Neural regulation of the structure of myosin

Mammalian skeletal muscle responds metabolically both to neural influences and to the demands of work. For example, the soleus exhibits increased speed of contraction when reinnervated by the peroneal nerve that normally supplies fast muscles, but this change can also be brought about merely by peroneal transection (a procedure which denervates the muscles antagonistic to soleus). We have investigated the effects of these two operative procedures on soleus myosin. Seven to eight months after reinnervation of rat soleus with peroneal nerve, the myosin Ca²⁺-ATPase had increased in specific activity, alkaline stability, and susceptibility to tryptic digestion. Tryptic digestion (25 C, 10 min, myosin: trypsin = 200:1) followed by SDS-gel electrophoresis produces seven distinct peptide bands in the case of normal soleus myosin. An additional peptide band (88,000 daltons) is characteristic of fast muscle myosin. This 88,000 dalton band is demonstrable in myosin prepared from cross-reinnervated soleus muscles. Qualitatively identical changes were produced in all of these properties of soleus myosin by transecting the peroneal nerves 7–8 months previously. However, the magnitudes of the changes were less than those occurring after cross-reinnervation. Cross-reinnervation experiments have been used as evidence for neurotrophic control of skeletal muscle myosin; however, the conversion of slow myosin to fast myosin when the peroneal nerve is merely transected renders this argument questionable. Since fast and slow muscle myosin heavy chains differ in primary or tertiary structure (or both), their changes after cross-reinnervation or after inactivation of antagonistic muscles must result from a qualitative transformation of myosin which is presumably accomplished by the regulation of myosin biosynthesis.     

Nimodipine Suppresses Preferential Reinnervation of Mouse Soleus Muscles by Slow α-Motoneurons

Denervation of mouse soleus muscle followed by self-reinnervation causes a significant increase in slow twitch (type I) muscle fiber content, suggesting preferential reinnervation by slow α-motoneurons. Since intracellular Ca²⁺influences both axonal elongation rate and branching, we examined the process of self-reinnervation in mouse soleus muscles in the presence of the L-type Ca²⁺channel blocker nimodipine. Soleus muscles in both control and experimental animals were denervated by crushing the soleus nerve where it enters the muscle. Experimental animals received a daily i.p. injection of a 0.1% nimodipine solution beginning 4 days prior to denervation and ending 2 weeks postdenervation. At 2 months postdenervation reinnervated and contralateral muscles from both control and experimental animals were sectioned and histochemically stained for myosin ATPase to determine the percentage of slow twitch fibers in the muscles. It was found that, in agreement with previous experiments, untreated reinnervated muscles had a significantly higher percentage of slow twitch fibers than did their contralateral controls (91.3 versus 74.6%). However, in nimodipine-treated animals only a small, but not statistically significant, difference between reinnervated and contralateral control muscles was observed (76.5 versus 72.8%). These results suggest that Ca²⁺influx through L-type calcium channels in growing neurites may play a role in the outcome of the reinnervation process.     

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     

Pattern Dependence in the Stimulation‐Induced Type Transformation of Rabbit Fast Skeletal Muscle

Little is known of the events that initiate the adaptive response of skeletal muscle to a sustained change in use. This study was designed to distinguish between the role of the electrical activity pattern and that of the resulting contractile force in driving different aspects of the response. A better understanding of these issues would lead to improved clinical protocols for functional electrical stimulation. Rabbit limb muscles were stimulated continuously for 12 weeks either at 2.5 Hz or with an equivalent optimized pattern producing peak forces three‐fold higher. The two patterns induced similar changes in shortening velocity, myosin isoforms, and fatigue resistance. They had markedly different effects on twitch dynamics and summation (“doublet effect”). This pointed to differences in activation that were not, however, attributable to sarcoplasmic reticulum Ca²⁺ transport ATPase activity. The optimized pattern maintained muscle bulk more effectively. We conclude that changes in myosin isoform composition and fatigue resistance are driven by aggregate impulse activity. Changes in Ca²⁺ transport and muscle bulk show a distinct pattern dependence.     

Myosin changes in experimental 2,4-dichlorophenoxyacetate myopathy

Young rats treated with 10 to 14 daily injections of 2,4-dichlorophenoxyacetate (2,4-D) developed a myopathy mainly involving fast muscles. Myosin isolated from the gastrocnemius muscles of treated and normal control animals differed in several respects. The Ca²⁺? and Mg²⁺ -mediated ATPases were higher in myopathic muscle myosin than in normals. Alkylation of thiols by N-ethylmaleimide (NEM) induced an increase of Ca²⁺ -activated ATPase that was higher in normal than in myopathic myosin. Trinitrophenylation of reactive amino groups by 2,4,6-trinitrobenzene sulfonate (TBS) induced an increase in Mg²⁺ -mediated ATPase in both preparations, but the increase was higher in normals. Although the heavy- and light-chain pattern was identical in normal and myopathic myosin, during storage at 0°C the relative amount of myopathic L₂ light chain decreased. Myosins fragmented either by limited proteolysis with trypsin and chymotrypsin or by specific cleavage at tryptophanyl and cysteinyl peptide bonds showed differences on sodium dodecylsulfate (SDS)-polyacrylamide-gel electrophoresis. The results indicate that there is a change in the heavy chains of myosin isolated from the gastrocnemius muscle in 2,4-D-induced rat myopathy.     

Malignant hyperthermia: Effects of sarcoplasmic reticulum Ca2+ pump inhibition

In porcine malignant hyperthermia-susceptible (MHS) skeletal muscles, calcium release is abnormal and resting calcium may be elevated. Thus MHS muscles may have prolonged twitch relaxation and lower fusion frequencies, which would be augmented by inhibition of sarcoplasmic reticulum (SR) Ca²⁺ adenosine triphosphatase (ATPase) activity; bundles of intact muscle cells from MHS and normal pigs were used to investigate this possibility. Cooling and low-frequency stimulation, in combination, enhanced twitch fusion and prolonged twitch relaxation significantly more in MHS than in normal muscles (e.g., 34 ± 4% versus 16 ± 4% fusion, and 82.4 ± 9.4 ms versus 43.2 ± 7.8 ms half-relaxation time, for MHS and normal muscles, respectively). Similarly, inhibition of the SR Ca²⁺ ATPase by cyclopiazonic acid resulted in significantly greater twitch fusion in MHS muscles. These results were consistent with predicted effects of enhanced SR Ca²⁺ release and/or elevated resting calcium in MHS muscles and indicate that cooling during a malignant hyperthermia crisis could actually increase the force of muscle contractures. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:361–366, 1998.     

Neural regulation of muscle activation

The effects of temperature and repetitive stimulation on isometric twitch contractions of normal, self-innervated and cross-innervated fast extensor digitorum longus and slow soleus muscles of the rat were studied in situ. Normal and self-innervated extensor digitorum longus showed a 2-fold increase in twitch tension following either a train of 200 stimuli at 35 C (post-tetanic potentiation) or lowering of temperature from 35 C to 20 C (cooling potentiation), while under these conditions the twitch tensions of normal and self-innervated slow soleus fell by about 15%. Post-tetanic and cooling potentiation were virtually abolished in cross-innervated extensor digitorum longus but appeared in cross-innervated soleus. The degree of potentiation in these and control muscles was inversely proportional to contraction time. These experiments suggest that mammalian motor nerves exert a controlling influence on the degree of activation of skeletal muscle during a twitch. An hypothesis of the molecular mechanism of post-titanic and cooling potentiation is proposed. According to this hypothesis, neural regulation of muscle activation results from the transformation of myosin after nerve cross-union.     

Stretch‐activated signaling is modulated by stretch magnitude and contraction

Introduction: Stretch therapy is commonly utilized to prevent shortening maladaptation of skeletal muscle. Stretch in combination with isometric contraction prevents shortening, but the signaling mechanisms are not understood. Methods: Using a soleus tenotomy + stretch rat model, the phosphorylation–activation of mechanosensitive kinases (Akt, p70^S6K, p38 MAPK, and ERK1/2) were measured for various stretch magnitudes, set relative to optimal soleus length (Lo). Results: The kinases were not activated by passive stretch until it exceeded the normal physiological range. Stretch + isometric contraction resulted in relatively strong phosphorylation, even at short lengths. Conclusions: Whereas passive stretch results in kinase phosphorylation only during extreme lengthening, isometric contraction generated pronounced phosphorylation of kinases at Lo and Lo + 25%, indicating stimulation of pathways that lead to the preservation or increase of muscle length. Understanding the effects of passive and active stretch with respect to Lo and contraction is essential for predicting therapeutic outcomes and influencing optimal muscle length. Muscle Nerve 49 : 98–107, 2014     

From excitation to intracellular Ca2+ movements in skeletal muscle: Basic aspects and related clinical disorders

In skeletal muscle fiber, excitation-contraction coupling corresponds to the sequence of events occurring from action potential firing to initiation of contraction by an increase in cytosolic Ca²⁺. These events are elicited in response to excitation of the motor neuron which induces trains of action potentials in the muscle cell that spread along the sarcolemma and in depth along the T-tubule membrane. Depolarization of the T-tubule membrane induces a conformational change in a protein complex, called the dihydropyridine receptor, which opens a calcium channel anchored in the membrane of the sarcoplasmic reticulum, called the ryanodine receptor, in charge of release of Ca²⁺ ions that activate contractile proteins. Ryanodine receptors shut upon return of the T-tubule membrane potential to its resting value and muscle cell relaxation results from the removal of cytosolic Ca²⁺ that is pumped back into the SR lumen through the sarcoplasmic reticulum Ca²⁺ ATPase. Mutations in genes encoding either plasma membrane ion channels, the main subunit of the dihydropyridine receptor, ryanodine receptor, sarcoplasmic reticulum Ca²⁺ ATPase or proteins interfering with trans-sarcolemmal Ca²⁺ influx or sarcoplasmic reticulum Ca²⁺ efflux lead to clinical disorders that manifest as myotonia, muscle weakness, paralysis or muscle wasting.     

Calmodulin content and Ca-activated protease activity in dystrophic hamster muscles

As part of a study on the implication of elevated Ca²⁺ levels in the myofibrillar degeneration seen in dystrophic muscle, the content of calmodulin and the activity of Ca²⁺ -activated neutral protease (CANP) have been measured in normal and dystrophic (UM-X7.1) hamsters. Calmodulin levels, expressed as micrograms ± SEM per gram wet weight were highest in brain (385 ± 24.7), followed by tongue (93.88 ± 3.93), heart (42.13 ± 2.93), and skeletal muscle (31.69 ± 1.42). No significant increases in calmodulin were observed in the dystrophic tissues thus suggesting that the Ca²⁺ accumulations observed in dystrophic muscles are unrelated to changes in calmodulin levels. Because of the complexity of regulation of CANP, a time-dependent study was done using extracts of skeletal, heart, and tongue muscles. Marginal increases in dystrophic CANP were seen in skeletal muscle at all times studied and in the heart and tongue at initial time points only. The data are discussed in terms of rising levels of Ca²⁺ in muscles of the UM-X7.1 hamster being sufficient to increase CANP activity (without increasing content) to where it causes Z-line dissolution.     

Global ischemia-induced inhibition of the coupling ratio of calcium uptake and ATP hydrolysis by rat whole brain microsomal Mg/Ca ATPase

Ischemia is associated with a loss of cytosolic calcium homeostasis. Intracellular stores, particularly in endoplasmic reticulum, are critical for the maintenance of calcium homeostasis. Recent studies have shown that ischemia significantly inhibited microsomal calcium uptake mediated by Mg²⁺/Ca²⁺ ATPase, the major mechanism of endoplasmic reticulum calcium sequestration. This study was initiated to determine whether the decreased calcium uptake caused by ischemia was the result of inhibition of Mg²⁺/Ca²⁺ ATPase activity or an uncoupling of calcium uptake from ATP hydrolysis. The microsomal Mg²⁺/Ca²⁺ ATPase specific inhibitor thapsigargin partially inhibited ATPase activity and completely inhibited calcium uptake. ATPase inhibited by thapsigargin was considered microsomal Mg²⁺/Ca²⁺ ATPase. Ischemia from 5 to 60 min had no significant effect on thapsigargin sensitive ATPase activity. However, under identical conditions, increasing ischemia from 5 to 60 min significantly inhibited microsomal calcium uptake. Comparing calcium uptake to ATP hydrolysis as ischemia increased from 5 to 60 min revealed that the coupling ratio of calcium molecules sequestered to ATP molecules hydrolyzed became significantly decreased. The results demonstrated that the effect of ischemia on microsomal calcium uptake was mediated by an uncoupling of calcium transport from Mg²⁺/Ca²⁺ ATPase activity.     

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.     

Experimental corticosteroid myopathy: Effect on myofibrillar ATPase activity and protein degradation

Corticosteroid myopathy was studied in young, mature New Zealand white rabbits given daily injections of betamethasone (0.3 mg/kg body weight/day) for two weeks. Control rabbits were pair-fed and received saline injections. Betamethasone treatment caused significant wasting of type 2 gluteus medius and psoas muscles but did not cause any atrophy of type 1 soleus and gluteus minimus muscles. The Mg²⁺- and Ca²⁺-activated myofibrillar ATPase activities of the corticosteroid-treated rabbits did not differ from controls despite a 30% reduction in muscle wet weight and pronounced reduction in cross-sectional area of fibers. SDS-polyacrylamide gel electrophoresis profiles of myofibrillar proteins did not differ quantitatively or qualitatively between experimental and control rabbits. Studies of net muscle protein degradation (using ³H-leucine) in betamethasone-treated and control rabbits indicate that both type 1 and type 2 muscle fiber proteins are degraded several times faster in the corticosteroid-treated group. This suggests that a compensatory mechanism exists for those type 1 and mixed fiber type muscles which have increased degradation but do not undergo wasting.     

Ca2+ sensitizers: An emerging class of agents for counterbalancing weakness in skeletal muscle diseases?

Ca²⁺ ions are key regulators of skeletal muscle contraction. By binding to contractile proteins, they initiate a cascade of molecular events leading to cross-bridge formation and ultimately, muscle shortening and force production. The ability of contractile proteins to respond to Ca²⁺ attachment, also known as Ca²⁺ sensitivity, is often compromised in acquired and congenital skeletal muscle disorders. It constitutes, undoubtedly, a major physiological cause of weakness for patients. In this review, we discuss recent studies giving strong molecular and cellular evidence that pharmacological modulators of some of the contractile proteins, also termed Ca²⁺ sensitizers, are efficient agents to improve Ca²⁺ sensitivity and function in diseased skeletal muscle cells. In fact, they compensate for the impaired contractile proteins response to Ca²⁺ binding. Currently, such Ca²⁺ sensitizing compounds are successfully used for reducing problems in cardiac disorders. Therefore, in the future, under certain conditions, these agents may represent an emerging class of agents to enhance the quality of life of patients suffering from skeletal muscle weakness.     

Regulation of basal LC20 phosphorylation by MYPT1 and CPI-17 in murine gastric antrum, gastric fundus, and proximal colon smooth muscles

Background Myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) govern myosin light chain (LC20) phosphorylation and smooth muscle contraction. Rho kinase (ROK) inhibits MLCP, resulting in greater LC20 phosphorylation and force generation at a given [Ca²⁺]_i. Here, we investigate the role of ROK in regulating LC20 phosphorylation and spontaneous contractions of gastric fundus, gastric antrum, and proximal colon smooth muscles. Methods Protein and phosphorylation levels were determined by western blotting. The effects of Y27632, nicardipine, and GF109203X on phosphorylation levels and contraction were measured. Key Results γ‐Actin expression is similar in all three smooth muscles. LC20 and pS19 are highest, but ROK1 and ROK2 are lowest, in antrum and proximal colon smooth muscles. LZ +/? myosin phosphatase targeting subunit 1 (MYPT1), CPI‐17, and pT696, pT853, and pT38 are highest in fundus and proximal colon smooth muscles. Myosin phosphatase‐rho interacting protein (M‐RIP) expression is lowest in fundus, and highest in antrum and proximal colon smooth muscles. Y27632 reduced pT853 in each smooth muscle, but reduced pT696 only in fundus smooth muscles. Nicardipine had no effect on pT38 in each smooth muscle, while GF109203X reduced pT38 in proximal colon and fundus smooth muscles. Y27632 or nicardipine reduced pS19 in proximal colon and fundus smooth muscles. Y27632 or nicardipine inhibited antrum and proximal colon smooth muscle spontaneous contractions, but only Y27632 reduced fundus smooth muscle tone. Zero external Ca²⁺ relaxed each smooth muscle and abolished LC20 phosphorylation. Conclusions & Inferences Organ‐specific mechanisms involving the MLCP interacting proteins LZ +/? MYPT1, M‐RIP, and CPI‐17 are critical to regulating basal LC20 phosphorylation in gastrointestinal smooth muscles.     

Differences between the heavy chains of fast and slow muscle myosin

The different speeds of contraction of slow-twitch and fast-twitch mammalian hind limb muscles correlate with their myosin ATPase activities. Consequently, the structural characterization of these two types of myosin is relevant to an examination of the genetic level at which the speed of contraction is controlled. The myosin molecule consists of two to four light chains (molecular weight approx, 20,000), and two heavy chains (molecular weight of 200,000 each). The light chains from fast and slow muscle myosin are known to differ in number and molecular weight. Tryptic digestion of cat gastrocnemius (a fast muscle) myosin revealed a distinct peptide band of 88,000 daltons in sodium dodecyl sulfate-gel electrophoresis. This peptide was altogether absent in tryptic digests of cat soleus (a slow muscle) myosin. This difference in heavy chains from the two types of myosin was still evident after removing the light chains with p-chloromercuriphenyl sulfonate. The fast muscle myosin heavy chain still retained its characteristic peptide band, but its molecular weight had decreased to 77,000. Thus, the myosins from fast and slow muscles are different with respect to heavy chains (as well as light chains). It is therefore suggested that two different sets of genes are responsible for the synthesis of heavy chains in fast and slow muscles.     

Direct electrical stimulation of rat soleus during denervation-reinnervation

The effect of direct, low-frequency electrical stimulation (at 10 Hz continuously 8 h daily) of muscle on isometric twitch contractile properties of adult rat soleus was observed during denervation and reinnervation. The normal and the bilaterally sciatic nerve crush-denervated groups were implanted with unilateral juxtamuscular electrodes to stimulate the soleus muscle in one limb. After 10, 15, 20, 25, and 30 days of electrical stimulation (2- to 4-mA pulses at 4 ms duration) the normal control, normal-stimulated, crush-denervated control, and crush-denervated-stimulated soleus muscles (N = 6) were evaluated in vitro by massively field stimulating the muscles in physiologic buffer (pH 7.2) at 23 to 24°C. The parameters of isometric twitch contraction measured were latent period (LP), maximum isometric twitch tension (P_t), contraction time (CT), maximum rate of isometric twitch tension (V_t^max), and half-relaxation time (HRT). In normal muscle, 25 and 30 days of electrical stimulation produced significant (P < 0.05) changes: muscle hypertrophy (26.5 and 16.6%, respectively), decline in the P_t (23.4 and 12.1%, respectively), and decrease in the (V_t^max) (17.3 and 21.6%, respectively). For the same periods, compared with the crush-denervated control, the crush-denervated-stimulated muscles also showed significant (P < 0.05) changes: prolongation of the LP. (22.9 and 26.5%), decline in the P_t (24.5 and 31.6%), and decrease in the V_t^max (27.7 and 33.3%). These results, therefore, suggest that the long-term (200 to 240 h) direct, lowfrequency (10 Hz) electrical stimulation may impair the mechanism of isometric twitch development in slow-twitch muscle of the rat. However, our study does not prove that this pattern of electrostimulation can significantly alter the course of self-reinnervation in muscle.     

Structural changes in myosin in dystrophic, denervated and regenerating muscle in mice

The technique of sodium dodecyl sulphate-polyacrylamide gel electrophoresis was used to resolve the large and small polypeptide chains of mouse myosin and to determine their molecular weights. In myosin preparations from dystrophic, denervated and regenerating muscle a decrease in amount of the large sub-unit (molecular weight 200,000) was accompanied by an increase in smaller protein chains. There was evidence that some of the smaller chains in dystrophic myosin were due to proteolytic activity. These smaller chains repeatedly co-precipitated with myosin at low ionic strength and were not removed by gel filtration through Sephadex G-200, indicating that they were tightly bound to the myosin molecule. Normal myosin contained three low molecular weight proteins (26,500, 19,000 and 16,500). The 19,000 molecular weight protein was not detectable in myosin preparations from denervated muscle, regenerating muscle and normal muscle treated with extraction medium previously used to extract dystrophic muscle, but Ca²⁺-activated ATPase was unaffected. In dystrophic myosin both the 19,000 and 16,500 molecular weight proteins were undetectable and the level of Ca²⁺-ATPase was reduced.     

Sarcoplasmic–endoplasmic–reticulum Ca2+‐ATPase and calsequestrin are overexpressed in spared intrinsic laryngeal muscles of dystrophin‐deficient mdx mice

In the mdx mouse model of Duchenne muscular dystrophy, the lack of dystrophin is associated with increased calcium levels and skeletal muscle myonecrosis. The intrinsic laryngeal muscles (ILM) are protected and do not undergo myonecrosis. We investigated whether this protection is related to an increased expression of calcium‐binding proteins, which may protect against the elevated calcium levels seen in dystrophic fibers. The expression of sarcoplasmic–endoplasmic–reticulum Ca²⁺‐ATPase and calsequestrin was examined in ILM and in nonspared limb muscles of control and mdx mice using immunofluorescence and immunoblotting. Dystrophic ILM presented a significant increase in the proteins studied when compared to controls. The increase of Ca²⁺‐handling proteins in dystrophic ILM may permit better maintenance of calcium homeostasis, with the consequent absence of myonecrosis. The results further support the concept that abnormal Ca²⁺‐handling is involved in dystrophinopathies. Muscle Nerve, 2009