Contractile properties of slow and fast skeletal muscles from protease activated receptor‐1 null mice

Introduction: Protease‐activated receptors (PARs) may play a role in skeletal muscle development. We compared the contractile properties of slow‐twitch soleus muscles and fast‐twitch extensor digitorum longus (EDL) muscles from PAR‐1 null and littermate control mice. Methods: Contractile function was measured using a force transducer system. Fiber type proportions were determined using immunohistochemistry. Results: Soleus muscles from PAR‐1 null mice exhibited longer contraction times, a leftward shift in the force–stimulation frequency relationship, and decreased fatiguability compared with controls. PAR‐1 null soleus muscles also had increased type 1 and decreased type IIb/x fiber numbers compared with controls. In PAR‐1 null EDL muscles, no differences were found, except for a slower rate of fatigue compared with controls. Conclusions: The absence of PAR‐1 results in a slower skeletal muscle contractile phenotype, likely due to an increase in type I and a decrease in type IIb/x fiber numbers. Muscle Nerve 50: 991–998, 2014     

Inflammatory response during slow‐ and fast‐twitch muscle regeneration

Introduction: Skeletal muscles are characterized by their unique ability to regenerate. Injury of a so‐called fast‐twitch muscle, extensor digitorum longus (EDL), results in efficient regeneration and reconstruction of the functional tissue. In contrast, slow‐twitch muscle (soleus) fails to properly reconstruct and develops fibrosis. This study focuses on soleus and EDL muscle regeneration and associated inflammation. Methods: We determined differences in the activity of neutrophils and M1 and M2 macrophages using flow cytometry and differences in the levels of proinflammatory cytokines using Western blotting and immunolocalization at different times after muscle injury. Results: Soleus muscle repair is accompanied by increased and prolonged inflammation, as compared to EDL. The proinflammatory cytokine profile is different in the soleus and ED muscles. Conclusions: Muscle repair efficiency differs by muscle fiber type. The inflammatory response affects the repair efficiency of slow‐ and fast‐twitch muscles. Muscle Nerve 55 : 400–409, 2017     

IGF-I treatment improves the functional properties of fast- and slow-twitch skeletal muscles from dystrophic mice

Although insulin-like growth factor-I (IGF-I) has been proposed for use by patients suffering from muscle wasting conditions, few studies have investigated the functional properties of dystrophic skeletal muscle following IGF-I treatment. 129P1 ReJ-Lama2(dy) (129 ReJ dy/dy) dystrophic mice suffer from a deficiency in the structural protein, laminin, and exhibit severe muscle wasting and weakness. We tested the hypothesis that 4 weeks of IGF-I treatment ( approximately 2 mg/kg body mass, 50 g/h via mini-osmotic pump, subcutaneously) would increase the mass and force producing capacity of skeletal muscles from dystrophic mice. IGF-I treatment increased the mass of the extensor digitorum longus (EDL) and soleus muscles of dystrophic mice by 20 and 29%, respectively, compared with untreated dystrophic mice (administered saline-vehicle only). Absolute maximum force (P(o)) of the EDL and soleus muscle was increased by 40 and 32%, respectively, following IGF-I treatment. Specific P(o) (sP(o)) was increased by 23% in the EDL muscles of treated compared with untreated mice, but in the soleus muscle sP(o) was unchanged. IGF-I treatment increased the proportion of type IIB and type IIA fibres and decreased the proportion of type I fibres in the EDL muscles of dystrophic mice. In the soleus muscles of dystrophic mice, IGF-I treatment increased the proportion of type IIA fibres and decreased the proportion of type I fibres. Average fibre cross-sectional area was increased in the EDL and soleus muscles of treated compared with untreated mice. We conclude that IGF-I treatment ameliorates muscle wasting and improves the functional properties of skeletal muscles of dystrophic mice. The findings have important implications for the role of IGF-I in ameliorating muscle wasting associated with the muscular dystrophies.     

3H]Ouabain binding in normal and dystrophic mouse skeletal muscles and the effect of age

The numbers of Na+-K+ ATPase sites in skeletal muscles of normal and dystrophic mice between 3 and 17 months of age have been estimated using [3H]ouabain binding assays. In normal mice, at all ages, slow twitch muscle, soleus (SOL), bound significantly more [3H]ouabain than fast-twitch muscle, extensor digitorum longus (EDL). [3H]Ouabain binding did not alter in either SOL or EDL from normal mice over the age range studied. The numbers of Na+-K+ ATPase sites did alter in muscles taken from dystrophic mice (C57BL/6J dy2J/dy2J). In EDL there was an increase and in SOL a decrease in [3H]ouabain binding. This may be related to a change in muscle fibre metabolism from glycolytic to oxidative or to an altered activity pattern. Increasing age resulted in a progressive reduction in [3H]ouabain binding of both SOL and EDL from dystrophic mice. Part of this reduction may be only apparent and due to an increase in connective tissue composition of dystrophic muscles. A limited study of muscles from neonate dystrophic mice indicated that abnormal [3H]ouabain binding was not present in EDL before two weeks of age.     

Comparison of contractile properties between developing and regenerating soleus muscle: Influence of external calcium concentration upon the contractility

In newborn rat skeletal extensor digitorum longus (EDL) muscle, it has been found that an influx of calcium from the extracellular medium is necessary for contraction, in contrast to the situation observed in adult EDL muscle. The aim of the present study was to determine the influence of the extracellular calcium concentration ([Ca]o) upon the contractile responses elicited in developing as well as in regenerating (notexin-injected) soleus (SOL) muscle. A morphological study was performed to follow the steps of postnatal development and regeneration in SOL muscle. In nominally calcium-free solution, the amplitudes of the twitch and tetanic tensions were greatly reduced in 1–14-day-old developing SOL muscles, as well as in notexin-injected SOL muscles. With longer times after birth, twitch and tetanic tensions of SOL muscle were less affected by the absence of calcium. This contrasts with notexin-injected SOL muscle in which the amplitudes of the contractions remained strongly dependent on [Ca]o. The present finding suggests that some functional characteristics are different in regenerating muscle fibers and may be of interest in the evaluation of the contractile properties of muscles in which injections of genetically engineered or not autologous myoblasts or viral vector have been performed. © John Wiley & Sons, Inc.     

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.     

Contractile properties, fiber types, and myosin isoforms in fast and slow muscles of hyperactive Japanese waltzing mice

This study focuses on the effects of neuromuscular hyperactivity on the contractile properties, fiber type composition, and myosin heavy chain (MHC) isoform expression of fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles in Japanese waltzing mice (JWM) of the C57BL/6J-v2J strain. The same properties were studied in the homologous muscle of control CBA/J mice (CM). In comparison to CM, the JWM exhibited (i) longer activity periods, prolonged bouts of running and a higher food intake, (ii) slower twitch and tetanic contractions of both EDL and SOL muscles, decreased cold and post-tetanic potentiation of the EDL, as well as increased cold and post-tetanic depressions of the SOL. Electrophoretic analyses of MHC isoform revealed a shift toward slower isoforms in both EDL and SOL muscles of JWM as compared to the homologous muscles of CM, namely, a shift from the fastest MHCIIb to the MHCIId/x isoform in the EDL muscle and a shift from MHCIIa to MHCI in the SOL muscle. The latter also contained a higher percentage of type I fibers and displayed a higher capillary density than the SOL muscle of CM. These findings show that the inherently enhanced motor activity of the JWM leads to fiber type transitions in the direction of slower phenotypes. JWM thus represent a suitable model for studying fast-to-slow fiber transitions under the influence of spontaneous motor hyperactivity.     

Changes in isometric contractile properties of fast-twitch and slow-twitch skeletal muscle of C57BL/6J dy2J/dy2J dystrophic mice during postnatal development

Our primary aim was to determine if there exists a preferential involvement of the fast-twitch or slow-twitch skeletal muscle fibers in the dy2J/dy2J strain of murine dystrophy. The changes in the contractile properties of the slow-twitch soleus (SOL) and the fast-twitch extensor digitorum longus (EDL) muscles of normal and dystrophic mice were studied at 4, 8, 12, and 32 weeks of age. Isometric twitch and tetanus tension were decreased in the 4- and 8-week-old dystrophic EDL compared with controls, this situation being reversed in the older animals. At 12 weeks, the dystrophic EDL generated 15% more tetanic tension than normal EDL and by 32 weeks no significant difference was seen between normal and dystrophic EDL twitch or tetanus tension. By 8 weeks, dystrophic EDL exhibited a prolonged time-to-peak twitch tension (TTP) and half-relaxation time (1/2RT) of the isometric twitch which continued to 32 weeks. For the dystrophic SOL, decreased twitch and tetanus tension was observed from 4 to 32 weeks. At 8 and 12 weeks, TTP and 1/2RT of dystrophic SOL were prolonged. However, by 32 weeks there was no longer a significant difference seen in TTP or 1/2RT between normal and dystrophic SOL. Our results appear to indicate that a loss of the primary control which is determining the fiber composition of the individual muscles is occurring as the dystrophic process advances.     

Long‐lasting in vivo inotropic effects of the K+ channel blocker 3,4‐diaminopyridine during fatigue‐inducing stimulation

Blocking K⁺ channels with aminopyridines enhances muscle contractile performance in vitro, but the improvements are relatively short‐lasting during fatigue‐inducing stimulation. We hypothesized that in vivo inotropic actions persist over long periods of fatigue‐inducing stimulation. The effects of 3,4‐diaminopyridine (DAP) were evaluated for rat extensor digitorum longus (EDL) muscle. DAP increased twitch force by 105%. There was a significant leftward shift in the force–frequency relationship, with force values being increased at frequencies up to and including 20 HZ . During repetitive fatigue‐inducing 20‐HZ stimulation, DAP‐induced force increases were large and persisted significantly for at least 30 minutes. Thus, DAP substantially improves contractile performance of EDL muscle in vivo for much longer periods during fatigue‐inducing contractions than in vitro. These data provide support for a potential role for aminopyridines as inotropic agents in applications such as functional electrical stimulation, in which low to medium stimulation frequencies are typically utilized. Muscle Nerve 38: 1616–1622, 2008     

Investigation of neuromuscular abnormalities in neurotrophin‐3‐deficient mice

Neurotrophin‐3 (NT‐3) is a trophic factor that is essential for the normal development and maintenance of proprioceptive sensory neurons and is widely implicated as an important modulator of synaptic function and development. We have previously found that animals lacking NT‐3 have a number of structural abnormalities in peripheral nerves and skeletal muscles. Here we investigated whether haploinsufficiency‐induced reduction in NT‐3 resulted in impaired neuromuscular performance and synaptic function. Motor nerve terminal function was tested by monitoring the uptake/release of the fluorescent membrane dye FM1‐43 by the electrophysiological examination of synaptic transmission and electron microscopic determination of synaptic vesicle density at the presynaptic active zone. We investigated skeletal muscle form and function by measuring force in response to both nerve‐mediated and direct muscle stimulation and by quantification of fiber number and area from transverse sections. Synaptic transmission was not markedly different between the two groups, although the uptake and release of FM1‐43 were impaired in mature NT‐3‐deficient mice but not in immature mice. The electron microscopic examination of mature nerve terminals showed no genotype‐dependent variation in the number of synaptic vesicles near the active zone. NT‐3^+/? mice had normal soleus muscle fiber numbers but their fibers had smaller cross‐sectional areas and were more densely‐packed than wild‐type littermates. Moreover, the muscles of adult NT‐3‐deficient animals were weaker than those of wild‐type animals to both nerve and direct muscle stimulation. The results indicate that a reduction in NT‐3 availability during development impairs motor nerve terminal maturation and synaptic vesicle recycling and leads to a reduction in muscle fiber diameter.     

Low dose formoterol administration improves muscle function in dystrophic mdx mice without increasing fatigue

The beta(2)-adrenoceptor agonist (beta(2)-agonist), formoterol, has been shown to cause muscle hypertrophy in rats even when administered at the micromolar dose of 25 micro g/kg/day. We investigated whether a similar low dose of formoterol could improve muscle function in the dystrophic mdx mouse. Ten-week-old male mdx and wild-type (C57BL/10) mice were administered formoterol (25 micro g/kg/day, i.p.) for 4 weeks. Formoterol treatment increased extensor digitorum longus (EDL) and soleus muscle mass, increased median muscle fibre size in diaphragm, EDL, and soleus muscles, and increased maximum force producing capacity in skeletal muscles of both wild-type and mdx mice. In contrast to other studies where beta(2)-agonists have been administered to mice and rats, generally at higher doses, low dose formoterol treatment did not increase the fatiguability of EDL, soleus or diaphragm muscles. Although others have found formoterol can decrease ubiquitin mRNA and proteasome activity when administered to tumour bearing rats at high doses (2mg/kg/day), in the present study low dose formoterol treatment did not alter ubiquitin or the E1 and E3 ubiquitin ligases in diaphragm muscles of wild-type or mdx mice, but it did reduce the level of ubiquitinated proteins in diaphragm of wild-type mice. The findings indicate that formoterol has considerably more powerful anabolic effects on skeletal muscle than older generation beta(2)-agonists (like clenbuterol and albuterol), and has considerable therapeutic potential for muscular dystrophies and other neuromuscular disorders where muscle wasting is indicated.     

The muscle spindle in cortisone-induced muscular atrophy

The muscle spindles in serially sectioned rat extensor digitorum longus (EDL) and soleus (SOL) muscles were studied histologically and histochemically after chronic cortisone administration. Nuclear chain fibers were found to be significantly atrophied in the EDL but not in the SOL. Nuclear bag fibers were not significantly affected in either muscle. Nuclear chain fibers all stained darkly with the pH 9.4 myofibrillar adenosine triphosphatase (ATPase) reaction (type II) and also stained darkly with the succine dehydrogenase (SDH) reaction. Nuclear bag fibers showed mostly light ATPase staining (type I) and dark, light, or intermediate SDH staining. Extrafusal fibers showed preferential type II fiber atrophy in EDL with relative sparing of type II fibers in SOL. The results suggest that histochemical characteristics that we have evaluated are alone insufficient to explain the differential susceptibility of skeletal muscle fibers to corticosteroids and, therefore, do not in themselves define either a myopathic or neurogenic etiology for the observed muscle wasting.     

Apigenin inhibits sciatic nerve denervation–induced muscle atrophy

Introduction: Apigenin (AP) has been reported to elicit anti‐inflammatory effects. In this study, we investigated the effect of AP on sciatic nerve denervation–induced muscle atrophy. Methods: Sciatic nerve–denervated mice were fed a 0.1% AP‐containing diet for 2 weeks. Muscle weight and cross‐sectional area (CSA), and the expression of atrophic genes and inflammatory cytokines in the gastrocnemius were analyzed. Results: Denervation significantly induced muscle atrophy. However, values for muscle weight and CSA were greater in the denervated muscle of the AP mice than the controls. AP suppressed the expression of MuRF1, but upregulated both myosin heavy chain (MHC) and MHC type IIb. AP also significantly suppressed expression of tumor necrosis‐alpha in the gastrocnemius and soleus muscles, and interleukin‐6 expression in the soleus muscle. Discussion: AP appears to inhibit denervation‐induced muscle atrophy, which may be due in part to its inhibitory effect on inflammatory processes within muscle. Muscle Nerve 58 : 314–318, 2018     

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.     

Effect of hind-limb suspension on young and adult skeletal muscle. I. Normal mice

The purpose of this study was to determine the effect of hind-limb suspension (HS) on morphometric, histologic, and contractile characteristics of fast extensor digitorum longus (EDL) and slow soleus (SOL) twitch muscles in adult and immature mice. Hind-limb suspension for 2 weeks was used to produce atrophy in two groups of mice, ages 4 and 12 weeks, with nonsuspended animals serving as controls. Young HS mice exhibited marked decreases in SOL weight, length, cross-sectional area (CSA), twitch and tetanic tensions, and rates of tension development and relaxation, with increases in fatigue resistance. HS reduced the diameter of both type I and IIA fibers, increased the percentage of type I fibers, and decreased the percentage of type IIA fibers in both young and adult SOL. Muscle weight, length, CSA, IIA and IIB fiber areas, and maximum rate of tetanic tension development were decreased in EDL of young HS mice; fatigue resistance and EDL half-relaxation times were increased. For most parameters evaluated, slow twitch muscle was more affected than fast twitch. HS affected contractile characteristics less than morphometric or histologic parameters. Rates of tension development and relaxation were the contractile parameters most affected by HS, and the time parameters of contraction were least affected. For all measurements young mice were more affected than adult mice.     

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.     

Loss of myostatin expression alters fiber-type distribution and expression of myosin heavy chain isoforms in slow- and fast-type skeletal muscle

Myostatin (Mstn) is a member of the transforming growth factor-beta family that negatively regulates skeletal muscle mass. Mstn knockout mice have greater skeletal muscle mass than wild-type littermates. We investigated the effect of Mstn on fiber type by comparing adult muscles from the murine Mstn knockout with wild-type controls. Based on myofibrillar ATPase staining, the soleus of Mstn knockout mice displays a larger proportion of fast type II fibers and a reduced proportion of slow type I fibers compared with wild-type animals. Based on staining for succinate dehydrogenase (SDH) activity, a larger proportion of glycolytic fibers and a reduced proportion of oxidative fibers occur in the extensor digitorum longus (EDL) of Mstn knockouts. These differences in distribution of fiber types are accompanied by differences in the expression of myosin heavy chain (MHC) isoforms. In both Mstn knockout soleus and EDL, larger numbers of faster MHC isoforms are expressed at the expense of slower isoforms when compared with wild-type littermates. Thus, the absence of Mstn in the knockout mouse leads to an overall faster and more glycolytic muscle phenotype. This muscle phenotype is likely a consequence of developmental processes, and inhibition of Mstn in adults does not cause a transformation to a more fast and glycolytic phenotype. Our findings suggest that myostatin has a critical role in regulating the formation, proliferation, or differentiation of fetal myoblasts and postnatal fibers.     

Effects of leukemia inhibitory factor on rat skeletal muscles are modulated by clenbuterol

Leukemia inhibitory factor (LIF) is implicated in skeletal muscle regeneration, but the effect of exogenous LIF on uninjured muscles is not known. We tested the hypothesis that LIF administration would stimulate muscle hypertrophy, with an increased effect during clenbuterol-induced fiber remodeling. Rats received daily injections of either saline or LIF, and either regular or clenbuterol-supplemented drinking water for 4 weeks. In the slow-twitch soleus muscles of LIF-treated rats, specific force (sP(o)) and muscle fiber size were increased by approximately 13% and approximately 26%, respectively, compared to saline-treated rats. In the soleus muscles of rats receiving LIF and clenbuterol, compared to rats receiving clenbuterol alone, maximum isometric tension (P(o)) was approximately 19% greater. LIF alone did not affect the properties of fast-twitch extensor digitorum longus (EDL) muscles, but in rats receiving LIF and clenbuterol, compared to clenbuterol alone, EDL fiber size and muscle mass were increased by approximately 20% and approximately 10%, respectively. The hypertrophic effects of exogenous LIF on uninjured skeletal muscles indicate that LIF may have application in the treatment of conditions characterized by muscle wasting.     

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