Unloading stress disturbs muscle regeneration through perturbed recruitment and function of macrophages

Skeletal muscle is one of the most sensitive tissues to mechanical loading, and unloading inhibits the regeneration potential of skeletal muscle after injury. This study was designed to elucidate the specific effects of unloading stress on the function of immunocytes during muscle regeneration after injury. We examined immunocyte infiltration and muscle regeneration in cardiotoxin (CTX)-injected soleus muscles of tail-suspended (TS) mice. In CTX-injected TS mice, the cross-sectional area of regenerating myofibers was smaller than that of weight-bearing (WB) mice, indicating that unloading delays muscle regeneration following CTX-induced skeletal muscle damage. Delayed infiltration of macrophages into the injured skeletal muscle was observed in CTX-injected TS mice. Neutrophils and macrophages in CTX-injected TS muscle were presented over a longer period at the injury sites compared with those in CTX-injected WB muscle. Disturbance of activation and differentiation of satellite cells was also observed in CTX-injected TS mice. Further analysis showed that the macrophages in soleus muscles were mainly Ly-6C-positive proinflammatory macrophages, with high expression of tumor necrosis factor-α and interleukin-1β, indicating that unloading causes preferential accumulation and persistence of proinflammatory macrophages in the injured muscle. The phagocytic and myotube formation properties of macrophages from CTX-injected TS skeletal muscle were suppressed compared with those from CTX-injected WB skeletal muscle. We concluded that the disturbed muscle regeneration under unloading is due to impaired macrophage function, inhibition of satellite cell activation, and their cooperation.     

Contractile properties,structure and fiber phenotype of intact and regenerating slow-twitch muscles of mice treated with cyclosporin A

We have studied the contractile properties, structure, fiber-type composition, and myosin heavy chain (MyHC) expression pattern of regenerating and intact soleus muscles of adult CBA/J mice treated with cyclosporin A (CsA) or vehicle solutions (Cremophor, saline). A comparison of muscles after 4-7 weeks drug application with those receiving vehicle showed that the isometric contractile force of intact drug-treated muscles was reduced (tetanus, -21%; twitch, -34%) despite normal mass and muscle cross-sectional area. The frequency of fast-twitch fibers was increased, whereas no innervation deficits, histopathological alterations, or changes in fiber numbers were observed. Regeneration after cryolesion of the contralateral soleus proceeded more slowly in CsA-treated than in vehicle-treated animals. Despite this, when muscle properties reached mature levels (4-7 weeks), muscle mass recovery was better in CsA-treated animals (30% higher weight, 50% more fiber profiles in cross-sections). The force production per unit cross-sectional area was deficient, but not the maximum tension. Twitch time-to-peak and half-relaxation time were shorter than controls correlating with a predominance of fast-twitch fibers (98% Type II fibers versus 16%-18% in control muscles) and fast MyHC isoforms. Partial reversal of this fast phenotype and an increase in muscle force were observed when the animals were left to recover without treatment for 5-8 weeks after CsA application over 7 weeks. The high numbers of fiber profiles in CsA-treated regenerated muscles and increased mass remained unchanged after withdrawal. Thus, CsA treatment has a hyperplastic effect on regenerating muscles, and drug-induced phenotype alterations are much more prominent in regenerated muscles.     

Abnormalities in structure and function of limb skeletal muscle fibres of dystrophic mdx mice.

In this study we have shown that the skeletal muscle fibres from adult (older than 26 weeks) mdx mice have gross structural deformities. We have characterized the onset and age dependence of this feature in mdx mice. The three dimensional structure of these deformities has been visualized in isolated fibres and the orientation of these deformities was determined within the muscle by confocal laser scanning microscopy. We have also shown that the occurrence of morphologically abnormal fibres is greater in muscles with longer fibres (extensor digitorum longus (EDL) and soleus, 6-7.3 mm long), than in muscles with shorter fibres (flexor digitorum brevis (FDB), 0.3-0.4 mm long). A population of post-degenerative fibres, with both central and peripheral nuclei coexistent along the length of the fibre, has also been identified in the muscles studied. We showed that a mild protocol of lengthening (eccentric) contractions (the muscle was stretched by 12% during a tetanic contraction) caused a major reduction in the maximal tetanic force subsequently produced by mdx EDL muscle. In contrast, maximal tetanic force production in normal soleus, normal EDL and mdx soleus muscles was not altered by this protocol. We suggest that the deformed fast glycolytic fibres which are found in adult mdx EDL but not in adult mdx soleus muscles are the population of fibres damaged by the lengthening protocol.     

MicroRNA-206 is highly expressed in newly formed muscle fibers: implications regarding potential for muscle regeneration and maturation in muscular dystrophy

miR-1, miR-133a, and miR-206 are muscle-specific microRNAs expressed in skeletal muscles and have been shown to contribute to muscle development. To gain insight into the pathophysiological roles of these three microRNAs in dystrophin-deficient muscular dystrophy, their expression in the tibialis anterior (TA) muscles of mdx mice and CXMD(J) dogs were evaluated by semiquantitative RT-PCR and in situ hybridization. Their temporal and spatial expression patterns were also analyzed in C2C12 cells during muscle differentiation and in cardiotoxin (CTX)-injured TA muscles to examine how muscle degeneration and regeneration affect their expression. In dystrophic TA muscles of mdx mice, miR-206 expression was significantly elevated as compared to that in control TA muscles of age-matched B10 mice, whereas there were no differences in miR-1 or miR-133a expression between B10 and mdx TA muscles. On in situ hybridization analysis, intense signals for miR-206 probes were localized in newly formed myotubes with centralized nuclei, or regenerating muscle fibers, but not in intact pre-degenerated fibers or numerous small mononucleated cells, possibly proliferating myoblasts and inflammatory infiltrates. Similar increased expression of miR-206 was also found in C2C12 differentiation and CTX-induced regeneration, in which differentiated myotubes or regenerating fibers showed abundant expression of miR-206. However, CXMD(J) TA muscles contained smaller amounts of miR-206, miR-1, and miR-133a than controls. They exhibited more severe and more progressive degenerative alterations than mdx TA muscles. Taken together, these observations indicated that newly formed myotubes showed markedly increased expression of miR-206, which might reflect active regeneration and efficient maturation of skeletal muscle fibers.     

Expression of nestin,desmin and vimentin in intact and regenerating muscle spindles of rat hind limb skeletal muscles

We describe the expression and distribution patterns of nestin, desmin and vimentin in intact and regenerating muscle spindles of the rat hind limb skeletal muscles. Regeneration was induced by intramuscular isotransplantation of extensor digitorum longus (EDL) or soleus muscles from 15-day-old rats into the EDL muscle of adult female inbred Lewis rats. The host muscles with grafts were excised after 7-, 16-, 21- and 29-day survival and immunohistochemically stained. Nestin expression in intact spindles in host muscles was restricted to Schwann cells of sensory and motor nerves. In transplanted muscles, however, nestin expression was also found in regenerating “spindle fibers”, 7 and 16 days after grafting. From the 21st day onwards, the regenerated spindle fibers were devoid of nestin immunoreactivity. Desmin was detected in spindle fibers at all developmental stages in regenerating as well as in intact spindles. Vimentin was expressed in cells of the outer and inner capsules of all muscle spindles and in newly formed myoblasts and myotubes of regenerating spindles 7 days after grafting. Our results show that the expression pattern of these intermediate filaments in regenerating spindle fibers corresponds to that found in regenerating extrafusal fibers, which supports our earlier suggestion that they resemble small-diameter extrafusal fibers.     

Expression of myosin isoforms during notexin-induced regeneration of rat soleus muscles

Myosin isozymes and their fiber distribution were studied during regeneration of the soleus muscle of young adult (4-6 week old) rats. Muscle degeneration and regeneration were induced by a single subcutaneous injection of a snake toxin, notexin. If reinnervation of the regenerating muscle was allowed to occur (functional innervation nearly complete by 7 days), then fiber diameters continued to increase and by 28 days after toxin treatment they attained the same values as fibers in the contralateral soleus. If the muscles were denervated at the time of toxin injection, the early phases of regeneration still took place but the fibers failed to continue to increase in size. Electrophoresis of native myosin showed multiple bands between 3 and 21 days of regeneration which could be interpreted as indicating the presence of embryonic, neonatal, fast and slow myosins in the innervated muscles. Adult slow myosin became the exclusive from in innervated regenerates. In contrast, adult fast myosin became the predominant form in denervated regenerating muscles. Immunocytochemical localization of myosin isozymes demonstrated that in innervated muscles the slow form began to appear in a heterogeneous fashion at about 7 days, and became the major form in all fibers by 21-28 days. Thus, the regenerated muscle was almost entirely composed of slow fibers, in clear contrast to the contralateral muscle which was still substantially mixed. In denervated regenerating muscles, slow myosin was not detected biochemically or immunocytochemically whereas fast myosin was detected in all denervated fibers by 21-28 days. The regenerating soleus muscle therefore is clearly different from the developing soleus muscle in that the former is composed of a uniform fiber population with respect to myosin transitions. Moreover the satellite cells which account for the regeneration process in the soleus muscle do not appear to be predetermined with respect to myosin heavy chain expression, since the fibers they form can express either slow or fast isoforms. The induction of the slow myosin phenotype is entirely dependent on a positive, extrinsic influence of the nerve.     

Distinctive patterns of basic fibroblast growth factor (bFGF) distribution in degenerating and regenerating areas of dystrophic (mdx) striated muscles

Mdx mice uniquely recover from degenerative dystrophic lesions by an intense myoproliferative (regenerative) response. To investigate a potential role of endogenous basic fibroblast growth factor (bFGF) in injury-repair processes, we investigated its localization in several striated muscles of mdx and control mice using immunofluorescence labeling with specific antibodies. Basic FGF was localized consistently to the myofiber periphery and nuclei of intact myofibers, as well as in single, dystrophin-positive cells in close association with the myofibers (potential myosatellite cells). In mdx mice, actively degenerating skeletal or cardiac muscle fibers presented intense cytoplasmic anti-bFGF staining prior to mononuclear infiltration. Small regenerating fibers in mdx skeletal muscle exhibited greater bFGF accumulation than adjacent larger myofibers. Strong nuclear anti-bFGF immunolabeling was frequently observed in mdx cardiac myocytes at the borders of necrotic regions. In agreement with differences in intensity of immunolabeling, extracts from slow-twitch muscles contained higher levels of bFGF compared to those from fast-twitch muscles, in both control and mdx mice. In addition, bFGF levels were consistently higher in extracts from all mdx tissues compared to those derived from their control counterparts. Our data suggest that bFGF participates in the degenerative and regenerative responses of striated muscle to dystrophic injury and also indicate a potential involvement of this factor with the physiology of different striated muscles.     

iNOS expression in dystrophinopathies can be reduced by somatic gene transfer of dystrophin or utrophin

BACKGROUND: Nitric oxide (NO) is an inorganic gas produced by a family of NO synthase (NOS) proteins. The presence and the distribution of inducible-NOS (NOS II or iNOS), and NADPH-diaphorase (NADPH-d), a marker for NOS catalytic activity, were determined in muscle sections from control, DMD, and BMD patients. MATERIALS AND METHODS: NADPH-d reactivity, iNOS- and nNOS (NOS I)-immunolocalization were studied in muscles from mdx mice before and after somatic gene transfer of dystrophin or utrophin. RESULTS: In control patients, few fibers (<2%) demonstrated focal accumulation of iNOS in sarcolemma. In DMD patients, a strong iNOS immunoreactivity was observed in some necrotic muscle fibers as well as in some mononuclear cells, and regenerating muscle fibers had diffusely positive iNOS immunoreactivity. In DMD patients, NADPH-d reactivity was increased and mainly localized in regenerating muscle fibers. In mdx mice quadriceps, iNOS expression was mainly observed in regenerating muscle fibers, but not prior to 4 weeks postnatal, and was still present 8 weeks after birth. The expression of dystrophin and the overexpression of utrophin using adenovirus-mediated constructs reduced the number of iNOS-positive fibers in mdx quadriceps muscles. The correction of some pathology in mdx by dystrophin expression or utrophin overexpression was independent of the presence of nNOS. CONCLUSIONS: These results suggest that iNOS could play a role in the physiopathology of DMD and that the abnormal expression of iNOS could be corrected by gene therapy.     

Susceptibility to sarcomere injury induced by single stretches of maximally activated muscles of mdx mice.

The purpose was to investigate the contribution of mechanical damage to sarcomeres to the greater susceptibility of dystrophic muscle fibers to contraction-induced injury. Single stretches provide an effective method for studying mechanical factors that contribute to the initiation of contraction-induced injury. We hypothesized that, after single stretches, the deficits in isometric force would be greater for muscles of mdx than C57BL/10 mice, whereas membrane damage would be minimal for all muscles. Extensor digitorum longus (EDL) and soleus muscles of mice were removed under anesthesia with Avertin (tribromoethanol). During the plateau of a maximum isometric contraction in vitro, muscles were stretched through single strains of 20-60% fiber length. Isometric force was remeasured 1 min later, and muscles were then incubated in procion orange dye to identify fibers with membrane damage. Force deficits at 1 min were two- to threefold greater for EDL muscles of mdx compared with C57BL/10 mice, whereas no significant differences were observed between soleus muscles of mdx and C57BL/10 mice. For all muscles, membrane damage was minimal and not significantly increased by single stretches for either strain of mice. These data support a critical role of dystrophin maintaining sarcomere stability in EDL muscles, whereas soleus muscles retain abilities, in the absence of dystrophin, not different from control muscles to resist sarcomere damage.     

PDGF-receptor concentration is elevated in regenerative muscle fibers in dystrophin-deficient muscle.

Dystrophin-deficient muscle undergoes sudden, postnatal onset of muscle necrosis that is either progressive, as in Duchenne muscular dystrophy, or successfully arrested and followed by regeneration, as in most muscles of mdx mice. The mechanisms regulating regeneration in mdx muscle are unknown, although the possibility that there is renewed expression of genes regulating embryonic muscle cell proliferation and differentiation may provide testable hypotheses. Here, we examine the possibility that necrotic and regenerating mdx muscles exhibit renewed or increased expression of PDGF-receptors. PDGF-binding to receptors on muscle has been shown previously to be associated with myogenic cell proliferation and delay of muscle differentiation. We find that PDGF-receptors are present in 4-week-old mdx mice in muscles that undergo brief, reversible necrosis (hindlimb muscles) or progressive necrosis (diaphragm), as well as in 4-week-old control mouse muscles. Immunoblots indicate that the concentrations of PDGF-receptors in 4-week-old dystrophic (necrotic) and control muscles are similar. Prenecrotic, dystrophic fibers and control fibers possess some cell surface labeling of fibers treated with anti-PDGF-receptor and viewed by indirect immunofluorescence. Necrotic fibers in dystrophic muscle show cytoplasmic labeling for PDGF-receptors and labeling of perinuclear regions at the muscle cell surface. Adult dystrophic muscle displays higher concentrations of PDGF-receptor in both regenerated muscle (hindlimb) and progressively necrotic muscle (diaphragm) than found in controls. Anti-PDGF-receptor labeling of regenerated, dystrophic muscle is observed primarily in granules surrounding central nuclei or surrounding nuclei located at the surface of regenerated fibers. No labeling of perinuclear regions of control muscle or prenecrotic fibers was observed. Myonuclei fractionated from adult mdx hindlimb muscles contained no PDGF-receptor, indicating that PDGF-receptor-positive structures are not tightly associated with nuclei or within nuclei. L6 myoblasts show PDGF-receptor distributed diffusely on the cell surface. Stimulation of L6 myoblasts with 10 ng/ml of PDGF-BB causes receptor internalization and concentration in granules at perinuclear regions. Thus, PDGF stimulation of myoblasts causes a redistribution of PDGF-receptors to resemble receptor localization observed during muscle regeneration. These findings implicate PDGF-mediated mechanisms in regeneration of dystrophic muscle.     

Increased expression of deltaCaMKII isoforms in skeletal muscle regeneration: Implications in dystrophic muscle disease

The expression of delta isoforms of calcium-calmodulin/dependent protein kinase II (CaMKII) has been reported in mammalian skeletal muscle; however, their functions in this tissue are largely unknown. This study was conducted to determine if deltaCaMKII expression was altered during regeneration of skeletal muscle fibers in two distinct models. In the first model, necrosis and regeneration were induced in quadriceps of normal mice by intramuscular administration of 50% glycerol. Immunostaining and confocal microscopy revealed that deltaCaMKII expression was clearly enhanced in fibers showing centralized nuclei. The second model was the mdx mouse, which undergoes enhanced muscle necrosis and regeneration due to a mutation in the dystrophin gene. sern blot analysis of hind leg extracts from 4 to 6 week old mdx mice revealed that deltaCaMKII content was decreased when compared to age-matched control mice. This loss in delta kinase content was seen in myofibrillar and membrane fractions and was in contrast to unchanged deltaCaMKII levels in cardiac and brain extracts from dystrophic mice. Confocal microscopy of mdx quadriceps and tibialis muscle showed that deltaCaMKII expression was uniformly decreased in most fibers from dystrophic mice; however, enhanced kinase expression was observed in regenerating muscle fibers. These data support a fundamental role for deltaCaMKII in the regeneration process of muscle fibers in normal and mdx skeletal muscle and may have important implications in the reparative process following muscle death.     

Influence of hindlimb suspension on calcium-induced contraction characteristics in dystrophin-deficient animals

The direct data concerning effects of unloading on dystrophic muscle were received in study of mdx mice, a model for Duchenne muscular dystrophy, muscles before and after hindlimb suspension. Experiments were performed on softer skinned soleus muscle fibers isolated from wild-type (C57black) as a control and mdx mice aged 2 weeks. Animals of two experimental groups were tail suspended during 21 days. In both groups of hindlimb suspended mice isolated soleus fibers were thinner than in the control groups. But there was a greater 37% significant decrease in fiber diameter in wild-type (CHS) suspended mice vs. 24% in mdx (MHS) suspended group. Values of absolute peak tension in CHS were less than in the control group by 33%, and in MHS mice suspended--by 39%. 21 days of hindlimb suspension resulted in reduction of mean peak specific tension by 28% in MHS and significantly less drop (15%) in CHS groups. We observed a similar rightward shift of the tension pCa curve in both mice strains.     

Stimulation of calcineurin Aalpha activity attenuates muscle pathophysiology in mdx dystrophic mice

Calcineurin activation ameliorates the dystrophic pathology of hindlimb muscles in mdx mice and decreases their susceptibility to contraction damage. In mdx mice, the diaphragm is more severely affected than hindlimb muscles and more representative of Duchenne muscular dystrophy. The constitutively active calcineurin Aalpha transgene (CnAalpha) was overexpressed in skeletal muscles of mdx (mdx CnAalpha*) mice to test whether muscle morphology and function would be improved. Contractile function of diaphragm strips and extensor digitorum longus and soleus muscles from adult mdx CnAalpha* and mdx mice was examined in vitro. Hindlimb muscles from mdx CnAalpha* mice had a prolonged twitch time course and were more resistant to fatigue. Because of a slower phenotype and a decrease in fiber cross-sectional area, normalized force was lower in fast- and slow-twitch muscles of mdx CnAalpha* than mdx mice. In the diaphragm, despite a slower phenotype and a approximately 35% reduction in fiber size, normalized force was preserved. This was likely mediated by the reduction in the area of the diaphragm undergoing degeneration (i.e., mononuclear cell and connective and adipose tissue infiltration). The proportion of centrally nucleated fibers was reduced in mdx CnAalpha* compared with mdx mice, indicative of improved myofiber viability. In hindlimb muscles of mdx mice, calcineurin activation increased expression of markers of regeneration, particularly developmental myosin heavy chain isoform and myocyte enhancer factor 2A. Thus activation of the calcineurin signal transduction pathway has potential to ameliorate the mdx pathophysiology, especially in the diaphragm, through its effects on muscle degeneration and regeneration and endurance capacity.     

Normal distribution of presenilin-1 and nicastrin in skeletal muscle and the differential responses of these proteins after denervation

Presenilin-1 and nicastrin, two components of gamma-secretase associated with Alzheimer's disease plaques, are present in the synapses of the brain and in various peripheral organs, including skeletal muscle. In the present study, we examined the expression pattern of presenilin-1 and nicastrin in normal and denervated hindlimb muscles of the rat. Using immunohistochemical approaches, we found that presenilin-1 and AChRalpha was co-localized at the neuromuscular junction in the normal skeletal muscles of rats. The immunoreactivities of both presenilin-1 and nicastrin were also observed at the sarcolemma of muscle fibers. We discovered that presenilin-1 mRNA and its protein are upregulated after denervation of the soleus and tibialis anterior muscles. Furthermore, clear co-localization between presenilin-1 and DAPI, but not nicastrin, was noted in several myonuclei in the denervated muscles. We recognized a few fibers possessing both ubiquitin and presenilin-1 protein in the cytosol. The amount of presenilin-1 in the nucleus and membrane fraction was more abundantly expressed in the denervated muscle fibers. In contrast, no significant difference in the nicastrin protein level was observed between normal and denervated muscle fibers. These data suggest that enhanced presenilin-1 protein may play a role in the degeneration and regeneration of skeletal muscle.     

Early metabolic changes measured by 1H MRS in healthy and dystrophic muscle after injury

Skeletal muscle injury is often assessed by clinical findings (history, pain, tenderness, strength loss), by imaging, or by invasive techniques. The purpose of this work was to determine if in vivo proton magnetic resonance spectroscopy ((1)H MRS) could reveal metabolic changes in murine skeletal muscle after contraction-induced injury. We compared findings in the tibialis anterior muscle from both healthy wild-type (WT) muscles (C57BL/10 mice) and dystrophic (mdx mice) muscles (an animal model for human Duchenne muscular dystrophy) before and after contraction-induced injury. A mild in vivo eccentric injury protocol was used due to the high susceptibility of mdx muscles to injury. As expected, mdx mice sustained a greater loss of force (81%) after injury compared with WT (42%). In the uninjured muscles, choline (Cho) levels were 47% lower in the mdx muscles compared with WT muscles. In mdx mice, taurine levels decreased 17%, and Cho levels increased 25% in injured muscles compared with uninjured mdx muscles. Intramyocellular lipids and total muscle lipid levels increased significantly after injury but only in WT. The increase in lipid was confirmed using a permeable lipophilic fluorescence dye. In summary, loss of torque after injury was associated with alterations in muscle metabolite levels that may contribute to the overall injury response in mdx mice. These results show that it is possible to obtain meaningful in vivo (1)H MRS regarding skeletal muscle injury.     

Presence of embryonic myosin in normal postural muscles of the adult rat

Indirect immunofluorescence was used to localize embryonic myosin heavy chains in soleus, adductor longus, tibialis anterior, plantaris, and extensor digitorum longus muscles of 6-month-old rats. A monoclonal antibody (2B6), specifically recognizing rat embryonic myosin, was applied to unfixed, transverse, frozen sections. The number of embryonic myosin-positive (EMP) extrafusal fibers was expressed as a percentage of the total number of fibers. EMP extrafusal fibers were only seen in the soleus and adductor longus muscles, both postural muscles. Approximately 1% of the soleus muscle fibers appeared positively stained for embryonic myosin. The majority of such fibers had a small diameter (<500 ), appeared intensely fluorescent, and typically contained central nuclei. Re-expression of embryonic myosin due to spontaneous fiber denervation is not a likely factor in this study, since alpha-bungarotoxin and N-CAM localization were restricted to the motor end-plate region of EMP fibers. Since embryonic myosin was shown to disappear in all normal-sized myofibers by 2 to 3 months of age, the results suggest that the EMP extrafusal fibers seen in postural muscles of 6 to 12-month-old animals are regenerating myofibers. We speculate that a small number of muscle fibers may be regenerating in normal, adult postural muscles, in response to fiber damage possibly caused by excessive recruitment or overloading.     

Calcineurin-A alpha activation enhances the structure and function of regenerating muscles after myotoxic injury

Calcineurin signaling is essential for successful muscle regeneration. Although calcineurin inhibition compromises muscle repair, it is not known whether calcineurin activation can enhance muscle repair after injury. Tibialis anterior (TA) muscles from adult wild-type (WT) and transgenic mice overexpressing the constitutively active calcineurin-A alpha transgene under the control of the mitochondrial creatine kinase promoter (MCK-CnA alpha*) were injected with the myotoxic snake venom Notexin to destroy all muscle fibers. The TA muscle of the contralateral limb served as the uninjured control. Muscle structure was assessed at 5 and 9 days postinjury, and muscle function was tested in situ at 9 days postinjury. Calcineurin stimulation enhanced muscle regeneration and altered levels of myoregulatory factors (MRFs). Recovery of myofiber size and force-producing capacity was hastened in injured muscles of MCK-CnA alpha* mice compared with control. Myogenin levels were greater 5 days postinjury and myocyte enhancer factor 2a (MEF2a) expression was greater 9 days postinjury in muscles of MCK-CnA alpha* mice compared with WT mice. Higher MEF2a expression in regenerating muscles of MCK-CnA alpha* mice 9 days postinjury may be related to an increase of slow fiber genes. Calcineurin activation in uninjured and injured TA muscles slowed muscle contractile properties, reduced fatigability, and enhanced force recovery after 4 min of intermittent maximal stimulation. Therefore, calcineurin activation can confer structural and functional benefits to regenerating skeletal muscles, which may be mediated in part by differential expression of MRFs.     

Differential adaptation of growth and differentiation factor 8/myostatin, fibroblast growth factor 6 and leukemia inhibitory factor in overloaded, regenerating and denervated rat muscles

Mice genetically deficient in growth and differentiation factor 8 (GDF8/myostatin) had markedly increased muscle fiber numbers and fiber hypertrophy. In the regenerating muscle of mice possessing FGF6 mutation, fiber remodeling was delayed. Although myostatin and FGF6 may be important for the maintenance, regeneration and/or hypertrophy of muscle, little work has been done on the possible role of these proteins in adult muscle in vivo. Using Western blot and immunohistochemical analysis, we investigated, in rats, the distribution of myostatin, FGF6 and LIF proteins between slow- and fast-type muscles, and the adaptive response of these proteins in mechanically overloaded muscles, in regenerating muscles following bupivacaine injection and in denervated muscles after section of the sciatic nerve. The amounts of myostatin and LIF protein were markedly greater in normal slow-type muscles. In the soleus muscle, myostatin and LIF proteins were detected at the site of the myonucleus in both slow-twitch and fast-twitch fibers. In contrast, FGF6 protein was selectively expressed in normal fast-type muscles. Mechanical overloading rapidly enhanced the myostatin and LIF but not FGF6 protein level. In the regenerating muscles, marked diminution of myostatin and FGF6 was observed besides enhancement of LIF. Denervation of fast-type muscles rapidly increased the LIF, but decreased the FGF6 expression. Therefore, the increased expressions of myostatin and LIF play an important role in muscle hypertrophy following mechanical overloading. The marked reduction of FGF6 in the hypertrophied and regenerating muscle would imply that FGF6 regulates muscle differentiation but not proliferation of satellite cells and/or myoblasts.     

A myosin isoform repressed in hypertrophied ALD muscle of the chicken reappears during regeneration following cold injury

A library of monoclonal antibodies specific for myosin heavy chain (HC) was used to study myosin expression in regenerating fibers. The response to cold injury of slow skeletal ALD muscle previously induced to eliminate SM1 myosin by weight overload was compared to that of its contralateral control. Native gel electrophoresis combined with immunoblotting demonstrated that slow SM1 myosin HC eliminated from hypertrophic muscle reappeared both at the site of active regeneration and unexpectedly, also distal to the site of injury. The regeneration response of hypertrophied muscles was similar to that of the controls. In addition to SM1 myosin HC, ventricular-like and embryonic/fast isoforms were also expressed in both muscles during the early stages of regeneration and disappeared as the muscle fibers matured. These observations demonstrate that regenerating slow muscle fibers reexpress myosins' characteristic of developing muscle irrespective of the myosin phenotype prior to injury. The reappearance of repressed myosin HC in the hypertrophied ALD muscle is consistent with the presence of newly differentiated myonuclei.