Myogenic Satellite Cell Differentiation Research Articles

270 Cellular Response to Heat Exposure: Influences on the Growth Potential of Bovine Skeletal Muscle and Adipose Tissue

Abstract Our lack of understanding regarding how heat exposure affects major tissue growth and meat attributes impedes our ability to counteract more frequent climate extremes and promote profitability in the beef cattle industry. The objective of the study was to determine pathways that heat shock proteins (HSPs) regulate in beef cattle’s skeletal muscle and adipose tissue growth and metabolism. Bovine satellite cells (BSCs) were isolated from the longissimus muscle of 3-month-old Holstein bulls (n = 5). Preadipose cells were isolated from the subcutaneous fat of 18-month-old Angus steers (n = 5) and induced adipogenic differentiation. Satellite, myofibers, and adipose cells were incubated at 38°C (control; CON), 39.5°C (moderate heat stress; MHS), and 41°C (extreme heat stress; EHS) and harvested for mRNA gene expressions, protein level, proteomics, immunostaining. Heat exposure cued bovine satellite cell proliferation and may increase the self-renewing satellite cell population. After 3 h of heat exposure, the mRNA gene expression and protein levels of Pax3 and Myf5 were downregulated, while HSPs 20, 27, 70, and 90 were upregulated. Small heat shock proteins, especially HSP27, appear to be essential in regulating satellite cell activity under heat stress. When HSP27 knock-down BSCs were exposed to EHS for 3 h, Myf5 gene expressions and protein levels returned to normal. This may indicate that HSP27 directly regulates Myf5 under heat stress and temporarily activates the self-renewal mechanism instead of committing myogenic differentiation. Results from proteomics showed that pathways related to oxidative phosphorylation, NADH dehydrogenase activity, and transmembrane transport were upregulated by EHS while MHS upregulated biosynthesis of amino acids, PI3K-Akt signaling pathway. Heat exposure during myogenic differentiation of satellite cells seemingly stimulates the commitment of myogenesis. Heat treatments increased mRNA gene expressions of IGF I, MHC I, MyoD, and myogenin and protein levels. Heat exposure also improved the muscle protein synthesis pathway by regulating the phosphorylation of mTOR, Akt, and P70S6. Heat stress triggered adipogenic differentiation and lipid storage, followed by increased expression of HSP. Extreme heat stress increased adipose cell growth by upregulating C/EBPα and PPARγ, key regulators of adipogenesis. Lipogenic genes FAS and SCD were also increased, accompanied by HSPs. Adipocytes exposed to EHS also increased intracellular lipid accumulation. Proteomic analysis revealed that carbon and pyruvate metabolism pathways were upregulated by MHS, whereas glycolysis/ gluconeogenesis, protein processing in the endoplasmic reticulum, RNA transport, and biosynthesis of amino acids were upregulated by EHS. Cellular mechanisms related to skeletal muscle hypertrophy and adipose tissue growth are responsive in in-vitro settings; however, the results are controversial depending on cell type and animal species. Various factors such as metabolic, hormonal, and immunologic regulation need to be considered to accurately assess the effect of HS on the postnatal growth of major tissues at the animal level.

Ex vivo adult stem cell characterization from multiple muscles in ambulatory children with cerebral palsy during early development of contractures

Cerebral palsy (CP) is one of the most common conditions leading to lifelong childhood physical disability. Literature reported previously altered muscle properties such as lower number of satellite cells (SCs), with altered fusion capacity. However, these observations highly vary among studies, possibly due to heterogeneity in patient population, lack of appropriate control data, methodology and different assessed muscle.In this study we aimed to strengthen previous observations and to understand the heterogeneity of CP muscle pathology. Myogenic differentiation of SCs from the Medial Gastrocnemius (MG) muscle of patients with CP (n = 16, 3–9 years old) showed higher fusion capacity compared to age-matched typically developing children (TD, n = 13). Furthermore, we uniquely assessed cells of two different lower limb muscles and showed a decreased myogenic potency in cells from the Semitendinosus (ST) compared to the MG (TD: n = 3, CP: n = 6). Longitudinal assessments, one year after the first botulinum toxin treatment, showed slightly reduced SC representations and lower fusion capacity (n = 4). Finally, we proved the robustness of our data, by assessing in parallel the myogenic capacity of two samples from the same TD muscle.In conclusion, these data confirmed previous findings of increased SC fusion capacity from MG muscle of young patients with CP compared to age-matched TD. Further elaboration is reported on potential factors contributing to heterogeneity, such as assessed muscle, CP progression and reliability of primary outcome parameters.

PAX7 Balances the Cell Cycle Progression via Regulating Expression of Dnmt3b and Apobec2 in Differentiating PSCs.

PAX7 transcription factor plays a crucial role in embryonic myogenesis and in adult muscles in which it secures proper function of satellite cells, including regulation of their self renewal. PAX7 downregulation is necessary for the myogenic differentiation of satellite cells induced after muscle damage, what is prerequisite step for regeneration. Using differentiating pluripotent stem cells we documented that the absence of functional PAX7 facilitates proliferation. Such action is executed by the modulation of the expression of two proteins involved in the DNA methylation, i.e., Dnmt3b and Apobec2. Increase in Dnmt3b expression led to the downregulation of the CDK inhibitors and facilitated cell cycle progression. Changes in Apobec2 expression, on the other hand, differently impacted proliferation/differentiation balance, depending on the experimental model used.

Regulation of Skeletal Muscle Satellite Cell Differentiation by Omega-3 Polyunsaturated Fatty Acids: A Critical Review.

Skeletal muscle is composed of multinuclear cells called myofibres, which are formed by the fusion of myoblasts during development. The size of the muscle fiber and mass of skeletal muscle are altered in response to several pathological and physiological conditions. Skeletal muscle regeneration is primarily mediated by muscle stem cells called satellite cells (SCs). In response to injury, these SCs replenish myogenic progenitor cells to form new myofibers to repair damaged muscle. During myogenesis, activated SCs proliferate and differentiate to myoblast and then fuse with one another to form muscle fibers. A reduced number of SCs and an inability to undergo myogenesis may contribute to skeletal muscle disorders such as atrophy, cachexia, and sarcopenia. Myogenic regulatory factors (MRF) are transcription factors that regulate myogenesis and determines whether SCs will be in the quiescent, activated, committed, or differentiated state. Mitochondria oxidative phosphorylation and oxidative stress play a role in the determination of the fate of SCs. The potential activation and function of SCs are also affected by inflammation during skeletal muscle regeneration. Omega-3 polyunsaturated fatty acids (PUFAs) show promise to reduce inflammation, maintain muscle mass during aging, and increase the functional capacity of the muscle. The aim of this critical review is to highlight the role of omega-3 PUFAs on the myogenic differentiation of SCs and pathways affected during the differentiation process, including mitochondrial function and inflammation from the current body of literature.

The application of bone marrow mesenchymal stem cells and biomaterials in skeletal muscle regeneration.

Skeletal muscle injuries have bothered doctors and caused great burdens to the public medical insurance system for a long time. Once injured, skeletal muscles usually go through the processes of inflammation, repairing and remodeling. If repairing and remodeling stages are out of balance, scars will be formed to replace injured skeletal muscles. At present, clinicians usually use conventional methods to restore the injured skeletal muscles, such as flap transplantation. However, flap transplantation sometimes needs to sacrifice healthy autologous tissues and will bring extra harm to patients. In recent years, stem cells-based tissue engineering provides us new treatment ideas for skeletal muscle injuries. Stem cells are cells with multiple differentiation potential and have ability to differentiate into adult cells under special condition. Skeletal muscle tissues also have stem cells, called satellite cells, but they are in small amount and new muscle fibers that derived from them may not be enough to replace injured fibers. Bone marrow mesenchymal stem cells (BM-MSCs) could promote musculoskeletal tissue regeneration and activate the myogenic differentiation of satellite cells. Biomaterial is another important factor to promote tissue regeneration and greatly enhance physiological activities of stem cells in vivo. The combined use of stem cells and biomaterials will gradually become a mainstream to restore injured skeletal muscles in the future. This review article mainly focuses on the review of research about the application of BM-MSCs and several major biomaterials in skeletal muscle regeneration over the past decades.

LncRNAs are regulated by chromatin states and affect the skeletal muscle cell differentiation.

ObjectiveThis study aims to clarify the mechanisms underlying transcriptional regulation and regulatory roles of lncRNAs in skeletal muscle cell differentiation.MethodsWe analysed the expression patterns of lncRNAs via time‐course RNA‐seq. Then, we further combined the ATAC‐seq and ChIP‐seq to investigate the governing mechanisms of transcriptional regulation of differentially expressed (DE) lncRNAs. Weighted correlation network analysis and GO analysis were conducted to identify the transcription factor (TF)‐lncRNA pairs related to skeletal muscle cell differentiation.ResultsWe identified 385 DE lncRNAs during C2C12 differentiation, the transcription of which is determined by chromatin states around their transcriptional start sites. The TF‐lncRNA correlation network showed substantially concordant changes in DE lncRNAs between C2C12 differentiation and satellite cell rapid growth stages. Moreover, the up‐regulated lncRNAs showed a significant decrease following the differentiation capacity of satellite cells, which gradually declines during skeletal muscle development. Notably, inhibition of the lncRNA Atcayos and Trp53cor1 led to the delayed differentiation of satellite cells. Those lncRNAs were significantly up‐regulated during the rapid growth stage of satellite cells (4‐6 weeks) and down‐regulated with reduced differentiation capacity (8‐12 weeks). It confirms that these lncRNAs are positively associated with myogenic differentiation of satellite cells during skeletal muscle development.ConclusionsThis study extends the understanding of mechanisms governing transcriptional regulation of lncRNAs and provides a foundation for exploring their functions in skeletal muscle cell differentiation.

Numb And Numb-like Responses To Exercise Induced Muscle Damage In Human Skeletal Muscle

Numb and Numb-Like (NumbL) are adaptor proteins. Among their functions is control of cell fate determination and progression of cell differentiation. While no role for NumbL has been found in cells of the myogenic lineage, Numb promotes myogenic differentiation of satellite cells. The roles these proteins in human skeletal muscle in response to exercise-induced muscle damage have yet to be examined. PURPOSE: The purpose of this investigation is to examine changes in the expression of Numb and NumbL in human skeletal muscle after a bout of muscle damage via eccentric exercise. METHODS: Twelve male subjects signed an informed consent approved by The University of Kansas’s Institutional Review Board and were randomly assigned to one of two groups: a control group (n = 6) or an damage group (n = 6). Subjects completed a one repetition maximum (1RM) in leg extension followed by seven sets of ten repetitions of eccentric leg extension at %120 of 1RM with a two minutes of rest period between sets. Three muscle biopsies of the vastus lateralis were collected at baseline, two days post-, and five days post-muscle damage and analyzed utilizing Western blot and quantitative reverse transcription polymerase chain reaction analyses. The results were analyzed using a 2 X 3 (Group x Time) repeated-measures ANOVA. RESULTS: No significant differences in mRNA expression were observed for Numb between groups two days post- and five days post-damage (p = 0.37 and p = 0.29, respectively). There was no significant difference in NumbL for the exercise group (3.101 ± 1.763) in comparison to the control group (0.838 ± 0.234) two days post-exercise induced muscle damage (p = 0.27). However, there was a significant increase in NumbL at five days post-exercise between the exercise group (1.773 ± 0.358) and the control group (0.726 ± 0.087) from baseline measures (p = 0.04). No significant differences in Numb or NumbL proteins were observed at any time point or between the control group and exercise group (p > 0.05). CONCLUSION: Numb expression was unaltered post-muscle damage, while NumbL mRNA expression was increased after muscle damage. These results indicate that NumbL may have a greater role in muscle repair after strenuous exercise in humans than previously thought. Funding provided by NIA grant 5R01AG060341-02 to CPC and the CSACSM Doctoral Grant.

Retinoic acid regulates the activity of satellite cells and promotes skeletal muscle regeneration impaired due to obesity in mice

BackgroundEctopic lipid accumulation in skeletal muscle induced by obesity leads to muscle atrophy and impaired regenerative capacity. Retinoic acid (RA) has been reported to enhance myogenesis in embryonic stem cells and promotes regeneration in adult skeletal muscle. However, the effects of RA on muscle regeneration impaired due to obesity have not been addressed. The objective of this study is to explore the effects of RA on activation and myogenic differentiation of satellite cells (SCs) in obese mice, which promote muscle regeneration.MethodsTransgenic Pax7cre/ROSAmT/mG mice were used for lineage tracing of SCs. Obesity was induced by feeding 60% high fat diet for 10 weeks. Two days following tamoxifen injection, 10 mg/kg body weight (BDW) RA or equivalent vehicle were injected subcutaneously into the tibialis anterior (TA) muscle of mice once per two days before injury, and TA muscle injury was induced by cardiotoxin injection. The effects of RA supplementation on SCs and corresponding skeletal muscle regeneration were evaluated at different times post‐injury.ResultsHistological analysis showed that RA supplementation improved muscle fiber regeneration and recovery in obese mice at day 7 post‐injury. The number of SCs in the muscle of obese mice at 3 days post‐injury were increased and the fusion of SCs into regenerated muscle fibers at 7 days post‐injury were promoted in RA treated group. Correspondingly, RA supplementation increased the expression of retinoic acid receptor α (RARα) in the TA muscle samples. The expression of myogenesis genes such as Pax7, Myf5, MyoD and myogenin were increased in muscles with RA treatment. Compared with the vehicle treated group, inflammatory responses including expression of IL6 and TNFα at day 3 and IL1β and IL18 at day 7 were decreased in the skeletal muscle of mice with RA treatment. RA supplementation also increased the expression of insulin like growth factor 1 (IGF1) in muscle at day 3 post‐injury.ConclusionsRetinoic acid promoted regeneration and repair of muscle fibers after injury in obese mice accompanied by increased retinoic acid signaling, elevated expression of IGF1 and resolution of inflammation in the regenerating muscle. These benefits could be attributed to enhanced proliferation and myogenic differentiation of SCs after RA treatment.Support or Funding InformationSupported by NIH Grant R01HD067449. Effects of retinoic acid (RA) supplementation on skeletal muscle regeneration. H&E staining (A, D) and fluorescence images (B, E) of tibialis anterior (TA) muscle at 3‐ and 7‐days post‐injury (dpi). Green fluorescence tracked Pax7+ satellite cells and derived muscle fibers (regenerated) after tamoxifen induction while red fluorescence showed original cells. C, F. mRNA expression. *p<0.05, ** p<0.01. Data are expressed as mean ±SEM (n=3).imageEffects of retinoic acid (RA) supplementation on skeletal muscle regeneration. H&E staining (A, D) and fluorescence images (B, E) of tibialis anterior (TA) muscle at 3‐ and 7‐days post‐injury (dpi). Green fluorescence tracked Pax7+ satellite cells and derived muscle fibers (regenerated) after tamoxifen induction while red fluorescence showed original cells. C, F. mRNA expression. *p<0.05, ** p<0.01. Data are expressed as mean ±SEM (n=3).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nandrolone, an Anabolic Steroid, Stabilizes Numb Protein Through Inhibition of mdm2 in C2C12 Myoblasts

Nandrolone, an anabolic steroid, slows denervation atrophy of rat muscle, prevents denervation-induced nuclear accumulation of intracellular domain of the Notch receptor, and elevates expression of Numb. Numb acts as an inhibitor of Notch signaling and promotes myogenic differentiation of satellite cells. Turnover of Numb is regulated by mdm2, an E3 ubiquitin ligase. With these considerations in mind, we investigated the effects of nandrolone on the expression of Numb and mdm2 proteins and determined the effect of mdm2 on nandrolone-induced alterations in Numb protein in C2C12 myoblasts. When C2C12 cells were cultured in a medium favoring differentiation (Dulbecco modified Eagle medium containing 2% horse serum), nandrolone up-regulated Numb protein levels in a time-dependent manner and prolonged Numb protein half-life from 10 to 18 hours. In contrast, nandrolone reduced the expression of mdm2 protein. To determine whether the decreased mdm2 expression induced by nandrolone was responsible for the increased levels and prolonged half-life of Numb protein in this cell line, mdm2-small interfering RNA (siRNA) was employed to inhibit mdm2 expression. Compared to cells transfected with scrambled siRNA (negative control), transfection with mdm2-siRNA increased basal Numb protein expression but abolished the further increase in Numb protein levels by nandrolone. In addition, transfection of mdm2-siRNA mimicked the effect of nandrolone to prolong the half-life of Numb protein. Moreover, when C2C12 cells were forced to overexpress mdm2, there was a significant decline in the expression of both basal and inducible Numb protein. Our data suggest that nandrolone, by a novel mechanism for this agent in a muscle cell type, increases Numb protein levels in C2C12 myoblasts by stabilizing Numb protein against degradation, at least in part, via suppression of mdm2 expression.

Gene expression profiles during differentiation and transdifferentiation of bovine myogenic satellite cells

The molecular mechanisms underlying myogenic satellite cells (MSCs) differentiation into myotube-formed cells (MFCs) and transdifferentiation into adipocyte-like cells (ALCs) are unclear. As a step towards understanding the molecular mechanisms underlying MSC differentiation and transdifferentiation, we attempted to identify the genes differentially expressed during differentiation and transdifferentiation using gene microarray analysis (GMA). Thirty oligonucleotide arrays were used with two technical replicates and nine and six biological replicates for MFCs vs. MSCs and ALCs vs. MSCs, respectively, to contrast expression profile differences. GMA identified 1,224 differentially expressed genes by at least 2-fold during differentiation and transdifferentiation of MSCs. To select the highly expressed genes for future functional study, genes with a 4-fold expression difference were selected for validation by real time RT-PCR and approximately 96.9% of the genes were validated. The up-regulation of marker genes for myogenesis (MYL2, MYH3) and adipogenesis (PPARγ, and FABP4) was observed during the differentiation and transdifferentiation of MSCs into MFCs and ALCs, respectively. KOG analysis revealed that the most of the genes up-regulated during differentiation and transdifferentiation of MSCs were related to signal transduction. Again the exact location of 109 differentially expressed genes by 4-fold were analyzed by chromosome mapping. Among those, co-localization of 29 genes up-regulated during transdifferentiation with QTL for marbling score and intramuscular fat percentage supports the involvement of these genes in cellular transdifferentiation. Interestingly, some genes with unknown function were also identified during the process. Functional studies on these genes may unfold the molecular mechanisms controlling MSC differentiation and transdifferentiation.

Heparan sulfate proteoglycans, syndecan-4 and glypican-1, differentially regulate myogenic regulatory transcription factors and paired box 7 expression during turkey satellite cell myogenesis: Implications for muscle growth

The heparan sulfate proteoglycans have been shown to play essential roles in the proliferation and differentiation of myogenic satellite cells. Myogenic regulatory factors (MRF) and paired box 7 (Pax7) are essential transcription factors for satellite cell myogenesis. The objective of the current study was to investigate whether the expression of the MRF and Pax7 is, in part, regulated by the heparan sulfate proteoglycans, syndecan-4, and glypican-1, whose expression has been shown to differentially affect satellite cell proliferation and differentiation. To test this objective, small interfering RNA was used to knockdown the gene expression of glypican-1 and syndecan-4. The effect on the expression of MRF and Pax7 was measured at the mRNA level by real-time quantitative PCR. The knockdown of the glypican-1 gene decreased mRNA expression of MyoD, myogenin, MRF4, and Pax7 expression during proliferation and differentiation of turkey satellite cells; whereas knockdown of the syndecan-4 gene increased mRNA expression of MyoD and MRF4 expression during cell proliferation but not during differentiation and had no effect on myogenin and Pax7 expression. These data suggested that the precise expression of the MRF are dependent upon the appropriate expression of glypican-1 and syndecan-4 during satellite cell proliferation and differentiation, and Pax7 expression is influenced by glypican-1.

Meat Science and Muscle Biology Symposium: stem cell niche and postnatal muscle growth.

Stem cell niche plays a critical role in regulating the behavior and function of adult stem cells that underlie tissue growth, maintenance, and regeneration. In the skeletal muscle, stem cells, called satellite cells, contribute to postnatal muscle growth and hypertrophy, and thus, meat production in agricultural animals. Satellite cells are located adjacent to mature muscle fibers underneath a sheath of basal lamina. Microenvironmental signals from extracellular matrix mediated by the basal lamina and from the host myofiber both impinge on satellite cells to regulate their activity. Furthermore, several types of muscle interstitial cells, including intramuscular preadipocytes and connective tissue fibroblasts, have recently been shown to interact with satellite cells and actively regulate the growth and regeneration of postnatal skeletal muscles. From this regard, interstitial adipogenic cells are not only important for marbling and meat quality, but also represent an additional cellular component of the satellite cell niche. At the molecular level, these interstitial cells may interact with satellite cells through cell surface ligands, such as delta-like 1 homolog (Dlk1) protein whose overexpression is thought to be responsible for muscle hypertrophy in callipyge sheep. In fact, extracellular Dlk1 protein has been shown to promote the myogenic differentiation of satellite cells. Understanding the cellular and molecular mechanisms within the stem cell niche that regulate satellite cell differentiation and maintain muscle homeostasis may lead to promising approaches to optimizing muscle growth and composition, thus improving meat production and quality.

돼지 고기의 아나볼릭 스테로이드가 Myogenic Satellite Cell의 증식과 분화에 미치는 영향

성호르몬은 근육의 발달(아나볼릭 효과)에 관여하는 것으로 잘 알려져 왔으며 인체의 치료제로 넓게 사용되고 있다. 돼지고기에는 주로 성선(정소와 난소)에서 생산되어 고기에 축적된 다양한 아나볼릭 효과를 나타내는 성호르몬이 많이 존재하는 것으로 알려져 있다. 따라서 본 연구는 돼지고기에 존재하는 아나볼릭 스테로이드를 추출하여 근육세포의 증식과 분화에 미치는 영향을 파악하고자 실시하였다. 본 연구에서 돼지의 각 조직으로부터 아나볼릭 스테로이드를 추출하는 3가지 방법(M1, M2, M3)을 개발하였다. 3가지 모든 추출방법에서 아나볼릭 스테로이드가 추출되었으며 특히 M3에서 조직 1g당 가장 많은 양을 획득할 수 있었다. M3를 이용하여 아나볼릭 스테로이드를 추출했을 때 돼지고기의 근육과 지방에서는 스테로이드 호르몬 양이 수컷에서 가장 높았고, 혈청에서는 nandrolone 과 testosterone의 양은 수컷에서 가장 높았으며, estrone와 <TEX>$17{\beta}$</TEX>-estradiol의 양은 비슷하였다. 근육세포 증식은 돼지 아나볼릭 스테로이드의 농도 의존적으로 촉진되었으며, M3에서 가장 빠르게 증식되었다. 근육세포의 분화는 추출된 아나볼릭 스테로이드 처리구가 대조구보다 더 효과적으로 분화가 잘 되는 것을 세포 모양과 유전자(AR, myoD, desmin, myogenin)를 통하여 확인할 수 있었다. 특히 M3에서 추출된 아나볼릭 스테로이드에서 myogenin과 AR 유전자가 유의적으로 높게 발현되었다(p<0.001). 전체적으로 돼지의 아나볼릭 스테로이드는 근육세포의 성장과 분화를 촉진하였다. 따라서 돼지고기에 있는 아나볼릭 스테로이드의 섭취를 통해 인체 근육의 성장과 발달에 긍정적 효과를 나타낼 수 있을 것으로 사료된다. Sex steroids are known to be involved in skeletal muscle development (anabolic effect) and are frequently used in medicines. It has been known that pork contains a variety of steroids that are mainly synthesized in the gonads (testis and ovary). Thus, the present study was conducted to evaluate the effects of anabolic steroids of pork on the proliferation and differentiation of myogenic satellite cells (MSC). Three different methods (M1, M2, and M3) were developed for the isolation and purification of steroids from porcine tissues. Among three extraction methods that we developed, M3 was the best method with respect to the quantities of steroids and the induction of MSC proliferation. Hormonal analysis showed that the steroid hormone levels were the highest in muscle and fat of intact male than those of castrated males and females. In addition, the highest serum levels of nandrolone and testosterone were detected in intact males, whereas estrone and <TEX>$17{\beta}$</TEX>-estradiol levels were similar in the entire experimental serum samples. Expression of androgen receptor (AR), myoD, desmin, and myogenin in bovine muscle cells were significantly up-regulated by the treatment of steroid extracts. The highest increas of myogenin and AR mRNA abundance were observed in the MSCs treated with M3 extract (p<0.001). Altogether, the present research showed the positive effect of steroids on MSC proliferation and differentiation in vitro. These results would certainly imply a beneficial effect of pork consumption on human muscle development.

Down-regulation of MyoD by Calpain 3 Promotes Generation of Reserve Cells in C2C12 Myoblasts

Calpain 3 is a calcium-dependent cysteine protease that is primarily expressed in skeletal muscle and is implicated in limb girdle muscular dystrophy type 2A. To date, its best characterized function is located within the sarcomere, but this protease is found in other cellular compartments, which suggests that it exerts multiple roles. Here, we present evidence that calpain 3 is involved in the myogenic differentiation process. In the course of in vitro culture of myoblasts to fully differentiated myotubes, a population of quiescent undifferentiated "reserve cells" are maintained. These reserve cells are closely related to satellite cells responsible for adult muscle regeneration. In the present work, we observe that reserve cells express higher levels of endogenous Capn3 mRNA than proliferating myoblasts. We show that calpain 3 participates in the establishment of the pool of reserve cells by decreasing the transcriptional activity of the key myogenic regulator MyoD via proteolysis independently of the ubiquitin-proteasome degradation pathway. Our results identify calpain 3 as a potential new player in the muscular regeneration process by promoting renewal of the satellite cell compartment.

Bex1 Participates in Muscle Regeneration by Regulating Myogenic Satellite Cell Differentiation

Bex1 protein is upregulated in regenerating muscle and interacts with calmodulin, a Ca 2+ -binding protein involved in cell cycle regulation. Following cardiotoxin-induced injury the regenerating muscle of Bex1 knock-out mice exhibits prolonged cell proliferation and delayed cell differentiation compared to wildtype mice. To gain insight into this process, we compared the regenerating myogenic morphologies of Bex1 knock-out and wild-type mice at several time points. Bex1-positive cells were identified by double immunofluorescence staining. These studies demonstrated that a population of cells that are Bex1- positive after injury are c-Met/basal lamina-positive and Mac-1-negative indicating that they are derived from at least a subset of myogenic progenitor/satellite cells but not invading immune cells. In addition, in regenerating muscle, Bex1 co-localizes with calmodulin in the cytoplasm of the late myoblast or early myotube stage of myogenesis. These results suggest that Bex1 participates in muscle regeneration through the regulation of satellite cell proliferation and differentiation by its interaction with calmodulin. Current studies of Bex1 may provide a new molecular tool for the identification of activated satellite cell and open the way to new or improved therapeutic regimens against progressive muscular atrophy.

The effects of hepatocyte growth factor and insulin‐like growth factor‐1 on the myogenic differentiation of satellite cells in human urethral rhabdosphincter

To examine the effects of hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) on the myogenic differentiation of human urethral rhabdosphincter (RS) satellite cells. Human RS was obtained from patients undergoing radical prostatectomy for prostate cancer. Selectively cultured RS satellite cells, transfected with temperature sensitive simian virus-40 T antigen (ts-SV40 Tag) to extend their lifespan, were cultured at 33 degrees C, and then incubated at 39 degrees C to induce myogenic differentiation. Varying concentrations of HGF and IGF-1 were added to the differentiation medium. Inactivation of SV40 Tag and evaluations of myogenic differentiation were examined by immunocytochemistry, western blotting and real-time RT-PCR. At 39 degrees C, ts-SV40 Tag-transfected RS satellite cells slowly proliferated, fused to form multinucleated myotubes, and expressed myosin heavy chain (MHC) in association with the temperature-dependent inactivation of SV40 Tag. IGF-1 significantly accelerated the MHC expression in a dose-dependent fashion through activation of the PI3-kinase pathway. Conversely, HGF did not promote MHC expression due a reduction in phosphorylation of both the MAP-kinase and the PI3-kinase pathways, a pattern of response different than the response of proliferating RS satellite cells to HGF. HGF did not induce myogenic differentiation of RS satellite cells. Conversely, IGF-1 promoted myogenic differentiation of RS satellite cells via the PI3-K pathway likewise proliferating RS satellite cells. These findings may be useful for developing novel techniques for regenerating the human RS to treat urinary incontinence.

The role of myostatin in chicken ( Gallus domesticus) myogenic satellite cell proliferation and differentiation

Myostatin (GDF-8) inhibits the activation, proliferation, and differentiation of myogenic satellite cells. The relative importance of this growth factor is demonstrated in myostatin-null mice and cattle possessing defective myostatin genes. These defects result in greatly enhanced musculature. In the present study, the effect of myostatin on chicken myogenic satellite cells derived from two different skeletal muscles was studied. The effect of anti-myostatin antibodies on cellular responses was also examined. Satellite cells isolated from the pectoralis major (PM) muscles were more responsive to the proliferation depressing effects of myostatin compared to cells from the biceps femoris (BF; P ⩽ 0.05). Myostatin inhibited differentiation of satellite cells derived from the PM muscle ( P ⩽ 0.05), but had no effect on cells from the BF ( P ⩾ 0.05). Myostatin administered to proliferating cells depressed the synthesis of decorin ( P ⩽ 0.05), an extracellular matrix proteoglycan. However, in differentiating cultures, only BF cells expressed lower levels of decorin ( P ⩽ 0.05). Decorin expression in PM cells was unchanged ( P ⩾ 0.05). Administration of anti-myostatin antibodies to proliferating cultures increased cell proliferation by 6–7% over 3 days. There was no effect on differentiation of either PM or BF cells. The present study demonstrates that there are differences in the responsiveness to myostatin of chicken satellite cells derived from different muscles. Evidence is also given to support the role of endogenous myostatin in autocrine regulation of muscle growth.

Effect of myostatin on turkey myogenic satellite cells and embryonic myoblasts

Myostatin (GDF-8) inhibits the activation, proliferation, and differentiation of myogenic satellite cells. The relative importance of this growth factor is demonstrated in myostatin-null mice and cattle possessing defective myostatin genes. These defects result in greatly enhanced musculature. In the present study, we examined the effect of myostatin on turkey myogenic satellite cells and embryonic myoblasts. Compared with controls ( P < 0.05), proliferation of both turkey embryonic myoblasts and satellite cells was inhibited between 26 and 45% in serum-free medium containing 20 ng/mL myostatin. While individual turkey satellite cell clones differed in their responsiveness to myostatin, there were no significant differences in the responsiveness of fast and slow growing cells as groups ( P > 0.05). A slow growing clone that exhibited the greatest response to myostatin also exhibited the greatest depression of differentiation with this growth factor ( P < 0.05). All other turkey satellite cell clones exhibited similar responses to the differentiation depressing effects of myostatin ( P > 0.05). However, myostatin had no effect on differentiation of turkey embryonic myoblasts ( P > 0.05). When exposed to myostatin, 4 of 6 proliferating clones and all differentiating clones increased their expression of decorin, a growth inhibitor ( P < 0.05). The present study demonstrates that myostatin inhibits the proliferation and differentiation of satellite cells and suggests a role for decorin in myostatin action in muscle development.

Dynamic distribution and formation of a para-sarcomeric banding pattern of prosomes during myogenic differentiation of satellite cells in vitro.

Myogenesis proceeds by fusion of proliferating myoblasts into myotubes under the control of various transcription factors. In adult skeletal muscle, myogenic stem cells are represented by the satellite cells which can be cultured and differentiate in vitro. This system was used to investigate the subcellular distribution of a particular type of prosomes at different steps of the myogenic process. Prosomes constitute the MCP core of the 26S proteasomes but were first observed as subcomplexes of the untranslated mRNPs; recently, their RNase activity was discovered. A monoclonal antibody raised against the p27K subunit showed that the p27K subunit-specific prosomes move transiently into the nucleus prior to the onset of myoblast fusion into myotubes; this represents possibly one of the first signs of myoblast switching into the differentiation pathway. Prior to fusion, the prosomes containing the p27K subunit return to the cytoplasm, where they align with the gradually formed lengthwise-running desmin-type intermediate filaments and the microfilaments, co-localizing finally with the actin bundles. The prosomes progressively form discontinuous punctate structures which eventually develop a pseudo-sarcomeric banding pattern. In myotubes just formed in vitro, the formation of this pattern seems to preceed that produced by the muscle-specific sarcomeric (alpha)-actin. Interestingly, this pattern of prosomes of myotubes in terminal in vitro differentiation was very similar to that of prosomes observed in vivo in foetal and adult muscle. These observations are discussed in relation to molecular myogenesis and prosome/proteasome function.

Expression of insulin-like growth factor II (IGF-II), IGF binding protein-2 and myogenin during differentiation of myogenic satellite cells derived from the turkey

Myogenic satellite cells are essential for the development of postnatal skeletal muscle. The proliferation and differentiation of these cells are, in part, regulated by the insulin-like growth factors (IGFs), and it has been shown that the IGF binding proteins (IGFBPs) are capable of modulating the actions of IGFs. We have examined the endogenous expression of IGF-II, IGFBP-2 and the myogenic regulatory factor, myogenin, during differentiation of clonally derived turkey muscle satellite cells. Cells were harvested at approximately 80% of confluent density. Additional cultures were rinsed, fed differentiation medium and harvested when approximately 20%, 60% and 80% differentiated (fused). Northern blot analyses were performed using total cellular RNA and labeled rat cDNAs specific for IGF-II, IGFBP-2 and myogenin. A single IGF-II mRNA transcript of approximately 4.0 kb was observed. The relative mRNA abundance was highest in proliferating cultures and decreased with the onset of differentiation, to approximately 60% of initial levels where it remained throughout differentiation. Use of the IGFBP-2 cDNA probe indicated a single mRNA transcript of approximately 2.0 kb. The level of expression of IGFBP-2 mRNA was highest in proliferating cells and decreased to 25%, 16% and 11% of initial levels as differentiation progressed. A single 1.8 kb mRNA transcript was detected with the myogenin probe. Expression of myogenin was undetectable in proliferating cultures and increased significantly as differentiation progressed. Serum-free medium was conditioned for 24 h (CM) at each time point and collected from similar cultures. An IGFBP species of M r approximately 30000 was detected in CM by probing western blots with [ 125I] IGF-I (ligand blot analysis). The intensity of this band decreased with differentiation to 35%, 24% and 18% of the level for proliferating cultures. Western blots were also probed with an antibody raised against the M r-34000 bovine IGFBP-2. This antibody specifically bound to the M r-30000 IGFBP, and the level of antibody binding decreased as differentiation progressed. It therefore appears that IGF-II, IGFBP-2 and myogenin are expressed in a differentiation-dependent manner by turkey myogenic satellite cells and may thus be involved in the process of differentiation of avian muscle cells.