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Injury of skeletal muscle is followed by muscle regeneration in which new muscle tissue is formed from the proliferating mononuclear myoblasts, and by systemic response to stress that exposes proliferating myoblasts to increased glucocorticoid (GC) concentration. Because of its various causes, hypoxia is a frequent condition affecting skeletal muscle, and therefore both processes, which importantly determine the outcome of the injury, often proceed under hypoxic conditions. It is therefore important to identify and characterize in proliferating human myoblasts: 1) response to hypoxia which is generally organized by hypoxia-inducible factor-1[alpha] (HIF-1[alpha]); 2) response to GCs which is mediated through the isoforms of glucocorticoid receptors (GRs) and 11[beta]-hydroxysteroid dehydrogenases (11[beta]-HSDs), and 3) the response to GCs under the hypoxic conditions and the influence of this combination on the factors controlling myoblast proliferation. Using real-time PCR, Western blotting, and HIF-1[alpha] small-interfering RNA silencing, we demonstrated that cultured human myoblasts possess both, the HIF-1[alpha]-based response to hypoxia, and the GC response system composed of GR[alpha] and types 1 and 2 11[beta]-HSDs. However, using combined dexamethasone and hypoxia treatments, we demonstrated that these two systems operate practically without mutual interactions. A seemingly surprising separation of the two systems that both organize response to hypoxic stress can be explained on the evolutionary basis: the phylogenetically older HIF-1[alpha] response is a protection at the cellular level, whereas the GC stress response protects the organism as a whole. This necessitates actions, like downregulation of IL-6 secretion and vascular endothelial growth factor, that might not be of direct benefit for the affected myoblasts. hypoxia-inducible factor-[alpha]; antisense version of hypoxia-inducible factor; vascular endothelial growth factor; glucocorticoid receptors; 11[beta]-hydroxysteroid dehydrogenases doi: 10.1152/ajpregu.00133.2010

Recent discoveries reveal complex interactions between skeletal muscle and the immune system that regulate muscle regeneration. In this review, we evaluate evidence that indicates that the response of myeloid cells to muscle injury promotes muscle regeneration and growth. Acute perturbations of muscle activate a sequence of interactions between muscle and inflammatory cells. The initial inflammatory response is a characteristic Th1 inflammatory response, first dominated by neutrophils and subsequently by [CD68.sup.+] M1 macrophages. M1 macrophages can propagate the Th1 response by releasing proinflammatory cytokines and cause further tissue damage through the release of nitric oxide. Myeloid cells in the early Th1 response stimulate the proliferative phase of myogenesis through mechanisms mediated by TNF-[alpha] and IL-6; experimental prolongation of their presence is associated with delayed transition to the early differentiation stage of myogenesis. Subsequent invasion by [CD163.sup.+]/[CD206.sup.+] M2 macrophages attenuates M1 populations through the release of anti-inflammatory cytokines, including IL-10. M2 macrophages play a major role in promoting growth and regeneration; their absence greatly slows muscle growth following injury or modified use and inhibits muscle differentiation and regeneration. Chronic muscle injury leads to profiles of macrophage invasion and function that differ from acute injuries. For example, mdx muscular dystrophy yields invasion of muscle by M1 macrophages, but their early invasion is accompanied by a subpopulation of M2a macrophages. M2a macrophages are IL-4 [receptor.sup.+]/[CD206.sup.+] cells that reduce cytotoxicity of M1 macrophages. Subsequent invasion of dystrophic muscle by M2c macrophages is associated with progression of the regenerative phase in pathophysiology. Together, these findings show that transitions in macrophage phenotype are an essential component of muscle regeneration in vivo following acute or chronic muscle damage. skeletal muscle; macrophage; neutrophil; muscular dystrophy; chemokines doi: 10.1152/ajpregu.00735.2009.

The bone morphogenetic protein (BMP) pathway is known to be involved in limb myogenesis during development, but whether it is involved in postnatal muscle regeneration is unclear. We have found that adult inhibitor of differentiation (Id)-mutant ([Id1.sup.+/-][Id3.sup.-/-]) mice display delayed and reduced skeletal muscle regeneration after injury compared with either wild-type littermates or Id3-null mice. Immunoblotting of wild-type muscle lysates revealed that, not only were Id1 and Id3 highly upregulated within 24 h after injury, but other upstream components of the BMP pathway were as well, including the BMP receptor type II and phosphorylated Smad1/5/8 (pSmad1/5/8). Inhibition of BMP signaling in injured skeletal muscle by Noggin injection reduced pSmad1/5/8, Id1, and Id3 protein levels. The mouse myoblast-derived cell line C2C12 also expressed Id1, Id3, BMP receptor type II, and pSmad1/5/8 during proliferation, but all were reduced upon differentiation into myotubes. In addition, these cells secreted mature BMP-4, and BMP signaling could be inhibited with exogenous Noggin, causing a reduction in pSmad1/5/8, Id1, and Id3 levels. Confocal immunofluorescence microscopy revealed that activated [Pax7.sup.+] myoblasts coexpressed nuclear pSmad1/5/8, Id1, and Id3 in injured mouse skeletal muscle sections. Although we did not observe differences in the numbers of quiescent [Pax7.sup.+] satellite cells in adult uninjured hindlimb muscles, we did observe a significant reduction in the number of proliferating [Pax7.sup.+] cells in the Id-mutant mice after muscle injury compared with either wild-type or Id3-null mice. These data suggest a model in which BMP signaling regulates Id1 and Id3 in muscle satellite cells, which directs their proper proliferation before terminal myogenic differentiation after skeletal muscle injury in postnatal animals. bone morphogenetic protein signaling; inhibitor of differentiation-1; inhibitor of differentiation-3; Pax7; satellite cells doi:10.1152/ajpcell.00388.2009.

Sphingosine l-phosphate (S1P) is a bioactive lipid known to control cell growth that was recently shown to act as atrophic factor for skeletal muscle, reducing the progress of denervation atrophy. The aim of this work was to investigate whether S1P is involved in skeletal muscle fiber recovery (regeneration) after myotoxic injury induced by bupivacaine. The postnatal ability of skeletal muscle to grow and regenerate is dependent on resident stem cells called satellite cells. Immunofluorescence analysis demonstrated that S1P-specific receptors S1[P.sub.1] and S1[P.sub.3] are expressed by quiescent satellite cells. Soleus muscles undergoing regeneration following injury induced by intramuscular injection of bupivacaine exhibited enhanced expression of S1[P.sub.1] receptor, while S1[P.sub.3] expression progressively decreased to adult levels. S1[P.sub.2] receptor was absent in quiescent cells but was transiently expressed in the early regenerating phases only. Administration of SIP (50 [micro]M) at the moment of myotoxic injury caused a significant increase of the mean crosssectional area of regenerating fibers in both rat and mouse. In separate experiments designed to test the trophic effects of S1P, neutralization of endogenous circulating SIP by intraperitoneal administration of anti-S1P antibody attenuated fiber growth. Use of selective modulators of S1P receptors indicated that S1[P.sub.1] receptor negatively and S1[P.sub.3] receptor positively modulate the early phases of regeneration, whereas S1[P.sub.2] receptor appears to be less important. The present results show that SIP signaling participates in the regenerative processes of skeletal muscle. sphingosine 1-phosphate receptors; satellite cells doi:10.1152/ajpcell.00072.2009

Ge Y, Wu A, Warnes C, Liu J, Zhang C, Kawasome H, Terada N, Boppart MD, Schoenherr CJ, Chen J. mTOR regulates skeletal muscle regeneration in vivo through kinase-dependent and kinase-independent mechanisms. Am J Physiol Cell Physiol 297: C1434-C1444, 2009. First published September 30, 2009; doi: 10.1152/ajpcell.00248.2009.--Rapamycin- sensitive signaling is required for skeletal muscle differentiation and remodeling. In cultured myoblasts, the mammalian target of rapamycin (roTOR) has been reported to regulate differentiation at different stages through distinct mechanisms, including one that is independent of roTOR kinase activity. However, the kinase-independent function of roTOR remains controversial, and no in vivo studies have examined those roTOR myogenic mechanisms previously identified in vitro. In this study, we find that rapamycin impairs injury-induced muscle regeneration. To validate the role of mTOR with genetic evidence and to probe the mechanism of roTOR function, we have generated and characterized transgenic mice expressing two mutants of roTOR under the control of human skeletal actin (HSA) promoter: rapamycin-resistant (RR) and RR/kinase-inactive (RR/KI). Our results show that muscle regeneration in rapamycin-administered mice is restored by RR-mTOR expression. In the RR/KI-mTOR mice, nascent myofiber formation during the early phase of regeneration proceeds in the presence of rapamycin, but growth of the regenerating myofibers is blocked by rapamycin. Igf2 mRNA levels increase drastically during early regeneration, which is sensitive to rapamycin in wild-type muscles but partially resistant to rapamycin in both RR-and RR/KI-mTOR muscles, consistent with roTOR regulation of Igf2 expression in a kinase-independent manner. Furthermore, systemic ablation of S6K1, a target of roTOR kinase, results in impaired muscle growth but normal nascent myofiber formation during regeneration. Therefore, roTOR regulates muscle regeneration through kinase-independent and kinase-dependent mechanisms at the stages of nascent myofiber formation and myofiber growth, respectively. rapamycin; rapamycin-resistant; s6kl; mice; myogenesis doi: 10.1152/ajpcell.00248.2009

Regenerative coordination and remodeling of the intramuscular motoneuron network and neuromuscular connections are critical for restoring skeletal muscle function and physiological properties. The regulatory mechanisms of such coordination remain unclear, although both attractive and repulsive axon guidance molecules may be involved in the signaling pathway. Here we show that expression of a neural secreted chemorepellent semaphorin 3A (Sema3A) is remarkably upregulated in satellite cells of resident myogenic stem cells that are positioned beneath the basal lamina of mature muscle fibers, when treated with hepatocyte growth factor (HGF), established as an essential cue in muscle fiber growth and regeneration. When satellite cells were treated with HGF in primary cultures of cells or muscle fibers, Sema3A message and protein were upregulated as revealed by reverse transcription-polymerase chain reaction and immunochemical studies. Other growth factors had no inductive effect except for a slight effect of epidermal growth factor treatment. Sema3A upregulation was HGF dose dependent with a maximum (about 7- to 8-fold units relative to the control) at 10-25 ng/ml and occurred exclusively at the early-differentiation stage, as characterized by the level of myogenin expression and proliferation (bromodeoxyuridine incorporation) of the cells. Neutralizing antibody to the HGF-specific receptor, c-met, did not abolish the HGF response, indicating that c-met may not mediate the Sema3A expression signaling. Finally, in vivo Sema3A was upregulated in the differentiation phase of satellite cells isolated from muscle regenerating following crush injury. Overall, the data highlight a heretofore unexplored and active role for satellite cells as a key source of Sema3A expression triggered by HGF, hence suggesting that regenerative activity toward motor innervation may importantly reside in satellite cells and could be a crucial contributor during postnatal myogenesis. hepatocyte growth factor; motor neuron; muscle regeneration; semaphorin 3A

When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the cell cycle. This process depends on nitric oxide (NO) production by NO synthase (NOS), matrix metalloproteinase activation, release of hepatocyte growth factor (HGF) from the extraeellular matrix, and presentation of HGF to the c-met receptor as demonstrated by a primary culture and in vivo assays. We now add evidence that calcium-calmodulin is involved in the satellite cell activation cascade in vitro. Conditioned medium from cultures that were treated with a calcium ionophore (A23187, ionomycin) for 2 h activated cultured satellite cells and contained active HGF, similar to the effect of mechanical stretch or NO donor treatments. The response was abolished by addition of calmodulin inhibitors (calmidazolium, W-13, W-12) or a NOS inhibitor [N.sup.G]-nitro-L-arginine methyl ester hydrochloride but not by its less inactive enantiomer [N.sup.G]-nitro-Darginine methyl ester hydrochloride. Satellite cells were also shown to express functional calmodulin protein having a calcium-binding activity at 12 h postplating, which is the time at which the calcium ionophore was added in this study and the stretch treatment was applied in our previous experiments. Therefore, results from these experiments provide an additional insight that calcium-calmodulin mediates HGF release from the matrix and that this step in the activation pathway is upstream from NO synthesis. muscle regeneration; stretch-activation

In the adult skeletal muscle, various kinds of trauma promote proliferation of satellite cells that differentiate into myoblasts forming new myofibers or to repair the damaged one. The aim of present work was to perform a comparative spatial and temporal analysis of connexin (Cx) 37, Cx39, Cx40, Cx43, and Cx45 expression in the adult regenerating skeletal muscle in response to crush injury. Within 24 h from injury, Cx37 expression was upregulated in the endothelial cells of blood vessels, and, 5 days after injury, Cx37-expressing cells were found inside the area of lesion and formed clusters generating new blood vessels with endothelial cells expressing Cx37. Three days after injury, Cx39 mRNA was selectively expressed in myogenin-positive cells, forming rows of closely apposed cell nuclei fusing in myotubes. Cx40 mRNA-labeled cells were observed within 24 h from injury in the endothelium of blood vessels, and, 5 days after lesion, Cx40-labeled cells were found inside the area of lesion-forming rows of myogenin-positive, closely apposed cells coexpressing Cx39. Within 24 h from lesion, both Cx43 and Cx45 mRNAs were upregulated in individual cells, and some of them were positive for M-cadherin. Three days after injury, a large number of both Cx43 and Cx45 mRNA-labeled and myogenin-positive cells were found inside the area of lesion. Taken together, these results show that at least four Cxs, out of five expressed in regenerating skeletal muscle, can be differentially involved in communication of myogenic cells during the process of cell proliferation, aggregation, and fusion to form new myotubes or to repair damaged myofibers. connexin 37; connexin 39; connexin 40; connexin 43; connexin 45; myogenic cells; muscle regeneration

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 A[alpha] transgene (CnA[alpha]) was overexpressed in skeletal muscles of mdx (mdx can[[alpha].sup.*]) 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 CnA[[alpha].sup.*] and mdx mice was examined in vitro. Hindlimb muscles from mdx CnA[[alpha].sup.*] 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 CnA[[alpha].sup.*] than mdx mice. In the diaphragm, despite a slower phenotype and a ~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 CnA[[alpha].sup.*] 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. muscle regeneration; muscular dystrophy; skeletal muscle; muscle contraction

The present study was designed to test the hypothesis that increasing physical activity by running exercise could favor the recovery of muscle mass after extensive injury and to determine the main molecular mechanisms involved. Left soleus muscles of female Wistar rats were degenerated by notexin injection before animals were assigned to either a sedentary group or an exercised group. Both regenerating and contralateral intact muscles from active and sedentary rats were removed 5, 7, 14, 21, 28 and 42 days after injury (n = 8 rats/group). Increasing contractile activity through running exercise during muscle regeneration ensured the full recovery of muscle mass and muscle cross-sectional area as soon as 21 days after injury, whereas muscle weight remained lower even 42 days postinjury in sedentary rats. Proliferator cell nuclear antigen and MyoD protein expression went on longer in active rats than in sedentary rats. Myogenin protein expression was higher in active animals than in sedentary animals 21 days postinjury. The Akt-mammalian target of rapamycin (roTOR) pathway was activated early during the regeneration process, with further increases of mTOR phosphorylation and its downstream ellectors, eukaryotic initiation factor-4E-binding protein-I and [p70.sup.s6k], in active rats compared with sedentary rats (days 7-14). The exerciseinduced increase in mTOR phosphorylation, independently of Akt, was associated with decreased levels of phosphorylated AMP-activated protein kinase. Taken together, these results provided evidence that increasing contractile activity during muscle regeneration ensured early and full recovery of muscle mass and suggested that these beneficial effects may be due to a longer proliferative step of myogenic cells and activation of mTOR signaling, independently of Akt, during the maturation step of muscle regeneration. muscle regeneration; myogenic regulatory factors; mitogen-activated protein kinases; mammalian target of rapamycin pathway; AMPactivated protein kinase

Stimulating the [beta]-adrenoceptor ([beta]-AR) signaling pathway can enhance the functional repair of skeletal muscle after injury, but long-term use of [beta]-AR agonists causes [beta]-AR downregulation, which may limit their therapeutic effectiveness. The aim was to examine [beta]-AR signaling during early regeneration in rat fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles after bupivacaine injury and test the hypothesis that, during regeneration, [beta]-agonist administration does not cause [beta]-AR desensitization. Rats received either the [beta]-AR agonist fenoterol (1.4 mg x [kg.sup.-1] x [day.sup.-1] ip) or saline for 7 days postinjury. Fenoterol reduced [beta]-AR density in regenerating soleus muscles by 42%. Regenerating EDL muscles showed a threefold increase in [beta]-AR density, and, again, these values were 43% lower with fenoterol treatment. An amplified adenylate cyclase (AC) response to isoproterenol was observed in cell membrane fragments from EDL and soleus muscles 7 days postinjury. Fenoterol attenuated this increase in regenerating EDL muscles but not soleus muscles. [beta]-AR signaling mechanisms were assessed using AC stimulants (NaF, forskolin, and [Mn.sup.2+]). Although [beta]-agonist treatment reduces [beta]-AR density in regenerating muscles, these muscles can produce large cAMP responses relative to healthy (uninjured) muscles. Desensitization of [beta]-AR signaling in regenerating muscles is prevented by altered rates of [beta]-AR synthesis and/or degradation, changes in G protein populations and coupling efficiency, and altered AC activity. These mechanisms have important therapeutic implications for modulating [beta]-AR signaling to enhance muscle repair after injury. muscle regeneration; [beta]-adrenoceptor; muscle function; muscle injury; adenylate cyclase; G protein

The hypothesis of this study was the urokinase-type plasminogen activator receptor (uPAR) is required for accumulation of inflammatory cells in injured skeletal muscle and for efficient muscle regeneration. Expression of uPAR was elevated at 1 and 3 days after cardiotoxin-induced muscle injury in wild-type mice before returning to baseline levels. Neutrophil accumulation peaked 1 day postinjury in muscle from both wild-type (WT) and uPAR null mice, while macrophage accumulation peaked between 3 and 5 days postinjury, with no differences between strains. Histological analyses confirmed efficient muscle regeneration in both wild-type and uPAR null mice, with no difference between strains in the formation or growth of regenerating fibers, or recovery of normal morphology. Furthermore, in vitro experiments demonstrated that chemotaxis is not different between WT and uPAR null macrophages. Finally, fusion of cultured satellite cells into multinucleated myotubes was not different between cells isolated from WT and uPAR null mice. These results demonstrate that uPAR is not required for the accumulation of inflammatory cells or the regeneration of skeletal muscle following injury, suggesting uPA can act independently of uPAR to regulate events critical for muscle regeneration. muscle repair; macrophage; chemotaxis

The regulation of vascular endothelial growth factor (VEGF) levels and angiogenic events during skeletal muscle regeneration remains largely unknown. This study examined angiogenesis, VEGF levels, and muscle regeneration after cardiotoxin (CT)-induced injury in mice lacking the CC chemokine receptor 2 (CCR2). Muscle regeneration was significantly decreased in CCR2-/- mice as was the early accumulation of macrophages after injury. In both mouse strains, tissue VEGF was similar at baseline (no injections) and significantly decreased at day 3 post-CT. Tissue VEGF in wild-type (WT) mice was restored within 7 days postinjury but remained significantly reduced in CCR2-/- mice until day 21. Capillary density (capillaries/[mm.sup.2]) within regenerating muscle was maximal in WT mice at day 7 and double that of baseline muscle. In comparison, maximal capillary density in CCR2-/- mice occurred at 21 clays postinjury. Maximal capillary density developed concurrent with the restoration of tissue VEGF in both strains. A highly significant, inverse relationship existed between the size of regenerated muscle fibers and capillaries per square millimeter. Although this relationship was comparable in WT and CCR2-/- animals, there was a significant decrease in the magnitude of this response in the absence of CCR2, reflecting the observation that regenerated muscle fiber size in CCR2-/- mice was only 50% of baseline at 42 days postinjury, whereas WT mice had attained baseline fiber size by day 21. Thus CCR2-dependent events in injured skeletal muscle, including impaired macrophage recruitment, contribute to restoration of tissue VEGF levels and the dynamic processes of capillary formation and muscle regeneration. adipogenesis; angiogenesis; CCL2; muscle regeneration; monocyte/ macrophage

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 (MCKCnA[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. myotoxic injury; skeletal muscle; muscle function

Muscle atrophy contributes to morbidity and mortality in aging and chronic disease, emphasizing the need to gain understanding of the mechanisms involved in muscle atrophy and (re)growth. We hypothesized that the magnitude of muscle regrowth during recovery from atrophy determines whether myonuclear accretion and myogenic differentiation are required and that insulin-like growth factor (IGF)-I/Akt/glycogen synthase kinase (GSK)-3[3 signaling differs between regrowth responses. To address this hypothesis we subjected mice to hindlimb suspension (HS) to induce atrophy of soleus (-40%) and plantaris (-27%) muscle. Reloading-induced muscle regrowth was complete after 14 days and involved an increase in IGF-IEa mRNA expression that coincided with Akt phosphorylation in both muscles. In contrast, phosphorylation and inactivation of GSK-3[beta] were observed during soleus regrowth only. Furthermore, soleus but not plantaris regrowth involved muscle regeneration based on a transient increase in expression of histone 3.2 and myosin heavy chain-perinatal, which are markers of myoblast proliferation and differentiation, and a strong induction of muscle regulatory factor (MRF) expression. Experiments in cultured muscle cells showed that IGF-I-induced MRF expression is facilitated by inactivation of GSK-3[beta] and selectively occurs in the myoblast population. This study suggests that induction of IGF-I expression and Akt phosphorylation during recovery from muscle atrophy is independent of the magnitude of muscle regrowth. Moreover, our data demonstrate for the first time that the regenerative response characterized by myoblast proliferation, differentiation, and increased MRF expression in recovering muscle is associated with the magnitude of regrowth and may be regulated by inactivation of GSK-3[beta]. glycogen synthase kinase-3[beta]; Akt; muscle growth; muscle atrophy doi:10.1152/ajpcell.00504.2006.

Although p38 MAPK activation is essential for myogenesis, the upstream signaling mechanism that activates p38 during myogenesis remains undefined. We recently reported that p38 activation, myogenesis, and regeneration in cardiotoxin-injured soleus muscle are impaired in TNF-[alpha] receptor double-knockout (p[55.sup.-/-]p[75.sup.-/-]) mice. To fully evaluate the role of TNF-[alpha] in myogenic activation of p38, we tried to determine whether p38 activation in differentiating myoblasts requires autocrine TNF-[alpha], and whether forced activation of p38 rescues impaired myogenesis and regeneration in the p[55.sup.-/-]p[75.sup.-/-] soleus. We observed an increase of TNF-[alpha] release from C2C12 or mouse primary myoblasts placed in low-serum differentiation medium. A TNF-[alpha]-neutralizing antibody added to differentiation medium blocked p38 activation and suppressed differentiation markers myocyte enhancer factor (MEF)-2C, myogenin, p21, and myosin heavy chain in C2C12 myoblasts. Conversely, recombinant TNF-[alpha] added to differentiation medium stimulated myogenesis at 0.05 ng/ml while inhibited it at 0.5 and 5 ng/ml. In addition, differentiation medium-induced p38 activation and myogenesis were compromised in primary myoblasts prepared from p[55.sup.-/-]p[75.sup.-/-] mice. Increased TNF-[alpha] release was also seen in cardiotoxin-injured soleus over the course of regeneration. Forced activation of p38 via the constitutive activator of p38, MKK6bE, rescued impaired myogenesis and regeneration in the cardiotoxin-injured p[55.sup.-/-]p[75.sup.-/-] soleus. These results indicate that TNF-[alpha] regulates myogenesis and muscle regeneration as a key activator of p38. myocyte enhancer factor-2C; myogenin; p21; myosin heavy chain; Akt; tumor necrosis factor-[alpha]; mitogen-activated protein kinase doi: 10.1152/ajpcell.00486.2006.

In the present study, we examined the roles of hepatocyte growth factor (HGF) and nitric oxide (NO) in the activation of satellite cells in passively stretched rat skeletal muscle. A hindlimb suspension model was developed in which the vastus, adductor, and gracilis muscles were subjected to stretch for 1 h. Satellite cells were activated by stretch determined on the basis of 5-bromo-2'-deoxyuridine (BrdU) incorporation in vivo. Extracts from stretched muscles stimulated BrdU incorporation in freshly isolated control rat satellite cells in a concentration-dependent manner. Extracts from stretched muscles contained the active form of HGF, and the satellite cell-activating activity could be neutralized by incubation with anti-HGF antibody. The involvement of NO was investigated by administering nitro-L-arginine methyl ester (L-NAME) or the inactive enantiomer [N.sup.G]-nitro-D-arginine methyl ester HC1 (D-NAME) before stretch treatment. In vivo activation of satellite cells in stretched muscle was not inhibited by D-NAME but was inhibited by L-NAME. The activity of stretched muscle extract was abolished by L-NAME treatment but could be restored by the addition of HGF, indicating that the extract was not inhibitory. Finally, NO synthase activity in stretched and unstretched muscles was assayed in muscle extracts immediately after 2-h stretch treatment and was found to be elevated in stretched muscle but not in stretched muscle from L-NAME-treated rats. The results of these experiments demonstrate that stretching muscle liberates HGF in a NO-dependent manner, which call activate satellite cells. muscle regeneration

Muscle precursor cells (MPCs) are required for the regrowth, regeneration, and/or hypertrophy of skeletal muscle, which are deficient in sarcopenia. In the present investigation, we have addressed the issue of age-associated changes in MPC differentiation. MPCs, including satellite cells, were isolated from both young and old rat skeletal muscle with a high degree of myogenic purity (>90% MyoD and desmin positive). MPCs isolated from skeletal muscle of 32-mo-old rats exhibited decreased differentiation into myotubes and demonstrated decreased myosin heavy chain (MHC) and muscle creatine kinase (CK-M) expression compared with MPCs isolated from 3-mo-old rats. [p27.sup.Kip1] is a cyclin-dependent kinase inhibitor that has been shown to enhance muscle differentiation in culture. Herein we describe our finding that [p27.sup.Kip1] protein was lower in differentiating MPCs from skeletal muscle of 32-mo-old rats than in 3-mo-old rat skeletal muscle. Although MHC and CK-M expression were ~50% lower in differentiating MPCs isolated from 32-mo-old rats, MyoD protein content was not different and myogenin protein concentration was twofold higher. These data suggest that there are inherent differences in cell signaling during the transition from cell cycle arrest to the formation of myotubes in MPCs isolated from sarcopenic muscle. Furthermore, there is an age-associated decrease in muscle-specific protein expression in differentiating MPCs despite normal MyoD and elevated myogenin levels. satellite cells; skeletal muscle; [p27.sup.Kip1]; myogenic regulatory factors

The successful use of myogenic progenitor cells for therapeutic applications requires an understanding of the intrinsic and extrinsic cues involved in their regulation. Herein we demonstrate the expression pattern and transcriptional regulation of Rad, a prototypical member of a family of novel Ras-related GTPases, during mammalian development and skeletal muscle regeneration. Rad was identified using microarray analysis, which revealed robust upregulation of its expression during skeletal muscle regeneration. Our current findings demonstrate negligible Rad expression with resting adult skeletal muscle; however, after muscle injury, Rad is expressed within the myogenic progenitor cell population. Rad expression is significantly increased and localized to the myogenic progenitor cell population during the early phases of regeneration and within the newly regenerated myofibers during the later phases of regeneration. Immunohistochemical analysis demonstrated that Rad and MyoD are coexpressed within the myogenic progenitor cell population of regenerating skeletal muscle. This expression profile of Rad during skeletal muscle regeneration is consistent with the proposed roles for Rad in the inhibition of L-type C[a.sup.2+] channel activity and the inhibition of Rho/RhoA kinase activity. We also have demonstrated that known myogenic transcription factors (MEF2, MyoD, and Myf-5) can increase the transcriptional activity of the Rad promoter and that this ability is significantly enhanced by the presence of the C[a.sup.2+]-dependent phosphatase calcineurin. Furthermore, this enhanced transcriptional activity appears to be dependent on the presence of a conserved NFAT binding motif within the Rad promoter. Taken together, these data define Rad as a novel factor within the myogenic progenitor cells of skeletal muscle and identify key regulators of its transcriptional activity. satellite cell; myoblast; stem cell; transcriptional regulation; RGK protein

Under normal circumstances, mammalian adult skeletal muscle is a stable tissue with very little turnover of nuclei. However, upon injury, skeletal muscle has the remarkable ability to initiate a rapid [...]

To investigate the cause of skeletal muscle weakening during aging we examined the sequence of cellular changes in murine muscles. Satellite cells isolated from single muscle fibers terminally differentiate progressively less well with increasing age of donor. This change is detected before decline in satellite cell numbers and all histological changes examined here. In MSVski transgenic mice, which show type IIb fiber hypertrophy, initial muscle weakness is followed by muscle degeneration in the first year of life. This degeneration is accompanied by a spectrum of changes typical of normal muscle aging and a more marked decline in satellite cell differentiation efficiency. On a myoD-null genetic background, in which satellite cell differentiation is defective, the MSVski muscle phenotype is aggravated. This suggests that, on a wild-type genetic background, satellite cells are capable of repairing MSVski fibers and preserving muscle integrity in early life. We propose that decline in myogenic cell differentiation efficiency is an early event in aging-related loss of muscle function, both in normal aging and in some late-onset muscle degenerative conditions. muscle regeneration; MyoD; myonuclear domain size

Researchers have investigated the temporal expression pattern of messenger RNAs for insulin-like growth factor (IGF)-I, IGF-I receptor and IGF-II in the regenerating tibialis anterior muscle following injection of the myotoxic anesthetic bupivacaine in young, adult and old rats. Five days after injection, IGF-II mRNA was boosted 46-fold in young rats, but only fourfold in adult rats. The results indicate that the impairment of muscle regeneration is a maturation effect, rather than an aging effect.

The specific role of weight bearing on the expression of myosin heavy chain (MHC) isoforms in regenerative fibers after muscle degeneration was investigated using rat soleus muscles. Immunocytochemical analysis showed that almost all fibers reacted positively with the MHC antibody, which has been reported to be associated with slow-twitch muscle fibers. However, removal of weight bearing resulted in the dramatic increase in MHC expression. These results suggested that the regulation of expression of MHC isoforms is more complete and homogenous in regenerating than degenerate muscles.

Hypertension in spontaneously hypertensive rats (SHR) has no effect on the revascularization of a nerve-intact graft of the extensor digitorum longus muscle, but muscle regeneration and maturation are severely impaired. Reinnervation and neuromuscular transmission of the graft in the SHR are similar to those of a graft in Wistar-Kyoto rats. The slower regeneration in the SHR may be due to insulin resistance. Muscle insulin receptors bind the insulin-like growth factor I, which helps in development.

Gamma irradiation-induced inhibition of muscle regeneration leads to loss of contractile strength in dytrophic muscles from mdx mice, which is stopped by treatment with clenbuterol, an adrenergic beta-receptor. Myosin concentrations and muscle mass are more in the clenbuterol-treated irradiated muscles than in non-irradiated muscles, indicating that beta2-agonists inhibit degenerative processes in dystrophin-depleted muscle fibers.

Myocardial infarction (MI) initiates adaptive tissue remodeling, which is essential for heart function (such as infarct healing) but is also important for maladaptive remodeling (for example, reactive fibrosis and left ventricular dilation). The effect of aldosterone receptor antagonism on these processes was evaluated in Sprague-Dawley rats using eplerenone, a selective aldosterone receptor antagonist. Infarct healing and left ventricular remodeling were evaluated at 3, 7, and 28 days after MI by determination of the diastolic pressure-volume relationship of the left ventricle, the infarct-thinning ratio, and the collagen-volume fraction. Eplerenone did not affect reparative collagen deposition as was evidenced by a similar collagen volume fraction in the infarcted myocardium between eplerenone and vehicle-treated groups at 7 and 28 days post-MI. In addition, the thinning ratio, which is an index of infarct expansion, was comparable between the eplerenone and vehicle-treated animals at 7 and 28 days post-MI. A protective effect of eplerenone was demonstrated at 28 days post-MI, where reactive fibrosis in the viable myocardium was reduced in eplerenone-treated animals compared with vehicle-treated animals. Thus aldosterone receptor antagonism does not retard infarct healing but rather protects against maladaptive responses after MI. fibrosis; left ventricular remodeling; eplerenone

Neutrophils injure cultured skeletal myotubes. Am J Physiol Cell Physiol 281: C335-C341, 2001.--The purpose of the study was to test the hypothesis that neutrophils can injure cultured skeletal myotubes. Human myotubes were grown and then cultured with human blood neutrophils. Myotube injury was quantitatively and qualitatively determined using a cytotoxicity ([sup.51]Cr) assay and electron microscopy, respectively. For the [sup.51]Cr assay, neutrophils, under non-in vitro-stimulated and N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated conditions, were cultured with myotubes at effector-to-target cell (E:T) ratios of 10, 30, and 50 for 6 h. Statistical analyses revealed that myotube injury was proportional to the E:T ratio and was greater in FMLP-stimulated conditions relative to non-in vitro-stimulated conditions. Transmission electron microscopy, using lanthanum as an extracellular tracer, revealed in cocultures a diffuse appearance of lanthanum in the cytoplasm of myotubes and a localized appearance within cytoplasmic vacuoles of myotubes. These observations and their absence in control cultures (myotubes only) suggest that neutrophils caused membrane rupture and increased myotube endocytosis, respectively. Myotube membrane blebs were prevalent in scanning and transmission electron micrographs of cultures consisting of neutrophils and myotubes (E:T ratio of 5) and were absent in control cultures. These data support the hypothesis that neutrophils can injure skeletal myotubes in vitro and may indicate that neutrophils exacerbate muscle injury and/or delay muscle regeneration in vivo. chromium assay; electron microscopy; muscle injury; muscle regeneration

Preconditioning improves function and recovery of single muscle fibers during severe hypoxia and reoxygenation. Am J Physiol Cell Physiol 281: C142-C146, 2001.--Reperfusion following prolonged ischemia induces cellular damage in whole skeletal muscle models. Ischemic preconditioning attenuates the deleterious effects. We tested whether individual skeletal muscle fibers would be similarly affected by severe hypoxia and reoxygenation (H/R) in the absence of extracellular factors and whether cellular damage could be alleviated by hypoxic preconditioning. Force and free cytosolic [Ca.sup.2+] ([[[Ca.sup.2+]].sub.c]) were monitored in Xenopus single muscle fibers (n = 24) contracting tetanically at 0.2 Hz during 5 min of severe hypoxia and 5 min of reoxygenation. Twelve cells were preconditioned by a shorter bout of H/R 1 h before the experimental trial. In preconditioned cells, force relative to initial maximal values (P/[P.sub.o]) and relative peak [[[Ca.sup.2+]].sub.c] fell (P [is less than] 0.05) during 5 min of hypoxia and recovered during reoxygenation. In contrast, P/[P.sub.o] and relative peak [[[Ca.sup.2+]].sub.c] fell more during hypoxia (P [is less than] 0.05) and recovered less during reoxygenation (P [is less than] 0.05) in control cells. The ratio of force to [[[Ca.sup.2+]].sub.c] was significantly higher in the pre-conditioned cells during severe hypoxia, suggesting that changes in [[[Ca.sup.2+]].sub.c] were not solely responsible for the loss in force. We conclude that 1) isolated skeletal muscle fibers contracting in the absence of extracellular factors are susceptible to H/R injury associated with changes in [Ca.sup.2+] handling; and 2) hypoxic preconditioning improves contractility, [Ca.sup.2+] handling, and cell recovery during subsequent hypoxic insult. skeletal muscle; postischemic injury; cytosolic calcium; ischemia; reperfusion