Glutamine supplementation accelerates functional recovery of EDL muscles after injury by modulating the expression of S100 calcium-binding proteins.

The aim of the current study was to investigate the effect of glutamine supplementation on the expression of HSP70 and the calcium-binding proteins from the S100 superfamily in the recovering extensor digitorum longus (EDL) muscle after injury. Two-month-old Wistar rats were subjected to cryolesion of the EDL muscle and then randomly divided into two groups (with or without glutamine supplementation). Starting immediately after the injury, the supplemented group received daily doses of glutamine (1 g/kg/day, via gavage) for 3 and 10 days orally. Then, muscles were subjected to histological, molecular, and functional analysis. Glutamine supplementation induced an increase in myofiber size of regenerating EDL muscles and prevented the decline in maximum tetanic strength of these muscles evaluated 10 days after injury. An accelerated upregulation of myogenin mRNA levels was detected in glutamine-supplemented injured muscles on day 3 post-cryolesion. The HSP70 expression increased only in the injured group supplemented with glutamine for 3 days. The increase in mRNA levels of NF-κB, the pro-inflammatory cytokines IL-1ß and TNF-α, and the calcium-binding proteins S100A8 and S100A9 on day 3 post-cryolesion in EDL muscles was attenuated by glutamine supplementation. In contrast, the decrease in S100A1 mRNA levels in the 3-day-injured EDL muscles was minimized by glutamine supplementation. Overall, our results suggest that glutamine supplementation accelerates the recovery of myofiber size and contractile function after injury by modulating the expression of myogenin, HSP70, NF-κB, pro-inflammatory cytokines, and S100 calcium-binding proteins.

microRNA-501 controls myogenin+/CD74+ myogenic progenitor cells during muscle regeneration.

OBJECTIVE: Skeletal muscle regeneration is markedly impaired during aging. How adult muscle stem cells contribute to this decrease in regenerative capacity is incompletely understood. We investigated mechanisms of age-related changes in myogenic progenitor cells using the tissue-specific microRNA 501. METHODS: Young and old C57Bl/6 mice were used (3 months or 24 months of age, respectively) with or without global or tissue-specific genetic deletion of miR-501. Muscle regeneration was induced using intramuscular cardiotoxin injection or treadmill exercise and analysed using single cell and bulk RNA sequencing, qRT-PCR and immunofluorescence. Muscle fiber damage was assessed with Evan`s blue dye (EBD). In vitro analysis was performed in primary muscle cells obtained from mice and humans. RESULTS: Single cell sequencing revealed myogenic progenitor cells in miR-501 knockout mice at day 6 after muscle injury that are characterized by high levels of myogenin and CD74. In control mice these cells were less in number and already downregulated after day 3 of muscle injury. Muscle from knockout mice had reduced myofiber size and reduced myofiber resilience to injury and exercise. miR-501 elicits this effect by regulating sarcomeric gene expression through its target gene estrogen-related receptor gamma (Esrrg). Importantly, in aged skeletal muscle where miR-501 was significantly downregulated and its target Esrrg significantly upregulated, the number of myog+/CD74+ cells during regeneration was upregulated to similar levels as observed in 501 knockout mice. Moreover, myog+/CD74+-aged skeletal muscle exhibited a similar decrease in the size of newly formed myofibers and increased number of necrotic myofibers after injury as observed in mice lacking miR-501. CONCLUSIONS: miR-501 and Esrrg are regulated in muscle with decreased regenerative capacity and loss of miR-501 is permissive to the appearance of CD74+ myogenic progenitors. Our data uncover a novel link between the metabolic transcription factor Esrrg and sarcomere formation and demonstrate that stem cell heterogeneity in skeletal muscle during aging is under miRNA control. Targeting Esrrg or myog+/CD74+ progenitor cells might improve fiber size and myofiber resilience to exercise in aged skeletal muscle.

Local application of a transcutaneous carbon dioxide paste prevents excessive scarring and promotes muscle regeneration in a bupivacaine-induced rat model of muscle injury.

In postoperative patients with head and neck cancer, scar tissue formation may interfere with the healing process, resulting in incomplete functional recovery and a reduced quality of life. Percutaneous application of carbon dioxide (CO2 ) has been reported to improve hypoxia, stimulate angiogenesis, and promote fracture repair and muscle damage. However, gaseous CO2 cannot be applied to the head and neck regions. Previously, we developed a paste that holds non-gaseous CO2 in a carrier and can be administered transdermally. Here, we investigated whether this paste could prevent excessive scarring and promote muscle regeneration using a bupivacaine-induced rat model of muscle injury. Forty-eight Sprague Dawley rats were randomly assigned to either a control group or a CO2 group. Both groups underwent surgery to induce muscle injury, but the control group received no treatment, whereas the CO2 group received the CO2 paste daily after surgery. Then, samples of the experimental sites were taken on days 3, 7, 14, and 21 post-surgery to examine the following: (1) inflammatory (interleukin [IL]-1ß, IL-6), and transforming growth factor (TGF)-ß and myogenic (MyoD and myogenin) gene expression by polymerase chain reaction, (2) muscle regeneration with haematoxylin and eosin staining, and (3) MyoD and myogenin protein expression using immunohistochemical staining. Rats in the CO2 group showed higher MyoD and myogenin expression and lower IL-1ß, IL-6, and TGF-ß expression than the control rats. In addition, treated rats showed evidence of accelerated muscle regeneration. Our study demonstrated that the CO2 paste prevents excessive scarring and accelerates muscle regeneration. This action may be exerted through the induction of an artificial Bohr effect, which leads to the upregulation of MyoD and myogenin, and the downregulation of IL-1ß, IL-6, and TGF-ß. The paste is inexpensive and non-invasive. Thus, it may be the treatment of choice for patients with muscle damage.

IGF-I and vitamin C promote myogenic differentiation of mouse and human skeletal muscle cells at low temperatures.

In a previous study investigating the effects of low temperature on skeletal muscle differentiation, we demonstrated that C2C12 mouse myoblasts cultured at 30°C do not express myogenin, a myogenic regulatory factor (MRF), or fuse into multinucleated myotubes. At this low temperature, the myoblasts continuously express Id3, a negative regulator of MRFs, and do not upregulate muscle-specific microRNAs. In this study, we examined if insulin-like growth factor-I (IGF-I) and a stable form of vitamin C (L-ascorbic acid phosphate) could alleviate the low temperature-induced inhibition of myogenic differentiation in C2C12 cells. Although the addition of either IGF-I or vitamin C alone could promote myogenin expression in C2C12 cells at 30°C, elongated multinucleated myotubes were not formed unless both IGF-I and vitamin C were continuously administered. In human skeletal muscle cells, low temperature-induced blockage of myogenic differentiation was also ameliorated by exogenous IGF-I and vitamin C. In addition, we demonstrated that satellite cells of IGF-I overexpressing transgenic mice in single-fiber culture expressed myogenin at a higher level than those of wild-type mice at 30°C. This study suggests that body temperature plays an important role in myogenic differentiation of endotherms, but the sensitivity to low temperature could be buffered by certain factors in vivo, such as IGF-I and vitamin C.

Permissive roles of phosphatidyl inositol 3-kinase and Akt in skeletal myocyte maturation.

Skeletal muscle differentiation, maturation, and regeneration are regulated by interactions between signaling pathways activated by hormones and growth factors, and intrinsic genetic programs controlled by myogenic transcription factors, including members of the MyoD and myocyte enhancer factor 2 (MEF2) families. Insulin-like growth factors (IGFs) play key roles in muscle development in the embryo, and in the maintenance and hypertrophy of mature muscle in the adult, but the precise signaling pathways responsible for these effects remain incompletely defined. To study mechanisms of IGF action in muscle, we have developed a mouse myoblast cell line termed C2BP5 that is dependent on activation of the IGF-I receptor and the phosphatidyl inositol 3-kinase (PI3-kinase)-Akt pathway for initiation of differentiation. Here, we show that differentiation of C2BP5 myoblasts could be induced in the absence of IGF action by recombinant adenoviruses expressing MyoD or myogenin, but it was reversibly impaired by the PI3-kinase inhibitor LY294002. Similar results were observed using a dominant-negative version of Akt, a key downstream component of PI3-kinase signaling, and also were seen in C3H 10T1/2 fibroblasts. Inhibition of PI3-kinase did not prevent accumulation of muscle differentiation-specific proteins (myogenin, troponin T, or myosin heavy chain), did not block transcriptional activation of E-box containing muscle reporter genes by MyoD or myogenin, and did not inhibit the expression or function of endogenous MEF2C or MEF2D. An adenovirus encoding active Akt could partially restore terminal differentiation of MyoD-expressing and LY294002-treated myoblasts, but the resultant myofibers contained fewer nuclei and were smaller and thinner than normal, indicating that another PI3-kinase-stimulated pathway in addition to Akt is required for full myocyte maturation. Our results support the idea that an IGF-regulated PI3-kinase pathway functions downstream of or in parallel with MyoD, myogenin, and MEF2 in muscle development to govern the late steps of differentiation that lead to multinucleated myotubes.

Reciprocal inhibition between MyoD and STAT3 in the regulation of growth and differentiation of myoblasts.

The development of myoblasts is regulated by various growth factors as well as by intrinsic muscle-specific transcriptional factors. In this study, we analyzed the roles for STAT3 in the growth and differentiation of myoblasts in terms of cell cycle regulation and interaction with MyoD using C2C12 cells. Here we found that STAT3 inhibited myogenic differentiation induced by low serum or MyoD as efficiently as the Ras/mitogen-activated protein kinase cascade. As for this mechanism, we found that STAT3 not only promoted cell cycle progression through the induction of c-myc but also inhibited MyoD activities through direct interaction. STAT3 inhibited not only DNA binding activities of MyoD but also its transcriptional activities. However, the inhibited transcriptional activities were restored by the supplement of p300/CBP and PCAF, suggesting that STAT3 might deprive MyoD of these transcriptional cofactors. In addition, we found that MyoD inhibited DNA binding activities of STAT3, thereby inhibiting STAT3-dependent cell growth and survival of Ba/F3 cells. These results suggest that the development of muscle cells is regulated by the coordination of cytokine signals and intrinsic transcription factors.

Transactivation of capn2 by myogenic regulatory factors during myogenesis.

The calcium-activated cysteine protease m-calpain plays a pivotal role during the earlier stages of myogenesis, particularly during fusion. The enzyme is a heterodimer, encoded by the genes capn2, for the large subunit, and capn4, for the small subunit. To study the regulation of m-calpain, the DNA sequence upstream of capn2 was analyzed for promoter elements, revealing the existence of five consensus-binding sites (E-box) for several myogenic regulatory factors and one binding site for myocyte enhancer factor-2 (MEF-2). Transient transfections with reporter gene constructs containing the E-box revealed that MyoD presents a high level of transactivation of reporter constructs containing this region, in particular the sequences including the MEF-2/E4-box. In addition, over-expression of various myogenic factors demonstrated that MyoD and myogenin with much less efficiency, can up-regulate capn2, both singly and synergistically, while Myf5 has no effect on synthesis of the protease. Experiments with antisense oligonucleotides directed against each myogenic factor revealed that MyoD plays a specific and pivotal role during capn2 regulation, and cannot be replaced wholly by myogenin and Myf5.

Myogenin induces higher oxidative capacity in pre-existing mouse muscle fibres after somatic DNA transfer.

Muscle is a permanent tissue, and in the adult pronounced changes can occur in pre-existing fibres without the formation of new fibres. Thus, the mechanisms responsible for phenotype transformation in the adult might be distinct from mechanisms regulating muscle differentiation during muscle formation and growth. Myogenin is a muscle-specific, basic helix-loop-helix transcription factor that is important during early muscle differentiation. It is also expressed in the adult, where its role is unknown. In this study we have overexpressed myogenin in glycolytic fibres of normal adult mice by electroporation and single-cell intracellular injection of expression vectors. Myogenin had no effects on myosin heavy chain fibre type, but induced a considerable increase in succinate dehydrogenase and NADH dehydrogenase activity, with some type IIb fibres reaching the levels observed histochemically in normal type IIx and IIa fibres. mRNA levels for malate dehydrogenase were similarly altered. The size of the fibres overexpressing myogenin was reduced by 30-50 %. Thus, the transfected fibres acquired a phenotype reminiscent of the phenotype obtained by endurance training in man and other animals, with a higher oxidative capacity and smaller size. We conclude that myogenin can alter pre-existing glycolytic fibres in the intact adult animal.

Interaction between the skeletal muscle type 1 Na+ channel promoter E-box and an upstream repressor element. Release of repression by myogenin.

We have defined how four elements that regulate expression of the rat skeletal muscle type 1 sodium channel (SkM1) gene cooperate to yield specific expression in differentiated muscle. A basal promoter region containing within it a promoter E-box (-31/-26) is broadly expressed in many cells, including myoblasts and myotubes; mutations within the promoter E-box that disrupt binding of the myogenic basic helix-loop-helix (bHLH) factors reduce expression in all cell types only slightly. Sequential addition of upstream elements to the wild-type promoter confer increasing specificity of expression in differentiated cells, even though all three upstream elements, including a positive element (-85/-57), a repressor E-box (-90/-85), and upstream repressor sequences (-135/-95), bind ubiquitously expressed transcription factors. Mutations in the promoter E-box that disrupt the binding of the bHLH factors counteract the specificity conferred by addition of the upstream elements, with the greatest interaction observed between the upstream repressor sequences and the promoter E-box. Forced expression of myogenin in myoblasts releases repression exerted by the upstream repressor sequences in conjunction with the wild-type, but not mutant, promoter E-box, and also initiates expression of the endogenous SkM1 protein. Our data suggest that particular myogenic bHLH proteins bound at the promoter E-box control expression of SkM1 by releasing repression exerted by upstream repressor sequences in differentiated muscle cells.

A dominant-negative form of transcription factor MEF2 inhibits myogenesis.

A biological role for MEF2 (myocyte enhancer factor 2) activity during mammalian myogenesis has been inferred but not directly proven because of its role in the transcriptional activation of many muscle-specific genes. Therefore, our purpose was to determine whether MEF2 activity is absolutely required for mammalian myogenesis. Using a dominant-negative approach to address this question, we constructed a mutated MEF2A protein comprised of the amino-terminal DNA binding/dimerization domain of MEF2A without its trans-activation domain as a bacterial fusion protein (GST-131) or in a eukaryotic expression vector (pcDNA-131). GST-131 and the protein encoded by pcDNA-131 bind specifically to the MEF2 cis element and abrogate trans-activation of a MEF2-responsive luciferase reporter gene by wild type MEF2A, thus serving a role as trans-dominant inhibitors of MEF2 function. In congruence with their ability to interfere with wild type MEF2 function, microinjection of GST-131 or pcDNA-131 into L6E9 or C2C12 myoblasts inhibited myotube formation. Immunofluorescence analysis showed that the expression of myogenin, myosin heavy chain, and MEF2A were inhibited in the GST-131 or pcDNA-131-injected cells compared with GST or pcDNA-injected controls. We also document that this trans-dominant MEF2 inhibitor impairs the myogenic conversion of C3H10T1/2 fibroblasts by MyoD. Thus, these data provide evidence that the trans-activation function of the MEF2 proteins during mammalian myogenesis is required for muscle-specific gene expression and differentiation.

Regulation of splicing is responsible for the expression of the muscle-specific 2a isoform of the sarco/endoplasmic-reticulum Ca(2+)-ATPase.

Tissue-specific alternative processing of sarco/endoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) transcripts generates functionally different Ca2+ pump isoforms in muscle compared with non-muscle tissues. In non-muscle cells, the SERCA2 pre-mRNA can be polyadenylated at a site located between the donor and acceptor splice site of an intron which is only removed in muscle tissues. To define the cis-active elements involved in differential processing, we constructed a minigene (pCM beta SERCA2) containing the 3' end of the SERCA2 gene. When stably transfected into a myogenic cell line, minigene transcripts were differentially processed depending on the differentiation state of the cells. This proves that the essential elements required for regulated processing are present in the construct. Furthermore, co-transfection of the pCM beta SERCA2 minigene and a myogenin expression vector in a fibroblast cell line induced muscle-specific splicing of transcripts from pCM beta SERCA2. This shows that trans-acting factor(s) responsible for muscle-specific processing can be induced by one of the important regulatory genes of muscle differentiation. Inactivation of the non-muscle poly(A) site did not induce splicing in non-muscle cells. This excludes a simple competition model between splicing and polyadenylation, but it is consistent with splicing being very inefficient in non-muscle cells. Moreover, splicing could be induced in non-muscle cells by optimizing the muscle-specific donor splice site and/or by shortening the intron length. We therefore propose that expression of the muscle-specific SERCA2a isoform is the result of activation of an otherwise inefficient splicing process.