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Muscle fibers are syncytial postmitotic cells that can acquire exogenous nuclei from resident muscle stem cells, called satellite cells. Myonuclei are added to muscle fibers by satellite cells during conditions such as load-induced hypertrophy. It is difficult to dissect the molecular contributions of resident versus satellite cell-derived myonuclei during adaptation due to the complexity of labeling distinct nuclear populations in multinuclear cells without label transference between nuclei. To sidestep this barrier, we used a genetic mouse model where myonuclear DNA can be specifically and stably labeled via nonconstitutive H2B-GFP at any point in the lifespan. Resident myonuclei (Mn) were GFP-tagged in vivo before 8 wk of progressive weighted wheel running (PoWeR) in adult mice (>4-mo-old). Resident + satellite cell-derived myonuclei (Mn+SC Mn) were labeled at the end of PoWeR in a separate cohort. Following myonuclear isolation, promoter DNA methylation profiles acquired with low-input reduced representation bisulfite sequencing (RRBS) were compared to deduce epigenetic contributions of satellite cell-derived myonuclei during adaptation. Resident myonuclear DNA has hypomethylated promoters in genes related to protein turnover, whereas the addition of satellite cell-derived myonuclei shifts myonuclear methylation profiles to favor transcription factor regulation and cell-cell signaling. By comparing myonucleus-specific methylation profiling to previously published single-nucleus transcriptional analysis in the absence (Mn) versus the presence of satellite cells (Mn+SC Mn) with PoWeR, we provide evidence that satellite cell-derived myonuclei may preferentially supply specific ribosomal proteins to growing myofibers and retain an epigenetic "memory" of prior stem cell identity. These data offer insights on distinct epigenetic myonuclear characteristics and contributions during adult muscle growth.

MicroRNA-1 (miR-1) has been shown to play an important role in muscle growth and development, however, it was mainly discovered in model animals. To explore the function and mechanism of miR-1 in goat, we firstly explored the expression profile of miR-1 in goat tissues and cells. Furthermore, the target gene of miR-1 was predicted, and the relationship between miR-1 and one of its target genes, histone deacetylase 4 ( HDAC4 ), was analyzed through double luciferase reporter assay, real-time PCR, and western blot. It was found that the miR-1 is most abundantly expressed in goat heart and skeletal muscle tissue. Meanwhile, the expression of miR-1 showed an increasing tendency from new-born goats to the 7-month-old goats, and then its expression decreases as the goats mature further. In addition, the expression levels of miR-1 decreased in goat skeletal muscle satellite cells with the algebraic increasing of cells. At last, the results showed that HDAC4 is a target gene of miR-1 in goat, and miR-1 can inhibit the post-transcriptional expression of HDAC4 , but had no significant influence on the mRNA level of HDAC4 . It was hypothesized that miR-1 promotes muscle development by inhibiting the post-transcriptional expression of HDAC4 in goat.

Although heterogeneity is recognized within the murine satellite cell pool, a comprehensive understanding of distinct subpopulations and their functional relevance in human satellite cells is lacking. We used a combination of single cell RNA sequencing and flow cytometry to identify, distinguish, and physically separate novel subpopulations of human PAX7+ satellite cells (Hu-MuSCs) from normal muscles. We found that, although relatively homogeneous compared to activated satellite cells and committed progenitors, the Hu-MuSC pool contains clusters of transcriptionally distinct cells with consistency across human individuals. New surface marker combinations were enriched in transcriptional subclusters, including a subpopulation of Hu-MuSCs marked by CXCR4/CD29/CD56/CAV1 (CAV1+). In vitro, CAV1+ Hu-MuSCs are morphologically distinct, and characterized by resistance to activation compared to CAV1- Hu-MuSCs. In vivo, CAV1+ Hu-MuSCs demonstrated increased engraftment after transplantation. Our findings provide a comprehensive transcriptional view of normal Hu-MuSCs and describe new heterogeneity, enabling separation of functionally distinct human satellite cell subpopulations., Competing Interests: EB, SG, KS, JW, SL, LB, AW, SX, ST, AB, JP No competing interests declared, (© 2020, Barruet et al.)

This study was conducted to determine the effects of long chain fatty acids (LCFAs) on triacylglycerol (TAG) content, as well as on genes associated with lipid synthesis and fatty acid composition in bovine satellite cells. Both saturated (palmitic and stearic) and unsaturated (oleic and linoleic) fatty acids stimulated the TAG accumulation at a concentration of 100 µM and oleate increased it significantly more than stearate and palmitate. The results revealed that the lipid droplet formation was markedly stimulated by linoleate and oleate at 100 µM. Compared to control, the expressions of adipose triglyceride lipase, carnitine acyltransferase 1 and the fatty acid translocase 36 were upregulated by LCFAs. All the fatty acids also significantly increased diacylglycerol acyltransferase 2 than the untreated control ( p  < 0.05). The monounsaturated fatty acids significantly increased ( p  < 0.05) in response to oleate and linoleate compared to the control as did the polyunsaturated fatty acids ( p  < 0.05), in addition to stearate, linoleate and oleate. In contrast, saturated fatty acids were significantly decreased in the oleate and linoleate-treated groups. The study results contribute to our enhanced understanding of LCFAs' regulatory roles on the bovine cell lipid metabolism.

Pig is a useful medical model for humans due to its similarity in size and physiology. Skeletal muscle plays an essential role in body movement. However, the skeletal muscle injuries are common. Skeletal muscle function maintenance largely depends on preserving the regenerative capacity of muscle. Muscle satellite cells proliferation plays an essential role in postnatal muscle growth and regeneration. Therefore, understanding the mechanisms associated with muscle satellite cells proliferation is essential for devising the alternative treatments for muscle injury. Previous studies showed JAG1-Notch1 signaling pathway and miRNAs regulate the skeletal muscle development. JAG1-Notch1 signal pathway regulates the transcription of certain types of miRNAs which further affects target gene expression. However, the specific relationship between JAG1-Notch1 signal pathway and miRNAs during muscle development has not been established. We found overexpression of intracellular domain of the Notch1 protein (N1ICD) in porcine muscle satellite cells (PSCs) decreased miR-199b level. We demonstrated that miR-199b inhibits PSCs proliferation using the overexpression and inhibition of miR-199b experiment. We also found JAG1, the miR-199b target gene, promotes PSCs proliferation through activating the Notch1 signal pathway. Furthermore, we demonstrated miR-199b forms a feedback loop with the JAG1-Notch1 signal pathway to maintain the PSCs niche homeostasis. Our results of miRNAs and genes work collaboratively in regulating PSCs proliferation expand our understanding in PSCs proliferation mechanism. Furthermore, this finding indicates miR-199b is a potential therapeutic target for muscle atrophy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Muscle stem cells, termed satellite cells (SC), and SC-derived myogenic progenitor cells (MPC) are involved in postnatal muscle growth, regeneration, and muscle adaptability. They can be released from their natural environment by mechanical disruption and tissue digestion. The literature contains several isolation protocols for porcine SC/MPC including various digestion procedures, but comparative studies are missing. In this report, classic trypsinization and a more complex trypsin, collagenase, and DNase (TCD) digestion were performed with skeletal muscle tissue from 4- to 5-d-old piglets. The two digestion procedures were compared regarding cell yield, viability, myogenic purity, and in vitro cell function. The TCD digestion tended to result in higher cell yields than digestion with solely trypsin (statistical trend p = 0.096), whereas cell size and viability did not differ. Isolated myogenic cells from both digestion procedures showed comparable proliferation rates, expressed the myogenic marker Desmin, and initiated myogenic differentiation in vitro at similar levels. Thus, TCD digestion tended to liberate slightly more cells without changes in the tested in vitro properties of the isolated cells. Both procedures are adequate for the isolation of SC/MPC from juvenile porcine muscles but the developmental state of the animal should always be considered.

Although skeletal muscle has a remarkable ability to repair/regenerate after most types of injuries, there is limited regeneration after volumetric muscle loss (VML). A number of scaffold materials have been used in the development of grafts to treat VML, however, there is still a need to better understand the most appropriate material with regards to its ability to maintain mechanical integrity while also supporting myogenesis. Five commonly used natural polymeric materials (Collagen I, Agarose, Alginate, Fibrin, and Collagen Chitosan) used in skeletal muscle tissue engineering grafts were evaluated for their mechanical properties and myogenic capacity. Rheological properties, water absorption rates, degradation stability, tensile characteristics, and the ability to support in vitro myogenesis were compared in all five materials. Collagen, Collagen Chitosan, and Fibrin demonstrated high elasticity and 100% stretch without failure, Agarose was the most brittle (20% max stretch), and Alginate demonstrated poor handleabilty. While Collagen was supportive of myogenesis, overall, Fibrin demonstrated the highest myogenic potential as indicated by the earliest and highest increases in myogenin and myosin heavy chain mRNA in satellite cells along with the most extensive myotube development as evaluated with immunohistochemistry. The findings herein support the notion that under the conditions used in this study, Fibrin is the most suitable scaffold for the development of scaffolds for skeletal muscle tissue engineering. Future studies are required to determine whether the differences in mechanical properties and myogenic potential observed in vitro in the current study translate to better skeletal muscle development in a VML injury model. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 672-679, 2018., (© 2017 Wiley Periodicals, Inc.)

Insulin-like growth factor-1 (IGF-1) regulates stem cell proliferation and differentiation in vitro. The aim of this study was to quantify the change in satellite cell (SC) specific IGF-1 colocalization following exercise. We observed a significant increase (p < 0.05) in the percentage of SC with IGF-1 colocalization from baseline to 72 h after a bout of resistance exercise. This strongly supports a role for IGF-1 in human SC function following exercise.

Satellite cell is a kind of myogenic stem cells, which plays an important role in muscle development and injury repair. Through proliferation, differentiation and fusion of muscle fiber can satellite cells make new myonuclear, leading to the hypertrophy of skeletal muscle and fiber type transformation, and this would further affect the meat quality. Here, we review the relationship between muscle fiber development and meat quality attributes as well as the influence of the satellite cell differentiation on muscle fiber character. Besides, we also summarize the classical signaling pathway (i.e., Notch etc.) and influence of epigenetic regulation (i.e. miRNA) on muscle quality.

We investigated whether myonuclear number increases in proportion to the increase in fiber size during maturational growth of skeletal muscle. Thoraco-abdominal muscle tissue was obtained from twenty 6-day to 15-year-old boys and girls during cardiothoracic surgery. Cross-sections were stained by anti-laminin for the basal lamina and by DAPI to identify nuclei. Basal lamina was traced on digital images to measure the fiber cross-sectional area (FCSA). Nuclei located within the basal lamina were considered myonuclei if pax7-negative and satellite cell nuclei if pax7-positive. Samples of two children were excluded from analysis because of clear signs of hypoxia as shown by positive carbonic anhydrase IX staining. Linear regression showed that FCSA increased with age by 187 μm(2) ·per annum (R(2) = 0.90; P < 0.001). Satellite cell density showed a dramatic decrease in the first months of life, but this was not accompanied by an increase in myonuclei per muscle fiber cross-section. Till four years of age the number of myonuclei per muscle fiber cross-section remained relatively constant but increased thereafter. Myonuclear domain size showed a steep increase during infancy and reached adult values in the young adolescent phase., (Copyright © 2012 Wiley Periodicals, Inc.)

We predicted that zilpaterol hydrochloride (ZH), a β-adrenergic receptor (AR) agonist, would depress mRNA and protein abundance of β-AR in bovine satellite cells. We also predicted that ZH would decrease total lipid synthesis in bovine adipose tissue. Bovine satellite cells isolated from the semimembranosus muscle were plated on tissue culture plates coated with reduced growth factor matrigel or collagen. Real-time quantitative PCR was used to measure specific gene expression after 48 h of ZH exposure in proliferating satellite cells and fused myoblasts. There was no effect of ZH dose on [(3)H]thymidine incorporation into DNA in proliferating myoblasts. Zilpaterol hydrochloride at 1 µM decreased (P < 0.05) β1-AR mRNA, and 0.01 and 1 µM ZH decreased (P < 0.05) β2-AR and β3-AR mRNA in myoblasts. The expression of IGF-I mRNA tended to increase (P = 0.07) with 1 µM ZH. There was no effect (P > 0.10) of ZH on the β-AR or IGF-I gene expression in fused myotube cultures at 192 h or on fusion percentage. The β2-AR antagonist ICI-118, 551 at 0.1 µM attenuated (P < 0.05) the effect of 0.1 µM ZH to reduce expression of β1- and β2-AR mRNA. The combination of 0.01 µM ZH and 0.1 µM ICI-118, 551 caused an increase (P < 0.05) in β1-AR gene expression. There was no effect (P > 0.10) of ICI-118, 551 or ZH on β3-AR or IGF-I. Western blot analysis revealed that the protein content of β2-AR in ZH-treated myotube cultures decreased (P < 0.05) relative to control. Total lipid synthesis from acetate was increased by ZH in bovine subcutaneous adipose tissue explants in the absence of theophylline but was decreased by ZH when theophylline was included in the incubation medium. These data indicate that ZH alters mRNA and protein concentrations of β-AR in satellite cell cultures, which in turn could affect responsiveness of cells to prolonged ZH exposure in vivo. Similar to other β-adrenergic agonists, ZH had only modest effects on lipid metabolism in adipose tissue explants.

The goal of this study was to provide evidence that melatonin improves muscle healing following blunt skeletal muscle injury. For this purpose, we used 56 rats and induced an open muscle injury. After injury, all animals received either daily melatonin or vehicle solution intraperitoneally. Subsequent observations were performed at day 1, 4, 7, and 14 after injury. After assessment of fast twitch and tetanic muscle force, we analyzed leukocyte infiltration, satellite cell number, and cell apoptosis. We further quantified the expression of the melatonin receptor and the activation of extracellular-signal-regulated kinase (ERK). Chronic treatment with melatonin significantly increased the twitch and tetanic force of the injured muscle at day 4, 7, and 14. At day 1, melatonin significantly reduced the leukocyte infiltration and significantly increased the number of satellite cells when compared to the control group. Consistent with this observation, melatonin significantly reduced the number of apoptotic cells at day 4. Furthermore, phosphorylation of ERK reached maximal values in the melatonin group at day 1 after injury. Additionally, we detected the MT1a receptor in the injured muscle and showed a significant up-regulation of the MT1a mRNA in the melatonin group at day 4. These data support the hypothesis that melatonin supports muscle restoration after muscle injury, inhibits apoptosis via modulation of apoptosis-associated signaling pathways, increases the number of satellite cells, and reduces inflammation., (© 2011 John Wiley & Sons A/S.)

It is hypothesized that repeated recruitment of low-threshold motor units is an underlying cause of chronic pain in trapezius myalgia. This study investigated the distribution of satellite cells (SCs), myonuclei, and macrophages in muscle biopsies from the trapezius muscle of 42 women performing repetitive manual work, diagnosed with trapezius myalgia (MYA; 44 ± 8 yr; mean ± SD) and 20 matched healthy controls (CON; 45 ± 9 yr). Our hypothesis was that muscle of MYA, in particular type I fibers, would demonstrate higher numbers of SCs, myonuclei, and macrophages compared with CON. SCs were identified on muscle cross sections by combined immunohistochemical staining for Pax7, type I myosin, and laminin, allowing the number of SCs associated with type I and II fibers to be determined. We observed a pattern of SC distribution in MYA previously only reported for individuals above 70 yr of age. Compared with CON, MYA demonstrated 19% more SCs per fiber associated with type I fibers (MYA 0.098 ± 0.039 vs. CON 0.079 ± 0.031; P < 0.05) and 40% fewer SCs associated with type II fibers (MYA 0.047 ± 0.017 vs. CON 0.066 ± 0.035; P < 0.05). The finding of similar numbers of macrophages between the two groups was not in line with our hypothesis and suggests that the elevated SC content of MYA was not due to heightened inflammatory cell contents, but rather to provide new myonuclei. The findings of greater numbers of SCs in type I fibers of muscle subjected to repeated low-intensity work support our hypothesis and provide new insight into stimuli capable of regulating SC content.

In humans, muscle satellite cell (SC) enumeration is an important measurement used to determine the myogenic response to various stimuli. To date, the standard practice for enumeration is immunohistochemistry (IHC) using antibodies against common SC markers (Pax7, NCAM). Flow cytometry (FC) analysis may provide a more rapid and quantitative determination of changes in the SC pool with potential for additional analysis not easily achievable with standard IHC. In this study, FC analysis revealed that the number of Pax7(+) cells per milligram isolated from 50 mg of fresh tissue increased 36% 24 h after exercise-induced muscle injury (300 unilateral maximal eccentric contractions). IHC analysis of Pax7 and neural cell adhesion molecule (NCAM) appeared to sufficiently and similarly represent the expansion of SCs after injury (28-36% increase). IHC and FC data illustrated that Pax7 was the most widely expressed SC marker in muscle cross-sections and represented the majority of positive cells, while NCAM was expressed to a lesser degree. Moreover, FC and IHC demonstrated a similar percentage change 24 h after injury (36% increase, Pax7; 28% increase, NCAM). FC analysis of isolated SCs revealed that the number of Pax7(+) cells per milligram in G(2)/M phase of the cell cycle increased 202% 24 h after injury. Number of cells per milligram in G(0)/G(1) and cells in S-phase increased 32% and 59% respectively. Here we illustrate the use of FC as a method for enumerating SC number on a per milligram tissue basis, providing a more easily understandable relation to muscle mass (vs. percentage of myonuclei or per myofibre). Although IHC is a powerful tool for SC analysis, FC is a fast, reliable and effective method for SC quantification as well as a more informative method for cell cycle kinetics of the SC population in humans.

Myoblast transfer therapy for Duchenne muscular dystrophy (DMD) largely fails due to cell death and inability of transplanted cells to engraft in diseased muscles. One method attempting to enrich for cell subpopulations is the Hoechst 33342 dye exclusion assay, yielding a side population (SP) thought to be progenitor enriched and a main population (MP). However, in vitro and transplant studies yielded inconsistent results relative to downstream progeny. Cell surface markers expressed by skeletal muscle-derived MP and SP cells have not been fully characterized directly ex vivo. Using flow cytometry, MP and SP cells were characterized based on their expression of several well-accepted progenitor cell antigens. Both the MP and SP populations are heterogeneous and overlapping in the cells they contain. The percentages of cells in each population vary with species and specific muscle examined. MP and SP populations contain both satellite and multipotent progenitor cells, based on expression of CD34, Sca-1, Pax7, and M-cadherin. Thus, isolation using this procedure cannot be used to predict downstream differentiation outcomes, and explains the conflicting literature on these cells. Hoechst dye also results in significant mortality of sorted cells. As defined subpopulations are easily obtained using flow cytometry, sorting immediately ex vivo based on accepted myogenic precursor cell markers will yield superior results in terms of cell homogeneity for transplantation therapy.

Presently applied methods to identify and quantify human satellite cells (SCs) give discrepant results. We introduce a new immunofluorescence method that simultaneously monitors two SC markers (NCAM and Pax7), the basal lamina and nuclei. Biopsies from power-lifters, power-lifters using anabolic substances and untrained subjects were re-examined. Significantly different results from those with staining for NCAM and nuclei were observed. There were three subtypes of SCs; NCAM(+)/Pax7(+) (94%), NCAM(+)/Pax7(-) (4%) and NCAM(-)/Pax7(+) (1%) but large individual variability existed. The proportion of SCs per nuclei within the basal lamina of myofibres (SC/N) was similar for all groups reflecting a balance between the number of SCs and myonuclei to maintain homeostasis. We emphasise that it is important to quantify both SC/N and the number of SCs per fibre. Our multiple marker method is more reliable for SC identification and quantification and can be used to evaluate other markers of muscle progenitor cells.

Satellite cells reside beneath the basal lamina of skeletal muscle fibers and include cells that act as precursors for muscle growth and repair. Although they share a common anatomical localization and typically are considered a homogeneous population, satellite cells actually exhibit substantial heterogeneity. We used cell-surface marker expression to purify from the satellite cell pool a distinct population of skeletal muscle precursors (SMPs) that function as muscle stem cells. When engrafted into muscle of dystrophin-deficient mdx mice, purified SMPs contributed to up to 94% of myofibers, restoring dystrophin expression and significantly improving muscle histology and contractile function. Transplanted SMPs also entered the satellite cell compartment, renewing the endogenous stem cell pool and participating in subsequent rounds of injury repair. Together, these studies indicate the presence in adult skeletal muscle of prospectively isolatable muscle-forming stem cells and directly demonstrate the efficacy of myogenic stem cell transplant for treating muscle degenerative disease.

Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. When satellite cells are activated by myotrauma, they proliferate, migrate, differentiate, and ultimately fuse to existing myofibers. The remainder of these cells do not differentiate, but instead return to quiescence and remain in a quiescent state until activation begins the process again. This ability to maintain their own population is important for skeletal muscle to maintain the capability to repair during postnatal life. However, the mechanisms by which satellite cells return to quiescence and maintain the quiescent state are still unclear. Here, we demonstrated that decorin mRNA expression was high in cell cultures containing a higher ratio of quiescent satellite cells when satellite cells were stimulated with various concentrations of hepatocyte growth factor. This result suggests that quiescent satellite cells express decorin at a high level compared to activated satellite cells. Furthermore, we examined the expression of decorin in reserve cells, which were undifferentiated myoblasts remaining after induction of differentiation by serum-deprivation. Decorin mRNA levels in reserve cells were higher than those in differentiated myotubes and growing myoblasts. These results suggest that decorin participates in the quiescence of myogenic cells.

A present debate in muscle biology is whether myonuclear addition is required during skeletal muscle hypertrophy. We utilized K-means cluster analysis to classify 66 humans after 16 wk of knee extensor resistance training as extreme (Xtr, n = 17), modest (Mod, n = 32), or nonresponders (Non, n = 17) based on myofiber hypertrophy, which averaged 58, 28, and 0%, respectively (Bamman MM, Petrella JK, Kim JS, Mayhew DL, Cross JM. J Appl Physiol 102: 2232-2239, 2007). We hypothesized that robust hypertrophy seen in Xtr was driven by superior satellite cell (SC) activation and myonuclear addition. Vastus lateralis biopsies were obtained at baseline and week 16. SCs were identified immunohistochemically by surface expression of neural cell adhesion molecule. At baseline, myofiber size did not differ among clusters; however, the SC population was greater in Xtr (P < 0.01) than both Mod and Non, suggesting superior basal myogenic potential. SC number increased robustly during training in Xtr only (117%; P < 0.001). Myonuclear addition occurred in Mod (9%; P < 0.05) and was most effectively accomplished in Xtr (26%; P < 0.001). After training, Xtr had more myonuclei per fiber than Non (23%; P < 0.05) and tended to have more than Mod (19%; P = 0.056). Both Xtr and Mod expanded the myonuclear domain to meet (Mod) or exceed (Xtr) 2,000 mum(2) per nucleus, possibly driving demand for myonuclear addition to support myofiber expansion. These findings strongly suggest myonuclear addition via SC recruitment may be required to achieve substantial myofiber hypertrophy in humans. Individuals with a greater basal presence of SCs demonstrated, with training, a remarkable ability to expand the SC pool, incorporate new nuclei, and achieve robust growth.

Skeletal muscle satellite cells play key roles in postnatal muscle growth and regeneration. To study molecular regulation of satellite cells, we directly prepared satellite cells from 8- to 12-week-old C57BL/6 mice and performed genome-wide gene expression analysis. Compared with activated/cycling satellite cells, 507 genes were highly upregulated in quiescent satellite cells. These included negative regulators of cell cycle and myogenic inhibitors. Gene set enrichment analysis revealed that quiescent satellite cells preferentially express the genes involved in cell-cell adhesion, regulation of cell growth, formation of extracellular matrix, copper and iron homeostasis, and lipid transportation. Furthermore, reverse transcription-polymerase chain reaction on differentially expressed genes confirmed that calcitonin receptor (CTR) was exclusively expressed in dormant satellite cells but not in activated satellite cells. In addition, CTR mRNA is hardly detected in nonmyogenic cells. Therefore, we next examined the expression of CTR in vivo. CTR was specifically expressed on quiescent satellite cells, but the expression was not found on activated/proliferating satellite cells during muscle regeneration. CTR-positive cells reappeared at the rim of regenerating myofibers in later stages of muscle regeneration. Calcitonin stimulation delayed the activation of quiescent satellite cells. Our data provide roles of CTR in quiescent satellite cells and a solid scaffold to further dissect molecular regulation of satellite cells. Disclosure of potential conflicts of interest is found at the end of this article.

The progression of Duchenne muscular dystrophy (DMD) is, in part, due to satellite cell senescence driven by high replicative pressure as these muscle stem cells repeatedly divide and fuse to damaged muscle fibers. We hypothesize that telomere shortening in satellite cells underlies their senescence. To test this hypothesis, we evaluated the diaphragm and a leg muscle from dystrophic mice of various ages for telomere dynamics. We found 30% telomere shortening in tibialis anterior muscles from 600-day-old mdx mice relative to age-matched wildtype mice. We also found a more severe shortening of telomere length in diaphragm muscles of old mdx mice. In those muscles, telomeres were shortened by approximately 15% and 40% in 100- and 600-day-old mdx mice, respectively. These findings indicate that satellite cells undergo telomere erosion, which may contribute to the inability of these cells to perpetually repair DMD muscle.

Recent research suggests that estrogen receptors (ERs) are of significance in skeletal muscle function. The aim of the present study was to investigate, whether ERalpha and ERbeta are expressed in different porcine skeletal muscles and in satellite cells derived from semimembranosus muscle (SM) at the protein and mRNA level. Immunohistochemistry demonstrated positive staining for ERalpha in the nuclei of skeletal muscle cells, while the ERbeta stain showed positive signals in nuclei and cytoplasm of skeletal myofibers and myoblasts derived from satellite cells. Additionally, a weak expression of both ER subtypes was seen in skeletal muscle tissue and SM satellite cells with Western blot analysis. A clear expression of the ERalpha mRNA and a weak expression of the ERbeta mRNA was seen in skeletal muscle tissue and SM satellite cell cultures, as determined by reverse transcription (RT)-PCR. The present study shows for the first time that both ERalpha and ERbeta are expressed in porcine skeletal muscle, which, consequently, could be considered as a target tissue for estrogens or estrogen-like compounds. However, more detailed studies on the functional impact of both receptor subtypes in skeletal muscle are necessary. The porcine SM satellite cell culture provides a suitable in vitro model to investigate estrogenic effects on pig skeletal muscle.

Objective: In this study, we determined the effects of graded doses of testosterone on muscle fiber cross-sectional area (CSA) and satellite cell number and replication in older men., Participants: Healthy men, 60-75 yr old, received a long-acting GnRH agonist to suppress endogenous testosterone production and 25, 50, 125, 300, or 600 mg testosterone enanthate im weekly for 20 wk., Methods: Immunohistochemistry, light and confocal microscopy, and electron microscopy were used to perform fiber typing and quantitate myonuclear and satellite cell number in vastus lateralis biopsies, obtained before and after 20 wk of treatment., Results: Testosterone administration in older men was associated with dose-dependent increases in CSA of both types I and II fibers. Satellite cell number increased dose dependently at the three highest doses (3% at baseline vs. 6.2, 9.2, and 13.0% at 125, 300, and 600 mg doses, P < 0.05). Testosterone administration was associated with an increase in the number of proliferating cell nuclear antigen+ satellite cells (1.8% at baseline vs. 3.9, 7.5, and 13% at 125, 300, and 600 mg doses, P < 0.005). The expression of activated Notch, examined only in the 300-mg group (baseline, 2.3 vs. 9.0% after treatment, P < 0.005), increased in satellite cells after testosterone treatment. The expression of myogenin (baseline, 6.2 vs. 20.7% after treatment, P < 0.005), examined only in the 300-mg group, increased significantly in muscle fiber nuclei after testosterone treatment, but Numb expression did not change., Conclusions: Older men respond to graded doses of testosterone with a dose-dependent increase in muscle fiber CSA and satellite cell number. Testosterone-induced skeletal muscle hypertrophy in older men is associated with increased satellite cell replication and activation.

Myoblast-extracellular matrix interactions play a pivotal role in skeletal muscle development. Transforming growth factor-beta (TGF-beta) is a key regulator of muscle cell proliferation and differentiation. The level of TGF-beta expressed will affect the concentration of the extracellular matrix proteoglycan decorin and the cell surface beta1 integrin subunit. The decorin proteoglycan is a regulator of cell growth as well as the organization of the extracellular matrix. The beta1 integrin plays a role in muscle cell attachment, migration, and the formation of multinucleated myotubes. In the current study, chicken myogenic satellite cells isolated from the pectoralis major muscle from the chicken genetic muscle weakness, low score normal (LSN), and normal pectoralis major muscle were used to investigate TGF-beta expression as it relates to decorin and beta1 integrin mRNA expression. The LSN muscle defect is characterized by altered myotube formation and sarcomere structure, and the satellite cells have reduced proliferation and differentiation. The mRNA expression was measured by real-time quantitative reverse transcription PCR. The LSN condition has elevated expression of TGF-beta2 and TGF-beta4 with increased expression of decorin and decreased beta1 integrin during myogenic satellite cell proliferation and differentiation. Normal satellite cell cultures were treated with the addition of exogenous TGF-beta during differentiation to determine if the altered expression of LSN decorin and beta1 integrin was associated with TGF-beta expression. The addition of exogenous TGF-beta decreased decorin expression during differentiation and reduced beta1 integrin expression at 24 and 48 h of differentiation. These results suggested that alteration of decorin expression in the LSN myogenic satellite cells may occur by a mechanism involving factors in addition to TGF-beta, but the addition of exogenous TGF-beta did affect both decorin and beta1 integrin expression. These data, therefore, suggested that TGF-beta might play a pivotal role in chicken skeletal muscle formation through modulation of the expression of both extracellular matrix molecules and cellular receptors important in the control of cell migration and growth regulation.

Understanding the complex role played by satellite cells in the adaptive response to exercise in human skeletal muscle has just begun. The development of reliable markers for the identification of satellite cell status (quiescence/activation/proliferation) is an important step towards the understanding of satellite cell behaviour in exercised human muscles. It is hypothesised currently that exercise in humans can induce (1) the activation of satellite cells without proliferation, (2) proliferation and withdrawal from differentiation, (3) proliferation and differentiation to provide myonuclei and (4) proliferation and differentiation to generate new muscle fibres or to repair segmental fibre injuries. In humans, the satellite cell pool can increase as early as 4 days following a single bout of exercise and is maintained at higher level following several weeks of training. Cessation of training is associated with a gradual reduction of the previously enhanced satellite cell pool. In the elderly, training counteracts the normal decline in satellite cell number seen with ageing. When the transcriptional activity of existing myonuclei reaches its maximum, daughter cells generated by satellite cell proliferation are involved in protein synthesis by enhancing the number of nuclear domains. Clearly, delineating the events and the mechanisms behind the activation of satellite cells both under physiological and pathological conditions in human skeletal muscles remains an important challenge.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by a selective degeneration of motor neurons, atrophy, and paralysis of skeletal muscle. Although a significant proportion of familial ALS results from a toxic gain of function associated with dominant SOD1 mutations, the etiology of the disease and its specific cellular origins have remained difficult to define. Here, we show that muscle-restricted expression of a localized insulin-like growth factor (Igf) -1 isoform maintained muscle integrity and enhanced satellite cell activity in SOD1(G93A) transgenic mice, inducing calcineurin-mediated regenerative pathways. Muscle-specific expression of local Igf-1 (mIgf-1) isoform also stabilized neuromuscular junctions, reduced inflammation in the spinal cord, and enhanced motor neuronal survival in SOD1(G93A) mice, delaying the onset and progression of the disease. These studies establish skeletal muscle as a primary target for the dominant action of inherited SOD1 mutation and suggest that muscle fibers provide appropriate factors, such as mIgf-1, for neuron survival.

Heparin affin regulatory peptide (HARP) is a heparin binding growth factor that belongs to a family of molecule whose biological function in myogenesis has been suspected without formal demonstration. In the present study, we investigated the expression and the distribution of HARP and its mRNA during soleus muscle regeneration using a crushed-induced regeneration model and also during differentiation of muscle satellite cells in primary cultures. We show that HARP mRNA and protein expression are increased during the regeneration process with a peak at day 5 after muscle crushing when new myotubes are formed. In situ hybridization and immunohistochemical studies showed that activated myoblasts expressed HARP at day two after crushing. Five days after muscle lesion, HARP is localised in newly formed myotubes as well as in prefused activated myoblasts. In regenerated myofibers, 15 days after crushing, expression of HARP was reduced. In vitro experiments using primary cultures of rat satellite cells indicated that HARP expression level increased during the differentiation process and peaked on fusion of myoblasts into myotubes. This is the first study demonstrating the presence of HARP in fusing myogenic cells suggests that this growth factor could play a function in myogenic differentiation.

Satellite cells (myogenic stem cells) dissociated from adult muscle tissue proliferate, fuse and form multinucleate myotubes when placed in culture. This study focused on the role of talin distribution during the differentiation of satellite cells. Talin plays a key role in anchoring actin filaments to integrins as well as to the plasma membrane in focal contacts. We demonstrated that there is a colocalization of talin and phosphoserine residues during the differentiation of satellite cells, and that it changes after TPA (a protein kinase C activator) treatment, and showed that talin existing in the cell-extracellular matrix and cell-cell contact area was not phosphorylated. In the presence of TPA (24 and 48 h exposure) the level of colocalization of both talin and phosphoserine residues was the same in the treated cells and in the control cells, but the level of talin phosphorylation was higher in the treated cells. We found that in myotubes from TPA treated cultures (144 h exposure to TPA), talin had localized near the cell membrane in the absence of phosphoserine residues, and that the level of talin phosphorylation was lower than in the control cells. We also demonstrated that the expression of talin during satellite cell differentiation was constant in both the control and TPA-treated cells.