cobra-cardiotoxin-proteins and Muscular-Diseases

The aim of the present study was to elucidate whether the recovery from muscle injury is impaired in marginal zinc deficiency. C57BL/6 male mice were fed a marginally zinc-deficient diet (MZD: 8 mg Zn/kg diet), a zinc-adequate diet (ZA: zinc 35 mg Zn/kg diet), and a zinc-high diet (ZH: 190 mg Zn/kg diet) for 4 weeks. Muscle injury was induced in the gastrocnemius muscles using cardiotoxin. The gastrocnemius muscles of these mice were harvested at 3, 5, 7, 10, 14, and 20 days after injury. We evaluated the regeneration of the skeletal muscle with hematoxylin and eosin staining and developmental myosin heavy-chain (dMHC: implicated in regeneration) immunostaining. The rate of dMHC-positive cells was significantly low in MZD mice compared with ZA mice at 3 days after cardiotoxin injection. The peak dMHC expression was found at 3 days after injection in ZA mice, 5 days in ZH mice, and 7 days in MZD mice. These results suggest that recovery from muscle injury might be partly impaired and delayed in MZD mice. Therefore, we strongly suggest the appropriate zinc intake to prevent the impairment of skeletal muscle regeneration.

Topics: Animals; Cobra Cardiotoxin Proteins; Diet; Immunohistochemistry; Male; Mice, Inbred C57BL; Muscle, Skeletal; Muscular Diseases; Myosin Heavy Chains; Organ Size; Regeneration; Zinc

Selenoprotein N (SelN) deficiency causes a group of inherited neuromuscular disorders termed SEPN1-related myopathies (SEPN1-RM). Although the function of SelN remains unknown, recent data demonstrated that it is dispensable for mouse embryogenesis and suggested its involvement in the regulation of ryanodine receptors and/or cellular redox homeostasis. Here, we investigate the role of SelN in satellite cell (SC) function and muscle regeneration, using the Sepn1(-/-) mouse model. Following cardiotoxin-induced injury, SelN expression was strongly up-regulated in wild-type muscles and, for the first time, we detected its endogenous expression in a subset of mononucleated cells by immunohistochemistry. We show that SelN deficiency results in a reduced basal SC pool in adult skeletal muscles and in an imperfect muscle restoration following a single injury. A dramatic depletion of the SC pool was detected after the first round of degeneration and regeneration that totally prevented subsequent regeneration of Sepn1(-/-) muscles. We demonstrate that SelN deficiency affects SC dynamics on isolated single fibres and increases the proliferation of Sepn1(-/-) muscle precursors in vivo and in vitro. Most importantly, exhaustion of the SC population was specifically identified in muscle biopsies from patients with mutations in the SEPN1 gene. In conclusion, we describe for the first time a major physiological function of SelN in skeletal muscles, as a key regulator of SC function, which likely plays a central role in the pathophysiological mechanism leading to SEPN1-RM.

Topics: Animals; Cell Proliferation; Cell Survival; Cells, Cultured; Cobra Cardiotoxin Proteins; Disease Models, Animal; Mice; Mice, Knockout; Muscle, Skeletal; Muscular Diseases; Mutation; Regeneration; Satellite Cells, Skeletal Muscle; Selenoproteins

The transcription factor Pax7 is a marker and regulator of muscle progenitors and satellite cells that contribute to the embryonic development and postembryonic growth of skeletal muscle in vertebrates, as well as to its repair and regeneration. Here, we identify Pax7(+ve) myogenic cells in the zebrafish and characterize their behavior in postembryonic stages. Mononucleate Pax7(+ve) cells can first be found associated with myofibers at 72 hours post fertilization (hpf). To follow the behavior of muscle progenitor cells in vivo, we generated transgenic lines expressing fluorescent proteins under the control of the pax7a or pax3a promoters. We established an injury model using cardiotoxin injection and monitored cell proliferation and myogenic regulatory factor expression in myogenic precursors cells and muscle fibers after injury using proliferation markers and the transgenic lines. We also analyzed Pax7(+ve) cells in animals with dystrophic phenotypes and found an increased number compared with wild-type.

Topics: Animals; Animals, Genetically Modified; Cobra Cardiotoxin Proteins; Disease Models, Animal; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Genes, Reporter; Humans; Muscle Development; Muscle, Skeletal; Muscular Diseases; Muscular Dystrophy, Duchenne; Myoblasts; PAX7 Transcription Factor; Zebrafish

The insulin-like family of factors are involved in the regulation of a variety of physiological processes, but the function of the family member termed insulin-like 6 (Insl6) in skeletal muscle has not been reported. We show that Insl6 is a myokine that is up-regulated in skeletal muscle downstream of Akt signaling and in regenerating muscle in response to cardiotoxin (CTX)-induced injury. In the CTX injury model, myofiber regeneration was improved by the intramuscular or systemic delivery of an adenovirus expressing Insl6. Skeletal muscle-specific Insl6 transgenic mice exhibited normal muscle mass under basal conditions but elevated satellite cell activation and enhanced muscle regeneration in response to CTX injury. The Insl6-mediated regenerative response was associated with reductions in muscle cell apoptosis and reduced serum levels of creatine kinase M. Overexpression of Insl6 stimulated proliferation and reduced apoptosis in cultured myogenic cells. Conversely, knockdown of Insl6 reduced proliferation and increased apoptosis. These data indicate that Insl6 is an injury-regulated myokine that functions as a myogenic regenerative factor.

Topics: Animals; Apoptosis; Blotting, Western; Cell Line; Cobra Cardiotoxin Proteins; Female; Gene Expression Profiling; Gene Expression Regulation; HEK293 Cells; Humans; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle, Skeletal; Muscular Diseases; Oligonucleotide Array Sequence Analysis; Proto-Oncogene Proteins c-akt; Regeneration; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Satellite Cells, Skeletal Muscle; Time Factors

Dystrophic calcifications often occur after injury, infection, or onset of certain rheumatic diseases. Treatment has been limited to surgical removal following failure of medical therapy. In an attempt to establish a reproducible animal model for dystrophic calcification that permitted the screening of potential interventions, we evaluated cardiotoxin (injury)-induced calcifications in three murine strains at both the cellular and ultrastructural levels. All osteopontin null mice and tumor necrosis factor receptor null mice on a C57B6 background had calcifications at days 3 and 7 after injury compared to 75% of wild-type C57B6 mice. There was no difference in mineral content among calcifications from the three mouse strains. Osteogenesis was suggested by the expression of osteocalcin, osterix, and alkaline phosphatase in calcified murine muscle tissue. Osteoclast-like cells facilitated the removal of transient dystrophic deposits (<28 days) in all models. However, none of the models showed an association of mineral crystals with collagen, suggesting that the deposits were not bone-like. The dystrophic mechanism was validated as cell death, and mitochondrial calcifications occurred soon after skeletal muscle injury in the three murine strains.

Topics: Alkaline Phosphatase; Animals; Bone Matrix; Calcinosis; Cell Death; Cobra Cardiotoxin Proteins; Collagen; Disease Models, Animal; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Mitochondrial Diseases; Muscle, Skeletal; Muscular Diseases; Osteogenesis; Osteopontin; Receptors, Tumor Necrosis Factor; Sp7 Transcription Factor; Transcription Factors

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 (p55(-/-)p75(-/-)) 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 p55(-/-)p75(-/-) 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 p55(-/-)p75(-/-) 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 p55(-/-)p75(-/-) soleus. These results indicate that TNF-alpha regulates myogenesis and muscle regeneration as a key activator of p38.

Topics: Animals; Autocrine Communication; Cell Differentiation; Cell Line; Cobra Cardiotoxin Proteins; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; MAP Kinase Kinase 6; Mice; Mice, Knockout; Muscle Development; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Diseases; Myoblasts; p38 Mitogen-Activated Protein Kinases; Receptors, Tumor Necrosis Factor, Type I; Receptors, Tumor Necrosis Factor, Type II; Regeneration; Tumor Necrosis Factor-alpha

Different neuromyotoxic agents are frequently used in rodent models of skeletal nerve/muscle injury and repair. However, their differential effects are not well known. Right Tibialis anterior muscles of mice were injured by one of three different neuromyotoxic agents: crude venom from Notechis scutatus, cardiotoxin from Naja atra or bupivacaine (local anesthetic). Mice were studied 5, 14 and 56 days after injury by analysing the recovery of in situ muscle isometric function in response to nerve stimulation, muscle weights and muscle histology. Our results show that at day 5 venom treatment had a more debilitating effect on muscle weights and maximal tetanic force than cardiotoxin and bupivacaine treatments (p<0.05). Moreover, the degree of recovery of muscle parameters 14 days after neuromyotoxic treatment varies as follow: venom

Topics: Analysis of Variance; Animals; Bupivacaine; Cobra Cardiotoxin Proteins; Elapid Venoms; Male; Mice; Muscular Diseases; Neuromuscular Junction; Peripheral Nervous System Diseases; Recovery of Function; Time Factors

Xin is a muscle-specific actin binding protein of which its role and regulation within skeletal muscle is not well understood. Here we demonstrate that Xin mRNA is robustly upregulated (>16-fold) within 12 h of skeletal muscle injury and is localized to the muscle satellite cell population. RT-PCR confirmed the expression pattern of Xin during regeneration, as well as within primary muscle myoblast cultures, but not other known stem cell populations. Immunohistochemical staining of single myofibers demonstrate Xin expression colocalized with the satellite cell marker Syndecan-4 further supporting the mRNA expression of Xin in satellite cells. In situ hybridization of regenerating muscle 5-7 days postinjury illustrates Xin expression within newly regenerated myofibers. Promoter-reporter assays demonstrate that known myogenic transcription factors [myocyte enhancer factor-2 (MEF2), myogenic differentiation-1 (MyoD), and myogenic factor-5 (Myf-5)] transactivate Xin promoter constructs supporting the muscle-specific expression of Xin. To determine the role of Xin within muscle precursor cells, proliferation, migration, and differentiation analysis using Xin, short hairpin RNA (shRNA) were undertaken in C2C12 myoblasts. Reducing endogenous Xin expression resulted in a 26% increase (P < 0.05) in cell proliferation and a 20% increase (P < 0.05) in myoblast migratory capacity. Skeletal muscle myosin heavy chain protein levels were increased (P < 0.05) with Xin shRNA administration; however, this was not accompanied by changes in myoglobin protein (another marker of differentiation) nor overt morphological differences relative to differentiating control cells. Taken together, the present findings support the hypothesis that Xin is expressed within muscle satellite cells during skeletal muscle regeneration and is involved in the regulation of myoblast function.

Topics: Animals; Cell Line; Cell Movement; Cell Proliferation; Cobra Cardiotoxin Proteins; Disease Models, Animal; DNA-Binding Proteins; Genes, Reporter; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Mice, Inbred mdx; Muscle Development; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Diseases; Myogenic Regulatory Factors; Nuclear Proteins; Promoter Regions, Genetic; Regeneration; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; RNA, Small Interfering; Satellite Cells, Skeletal Muscle; Syndecan-4; Time Factors; Transcriptional Activation; Up-Regulation

The purpose of this study was to investigate the effects of functional overload on the regeneration of injured skeletal muscles of male C57BL/6J mice. To activate a necrosis-regeneration cycle, cardiotoxin (CTX) was injected into soleus muscles both control and functionally overloaded groups. The recovery of muscle protein content, which was decreased by CTX injection, was significantly stimulated by application of functional overloading. The CTX-injection-related increment of satellite cell number in the overloaded groups was also greater than that in the group without overloading. Evidences suggest that the application of a mechanical stress on the injured skeletal muscles could activate satellite cells and facilitate the regeneration of the muscle.

Topics: Animals; Cell Proliferation; Cobra Cardiotoxin Proteins; Disease Models, Animal; Male; Mice; Mice, Inbred C57BL; Muscle Proteins; Muscle, Skeletal; Muscular Diseases; Necrosis; Organ Size; PAX7 Transcription Factor; Regeneration; Satellite Cells, Skeletal Muscle; Stress, Mechanical; Time Factors

Recent data suggest a physiological role for the proinflammatory cytokine TNF-alpha in skeletal muscle regeneration. However, the underlying mechanism is not understood. In the present study, we analyzed TNF-alpha-activated signaling pathways involved in myogenesis in soleus muscle injured by cardiotoxin (CTX) in TNF-alpha receptor double-knockout mice (p55(-/-)p75(-/-)). We found that activation of p38MAPK, which is critical for myogenesis, was blocked in CTX-injured p55(-/-)p75(-/-) soleus on day 3 postinjury when myogenic differentiation was being initiated, while activation of ERK1/2 and JNK MAPK, as well as transcription factor NF-kappaB, was not reduced. Consequently, the phosphorylation of transcription factor myocyte enhancer factor-2C, which is catalyzed by p38 and crucial for the expression of muscle-specific genes, was blunted. Meanwhile, expression of p38-dependent differentiation marker myogenin and p21 were suppressed. In addition, expression of cyclin D1 was fivefold that in wild-type (WT) soleus. These results suggest that myogenic differentiation is blocked or delayed in the absence of TNF-alpha signaling. Histological studies revealed abnormalities in regenerating p55(-/-)p75(-/-) soleus. On day 5 postinjury, new myofiber formation was clearly observed in WT soleus but not in p55(-/-)p75(-/-) soleus. To the contrary, p55(-/-)p75(-/-) soleus displayed renewed inflammation and dystrophic calcification. On day 12 postinjury, the muscle architecture of WT soleus was largely restored. Yet, in p55(-/-)p75(-/-) soleus, multifocal areas of inflammation, myofiber death, and myofibers with smaller cross-sectional area were observed. Functional studies demonstrated an attenuated recovery of contractile force in injured p55(-/-)p75(-/-) soleus. These data suggest that TNF-alpha signaling plays a critical regulatory role in muscle regeneration.

Topics: Animals; Cobra Cardiotoxin Proteins; Enzyme Activation; Mice; Mice, Knockout; Muscle Contraction; Muscle Development; Muscle, Skeletal; Muscular Diseases; p38 Mitogen-Activated Protein Kinases; Receptors, Tumor Necrosis Factor; Regeneration; Signal Transduction; Tumor Necrosis Factor-alpha