. © 1994 John Wiley & Sons, Inc.     

Aging impairs regulation of ryanodine receptors from extensor digitorum longus but not soleus muscles

ABSTRACT: Introduction: Because impaired excitation‐contraction coupling and reduced sarcoplasmic reticulum (SR) Ca²⁺ release may contribute to the age‐associated decline in skeletal muscle strength, we investigated the effect of aging on regulation of the skeletal muscle isoform of the ryanodine receptor (RyR1) by physiological channel ligands. Methods: [³H]Ryanodine binding to membranes from 8‐ and 26‐month‐old Fischer 344 extensor digitorum longus (EDL) and soleus muscles was used to investigate the effects of age on RyR1 modulation by Ca²⁺ and calmodulin (CaM). Results: Aging reduced maximal Ca²⁺‐stimulated binding to EDL membranes. In 0.3 μM Ca²⁺, age reduced binding and CaM increased binding to EDL membranes. In 300 μM Ca²⁺, CaM reduced binding, but the age effect was not significant. Aging did not affect Ca²⁺ or CaM regulation of soleus RyR1. Discussion: In aged fast‐twitch muscle, impaired RyR1 Ca²⁺ regulation may contribute to lower SR Ca²⁺ release and reduced muscle function. Muscle Nerve 57 : 1022–1025, 2018     

Observations on the efficiency of dystrophic muscle in vitro

A study of muscles of the dystrophic mouse has failed to substantiate earlier claims that these muscles were especially resistant to fatigue in vitro or that fast muscles are preferentially damaged. It has been found that the fast muscle selected for previous studies is very often unable to withstand isolation in an organ bath if it is working, and both the difficulty in removing the normal gastrocnemius muscle intact and the need to trim it surgically contribute independently toward its deterioration in vitro. The smaller dystrophic gastrocnemius muscle is less liable to excision damage, is able to satisfy its resting metabolic needs in nutrient solution, and requires no damaging dissection, but is nevertheless unable to recover normally from fatigue. Using EDL and soleus muscles which are small enough to withstand isolation in vitro, no differences are found between fatigue patterns of normal and dystrophic specimens. Responses to rest, KCl, and 2 mM caffeine are also quite similar, and the only distinguishing biomechanical characteristic we have found in dystrophic mouse muscle is a weaker contraction and a longer total twitch time.     

Staircase, fatigue, and caffeine in skeletal muscle in situ

The specific locus of impairment in excitation-contraction coupling that is associated with skeletal muscle fatigue has not been identified. In the present study the phenomena of staircase and fatigue were studied in the rat gastrocnemius muscle in situ, and the effect of caffeine (50 mg kg-1) given prior to or during 5 minutes of stimulation was observed. A 10 Hz indirect stimulation resulted in a staircase response that proceeded for 10.4 +/- 1.6 (mean +/- SD) seconds, reaching a peak force value that was 70-75% higher than the initial contraction. After 5 minutes of stimulation and 20 minutes of rest, the staircase response was longer (17 +/- 3.1 seconds) and proceeded more slowly when the stimulation regimen was repeated. Caffeine accelerated the fatigue and reversed the effect of fatigue on the staircase response. Since caffeine enhances the release of Ca2+ from terminal cisternae, it is postulated that the accelerated fatigue in the presence of caffeine is indicative of a reduced availability of Ca2+ for release. This hypothesis would also explain the slower progression of staircase in the fatigued muscle.     

Effect of muscle creatine content manipulation on contractile properties in mouse muscles

The effects of muscle creatine manipulation on contractile properties in oxidative and glycolytic muscles were evaluated. Whereas control mice (NMRi; n = 12) received normal chow (5 g daily), three experimental groups were created by adding creatine monohydrate (CR group; 5%, 1 week; n = 13); beta-guanidinoproprionic acid, an inhibitor of cellular creatine uptake (beta-GPA group; 1%, 2 weeks; n = 12); or CR following beta-GPA (beta-GPA+CR group; n = 11). Total creatine (TCr) and the contractile properties of incubated soleus and extensor digitorum longus (EDL) muscles were determined. For the soleus, compared with control, TCr increased in the CR group (+25%), decreased in beta-GPA group (-50%), and remained stable in the beta-GPA+CR group, whereas, for the EDL, TCr was similar in the CR, and lower in the beta-GPA (-40%) and beta-GPA+CR (-15%) groups. None of the experimental groups (CR, beta-GPA, or beta-GPA+CR) showed changes in peak tension (P(peak)), time to peak tension, or relaxation in soleus or EDL during twitch or tetanic stimulation. For the soleus, fatigue reduced P(peak) to approximately 60% of initial P(peak); 5 min of recovery restored P(peak) to values approximately 15% higher in CR than in controls. P(peak) recovery was not affected by beta-GPA or beta-GPA+CR in the soleus or any treatment in the EDL. Thus, peak tension recovery is enhanced by creatine intake in oxidative but not glycolytic muscles. This may be implicated in the beneficial action of creatine loading.     

Specific impulse patterns regulate acetylcholinesterase activity in skeletal muscles of rats and rabbits

In rats, acetylcholinesterase (AChE) activity in the fast muscles is several times higher than in the slow soleus muscle. The hypothesis that specific neural impulse patterns in fast or slow muscles are responsible for different AChE activities was tested by altering the neural activation pattern in the fast extensor digitorum longus (EDL) muscle by chronic low-frequency stimulation of its nerve. In addition, the soleus muscle was examined after hind limb immobilization, which changed its neural activation pattern from tonic to phasic. Myosin heavy-chain (MHC) isoforms were analyzed by gel electrophoresis. Activity of the molecular forms of AChE was determined by velocity sedimentation. Low-frequency stimulation of the rat EDL for 35 days shifted the profile of MHC II isoforms toward a slower MHCIIa isoform. Activity of the globular G₁ and G₄ molecular forms of AChE decreased by a factor of 4 and 10, respectively, and became comparable with those in the soleus muscle. After hind limb immobilization, the fast MHCIId isoform, which is not normally present, appeared in the soleus muscle. Activity of the globular G₁ form of AChE increased approximately three times and approached the levels in the fast EDL muscle. In the rabbit, on the contrary to the rat, activity of the globular forms of AChE in a fast muscle increased after low-frequency stimulation. The results demonstrate that specific neural activation patterns regulate AChE activity in muscles. Great differences, however, exist among different mammalian species in regard to muscle AChE regulation. J. Neurosci. Res. 47:49–57, 1997. © 1997 Wiley-Liss, Inc.     

Postnatal development of nitric oxide synthase activity in fast and slow muscles of the rat

Nitric oxide synthase (NOS) activity was measured in extensor digitorum longus (EDL) and soleus muscles during postnatal development in the rat. At 1 and 2 weeks of age, similar low levels were found in both muscles. After 2 weeks, activity increased significantly only in EDL. Adult NOS activity was significantly higher in EDL than soleus. Thus, the preferential expression of NOS in fast muscle only occurs once the adult pattern of motor activity is established. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:1344–1346, 1998.     

Factors affecting muscle fiber transformation in cross. Reinnervated muscle

Nerves of two fast muscles [peroneus longus (PL) and extensor digitorum longus (EDL)], having different type 2 muscle fiber compositions, were used to cross-reinnervate the slow soleus muscle in the rat. Contraction characteristics, histochemical muscle fiber type compsotions and myosin heavy chain (MHC) isoform compositions were determined for the reinnervated muscles. Shortening velocity increased in soleus muscles crossreinnervated with EDL nerve [X-SOL(EDL)] but not in muscles cross-reinnervated with PL nerve [X-SOL(PL)]. Type 2A MHC isoform content was increased in X-SOL(EDL) but not in X-SOL(PL), where MHC isoform composition remained similar to normal soleus. The complement of type 1 (slow) muscle fibers was reduced and that of type 2 (fast) fibers increased in both types of X-SOL muscle, but this change was significantly greater in X-SOL(EDL); the majority of the type 2 fibers in X-SOL muscles were of type 2A. Results show that “the type 2 composition” of the reinnervating motoneuron pool is an important factor in determining the transformation of a target slow muscle after cross-reinnervation. © 1993 John Wiley & Sons, Inc.     

Recovery of rat skeletal muscles after partial denervation is enhanced by treatment with nifedipine

Following partial denervation of adult rat skeletal muscle intact axons sprout to reinnervate denervated muscle fibres and increase their territory. The extent of this increase is limited and may depend on the ability of axon terminals to form and maintain synaptic contacts with the denervated muscle fibres. Here we tested the possibility whether reducing Ca²⁺ entry into presynaptic nerve terminals through dihydropyridine sensitive channels may allow more nerve–muscle contacts to be formed and maintained. Hindlimb muscles of adult Wistar rats were partially denervated by removing a small segment of the L4 or L5 spinal nerve on one side. A nifedipine-containing silastic rubber strip was subsequently implanted close to the partially denervated soleus or extensor digitorum longus (EDL) muscles in some animals. In control experiments silastic strips which did not contain nifedipine were used. Several weeks later isometric contractions were recorded, to determine the effect of (a) partial denervation and (b) nifedipine treatment on force output and motor unit numbers. The tension produced by nifedipine treated partially denervated muscles was 82% and 79% of the unoperated contralateral value for soleus and EDL, respectively. This was significantly greater than in untreated muscles, which only produced 61% and 48%, respectively. Mean motor unit force was also significantly larger with nifedipine treatment. Histological analysis revealed that a significantly larger proportion of the total number of muscle fibres remained in nifedipine-treated partially denervated muscles (soleus, 90% and EDL, 101%) compared with untreated muscles (soleus, 51% and EDL, 66%). Thus the number of neuromuscular contacts was increased with nifedipine treatment.     

Skeletal muscle fatigue and physical endurance of young and old mice

In order to evaluate the role played by muscular and extramuscular factors in the development of fatigue in old age, the time course of fatigue in isolated skeletal muscles and spontaneous motor activity and endurance of whole animals were monitored using young (3–6 months) and old (34–36 months) CF57BL/6J mice. The isolated extensor digitorum longus (EDL) and soleus muscles from old mice had smaller (P < 0.05) mass and developed lower (P < 0.02) maximal tetanic tension at 100-Hz stimulation than the muscles of young mice. During stimulation at 30 Hz every 2.5 s, a 50% decline in original tetanic tension occurred by 109 s in young EDL and 129 s in old EDL, but by 482 s in young soleus and 1134 s (projected) in old soleus, indicating more (P < 0.05) resistance to fatigue in old than young soleus. However, the old mice showed significantly fewer (P < 0.002) spontaneous ambulatory movements than the young mice. On a treadmill with a belt speed of 10 m/min at an inclination of 0°, the old mice could only run for 22 min compared to 39 min ran by young mice (P < 0.02). They took more rest periods (P < 0.02) than the young mice. In a quantitative swimming monitor, the old mice swam for a shorter (P < 0.05) time than young mice (20.4 min compared to 28.6 min). Integrated swimming activity at 20 min was smaller (P < 0.05) in old mice than in young mice (413 g/s compared to 628 g/s). Hence increased fatigue in old age is not caused by impairment of processes within the muscles, but by impairment of central or extramuscular processes. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21: 1729–1739, 1998     

Effects of long-term administration of ambenonium chloride on motor end-plate fine structure and acetylcholine receptor in rat

Ambenonium chloride was administered orally in a dosage of 6 mg/kg/day to rats for 14–360 days.Motor end-plate fine structure and junctional AChR were quantitatively analyzed in red (soleus) and white (EDL) muscle fibers. In treated animals, degeneration and simplification of postsynaptic folds and widening of synaptic clefts were often observed in soleus end-plates, but infrequently in EDL end-plates. On the other hand, the postsynaptic AChR was reduced markedly in both soleus and EDL end-plates. No presynaptic changes were observed.These results show that long-term administration of Anti-ChE agents in myasthenia gravis may have an adverse effect on neuromuscular transmission.