Chemokine-like receptor 1 regulates skeletal muscle cell myogenesis

The chemokine-like receptor-1 (CMKLR1) is a G protein-coupled receptor that is activated by chemerin, a secreted plasma leukocyte attractant and adipokine. Previous studies identified that CMKLR1 is expressed in skeletal muscle in a stage-specific fashion during embryogenesis and in adult mice; however, its function in skeletal muscle remains unclear. Based on the established function of CMKLR1 in cell migration and differentiation, we investigated the hypothesis that CMKLR1 regulates the differentiation of myoblasts into myotubes. In C(2)C(12) mouse myoblasts, CMKLR1 expression increased threefold with differentiation into multinucleated myotubes. Decreasing CMKLR1 expression by adenoviral-delivered small-hairpin RNA (shRNA) impaired the differentiation of C(2)C(12) myoblasts into mature myotubes and reduced the mRNA expression of myogenic regulatory factors myogenin and MyoD while increasing Myf5 and Mrf4. At embryonic day 12.5 (E12.5), CMKLR1 knockout (CMKLR1(-/-)) mice appeared developmentally delayed and displayed significantly lower wet weights and a considerably diminished myotomal component of somites as revealed by immunolocalization of myosin heavy chain protein compared with wild-type (CMKLR1(+/+)) mouse embryos. These changes were associated with increased Myf5 and decreased MyoD protein expression in the somites of E12.5 CMKLR1(-/-) mouse embryos. Adult male CMKLR1(-/-) mice had significantly reduced bone-free lean mass and weighed less than the CMKLR1(+/+) mice. We conclude that CMKLR1 is essential for myogenic differentiation of C(2)C(12) cells in vitro, and the CMKLR1 null mice have a subtle skeletal muscle deficit beginning from embryonic life that persists during postnatal life.     

Apoptosis-inducing factor regulates death in peripheral T cells

Apoptosis-inducing factor (Aif) is a mitochondrial flavoprotein with multiple roles in apoptosis as well as in cellular respiration and redox regulation. The harlequin (Hq) mouse strain carries an aif locus modification causing reduced Aif expression. We demonstrate that activated CD4(+) and CD8(+) peripheral T cells from Hq mice show resistance to neglect-induced death (NID) triggered by growth factor withdrawal, but not to death induced by multiple agents that trigger DNA damage. Aif translocates to the nucleus in cells undergoing NID, and, in Hq T cell blasts, resistance to NID is associated with reduced cytosolic release of mitochondrial cytochrome c, implicating Aif in this event. In contrast, Hq T cell blasts express higher levels of CD95L, demonstrating increased susceptibility to activation-induced cell death (AICD) and apoptosis triggered by hydrogen peroxide. Superoxide scavenging protects from AICD in wild-type, but not Hq, T cell blasts, suggesting that Aif plays a crucial superoxide-scavenging role to regulate T cell AICD. Finally, the altered pattern of death susceptibility is reproduced by siRNA-mediated reduction of Aif expression in normal T cells. Thus, Aif serves nonredundant roles, both proapoptotic and antiapoptotic, in activated peripheral T cells.     

Negative elongation factor regulates muscle progenitor expansion for efficient myofiber repair and stem cell pool repopulation

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Myostatin directly regulates skeletal muscle fibrosis

Skeletal muscle fibrosis is a major pathological hallmark of chronic myopathies in which myofibers are replaced by progressive deposition of collagen and other extracellular matrix proteins produced by muscle fibroblasts. Recent studies have shown that in the absence of the endogenous muscle growth regulator myostatin, regeneration of muscle is enhanced, and muscle fibrosis is correspondingly reduced. We now demonstrate that myostatin not only regulates the growth of myocytes but also directly regulates muscle fibroblasts. Our results show that myostatin stimulates the proliferation of muscle fibroblasts and the production of extracellular matrix proteins both in vitro and in vivo. Further, muscle fibroblasts express myostatin and its putative receptor activin receptor IIB. Proliferation of muscle fibroblasts, induced by myostatin, involves the activation of Smad, p38 MAPK and Akt pathways. These results expand our understanding of the function of myostatin in muscle tissue and provide a potential target for anti-fibrotic therapies.     

O(2) regulates skeletal muscle progenitor differentiation through phosphatidylinositol 3-kinase/AKT signaling

Skeletal muscle stem/progenitor cells, which give rise to terminally differentiated muscle, represent potential therapies for skeletal muscle diseases. Delineating the factors regulating these precursors will facilitate their reliable application in human muscle repair. During embryonic development and adult regeneration, skeletal muscle progenitors reside in low-O(2) environments before local blood vessels and differentiated muscle form. Prior studies established that low O(2) levels (hypoxia) maintained muscle progenitors in an undifferentiated state in vitro, although it remained unclear if progenitor differentiation was coordinated with O(2) availability in vivo. In addition, the molecular signals linking O(2) to progenitor differentiation are incompletely understood. Here we show that the muscle differentiation program is repressed by hypoxia in vitro and ischemia in vivo. Surprisingly, hypoxia can significantly impair differentiation in the absence of hypoxia-inducible factors (HIFs), the primary developmental effectors of O(2). In order to maintain the undifferentiated state, low O(2) levels block the phosphatidylinositol 3-kinase/AKT pathway in a predominantly HIF1α-independent fashion. O(2) deprivation affects AKT activity by reducing insulin-like growth factor I receptor sensitivity to growth factors. We conclude that AKT represents a key molecular link between O(2) and skeletal muscle differentiation.     

TRIM32 regulates skeletal muscle stem cell differentiation and is necessary for normal adult muscle regeneration

Limb girdle muscular dystrophy type 2H (LGMD2H) is an inherited autosomal recessive disease of skeletal muscle caused by a mutation in the TRIM32 gene. Currently its pathogenesis is entirely unclear. Typically the regeneration process of adult skeletal muscle during growth or following injury is controlled by a tissue specific stem cell population termed satellite cells. Given that TRIM32 regulates the fate of mammalian neural progenitor cells through controlling their differentiation, we asked whether TRIM32 could also be essential for the regulation of myogenic stem cells. Here we demonstrate for the first time that TRIM32 is expressed in the skeletal muscle stem cell lineage of adult mice, and that in the absence of TRIM32, myogenic differentiation is disrupted. Moreover, we show that the ubiquitin ligase TRIM32 controls this process through the regulation of c-Myc, a similar mechanism to that previously observed in neural progenitors. Importantly we show that loss of TRIM32 function induces a LGMD2H-like phenotype and strongly affects muscle regeneration in vivo. Our studies implicate that the loss of TRIM32 results in dysfunctional muscle stem cells which could contribute to the development of LGMD2H.     

The thymus regulates skeletal muscle regeneration by directly promoting satellite cell expansion

The thymus is the central immune organ, but it is known to progressively degenerate with age. As thymus degeneration is paralleled by the wasting of aging skeletal muscle, we speculated that the thymus may play a role in muscle wasting. Here, using thymectomized mice, we show that the thymus is necessary for skeletal muscle regeneration, a process tightly associated with muscle aging. Compared to control mice, the thymectomized mice displayed comparable growth of muscle mass, but decreased muscle regeneration in response to injury, as evidenced by small and sparse regenerative myofibers along with inhibited expression of regeneration-associated genes myh3, myod, and myogenin. Using paired box 7 (Pax7)-immunofluorescence staining and 5-Bromo-2′-deoxyuridine-incorporation assay, we determined that the decreased regeneration capacity was caused by a limited satellite cell pool. Interestingly, the conditioned culture medium of isolated thymocytes had a potent capacity to directly stimulate satellite cell expansion in vitro. These expanded cells were enriched in subpopulations of quiescent satellite cells (Pax7^highMyoD^lowEdU^pos) and activated satellite cells (Pax7^highMyoD^highEdU^pos), which were efficiently incorporated into the regenerative myofibers. We thus propose that the thymus plays an essential role in muscle regeneration by directly promoting satellite cell expansion and may function profoundly in the muscle aging process.     

Neural crest cell lineage restricts skeletal muscle progenitor cell differentiation through Neuregulin1-ErbB3 signaling

Coordinating the balance between progenitor self-renewal and myogenic differentiation is required for a regulated expansion of the developing muscles. Previous observation that neural crest cells (NCCs) migrate throughout the somite regions, where trunk skeletal muscles first emerge, suggests a potential role for these cells in influencing early muscle formation. However, specific signaling interactions between NCCs and skeletal muscle cells remain unknown. Here we show that mice with specific NCC and peripheral nervous system defects display impaired survival of skeletal muscle and show skeletal muscle progenitor cell (MPC) depletion due to precocious commitment to differentiation. We show that reduced NCC-derived Neuregulin1 (Nrg1) in the somite region perturbs ErbB3 signaling in uncommitted MPCs. Using a combination of explant culture experiments and genetic ablation in the mouse, we demonstrate that Nrg1 signals provided by the NCC lineage play a critical role in sustainable myogenesis, by restraining MPCs from precocious differentiation.     

Adiponectin promotes endothelial progenitor cell number and function

Obesity-linked diseases are associated with suppressed endothelial progenitor cell (EPC) function. Adiponectin is an adipose-derived protein that is downregulated in obese and diabetic subjects. Here, we investigated the effects of adiponectin on EPCs. EPC levels did not increase in adiponectin deficient (APN-KO) in response to hindlimb ischemia. Adenovirus-mediated delivery of adiponectin increased EPC levels in both WT and APN-KO mice. Incubation of human peripheral blood mononuclear cells with adiponectin led to an increase of the number of EPCs. Adiponectin induced EPC differentiation into network structures and served as a chemoattractant in EPC migration assays. These data suggest that hypoadiponectinemia may contribute to the depression of EPC levels that are observed in patients with obesity-related cardiovascular disorders.     

p21 is essential for normal myogenic progenitor cell function in regenerating skeletal muscle

Despite the ability of myogenic progenitor cells (MPCs) to completely regenerate skeletal muscle following injury, little is known regarding the molecular program that regulates their proliferation and differentiation. Although mice lacking the cyclin-dependent kinase inhibitor p21 (p21-/-), develop normally, we report here that p21-/- MPCs display increased cell number and enhanced cell cycle progression compared with wild-type MPCs. Therefore, we hypothesized that p21-/- mice would demonstrate temporally enhanced regeneration following myotrauma. In response to cardiotoxin-induced injury, p21-/- skeletal muscle regeneration was significantly attenuated vs. regenerating wild-type muscle, contrary to the hypothesis. Regenerating p21-/- skeletal muscle displayed increased proliferative (PCNA positive) nuclei coincident with increased apoptotic nuclei (TUNEL positive) compared with wild-type muscle up to 3 wk after injury. Differentiation of p21-/- MPCs was markedly impaired and associated with increased apoptosis compared with wild-type MPCs, confirming that the impaired differentiation of the p21-/- MPCs was a cell autonomous event. No dysregulation of p27, p53, or p57 protein expression in differentiating p21-/- MPCs compared with wild-type MPCs was observed, suggesting that other compensatory mechanisms are responsible for the regeneration that ultimately occurs. On the basis of these findings, we propose that p21 is essential for the coordination of cell cycle exit and differentiation in the adult MPC population and that in the absence of p21, skeletal muscle regeneration is markedly impaired. myoblasts; stem cells; apoptosis; differentiation     

Developmental programming of skeletal muscle phenotype/metabolism

Skeletal muscle is a highly dynamic and malleable tissue that is able to adapt to different stimuli placed upon it, both during gestation and after birth, ultimately resulting in anatomical changes to muscle fibre composition. Variation in nutrient supply throughout gestation is common, whether in livestock or in the human. The specific effects of maternal nutrition on foetal development are at the forefront of scientific research. However, results describing how different maternal feeding strategies affect skeletal muscle fibre development in the offspring are not fully consistent, even where the same time windows during gestation have been examined. The aim of this study is to determine the effects of increased maternal nutrition (above the recommended levels) on the Musculus semitendinosus phenotype of progeny. In all, 24 pregnant sows were assigned to one of four feeding regimes during gestation; T1 (control group): 30 MJ digestible energy per day (MJ DE/day) throughout gestation, T2: same as that for T1 but increased to 60 MJ DE/day from 25 to 50 days of gestation (dg), T3: same as that for T1 but increased to 60 MJ DE/day from 50 to 80 dg, T4: same as that for T1 but increased nutrition to 60 MJ DE/day from 25 to 80 dg. Light- and heavy-weight littermate pairs of the same sex were selected at birth and individually fed to slaughter (c. 158 days). Histochemical and immunohistochemical staining were used to identify the predominantly oxidative (deep) and less oxidative (superficial) regions of the M. semitendinosus, and to determine total fibre number and proportions of fibre types. The results demonstrate that increased maternal nutrition alters skeletal muscle phenotype in the offspring by changing fibre-type proportions, leading to an increased oxidative capacity due to an increase in Type IIA fibres. No change in total muscle area, total muscle fibre number, or fibre cross-sectional area is observed. The precise molecular mechanism(s) by which these findings occur is being investigated.     

Cell density regulates the number of cell surface receptors for fibroblast growth factor

Previous studies have shown that the binding of fibroblast growth factor to several different non-transformed cell lines decreases as cell density increases. However, it was not determined whether this is due to a reduction in receptor number or to a decrease in receptor affinity. In this study, we demonstrate that the reduction in fibroblast growth factor binding is due to a reduction in receptor number. In addition, flow cytometric analysis indicated little or no difference in the cell cycle distribution of the cells at low and high cell densities, yet the binding of fibroblast growth factor was reduced substantially at high cell densities. Thus, the reduction in growth factor binding observed in this study does not appear to result from cell cycle-dependent regulation of growth factor receptors.     

Adiponectin is expressed by skeletal muscle fibers and influences muscle phenotype and function

Adiponectin (Ad) is linked to various disease states and mediates antidiabetic and anti-inflammatory effects. While it was originally thought that Ad expression was limited to adipocytes, we demonstrate here that Ad is expressed in mouse skeletal muscles and within differentiated L6 myotubes, as assessed by RT-PCR, Western blot, and immunohistochemical analyses. Serial muscle sections stained for fiber type, lipid content, and Ad revealed that muscle fibers with elevated intramyocellular Ad expression were consistently type IIA and IID fibers with detectably higher intramyocellular lipid (IMCL) content. To determine the effect of Ad on muscle phenotype and function, we used an Ad-null [knockout (KO)] mouse model. Body mass increased significantly in 24-wk-old KO mice [+5.5 +/- 3% relative to wild-type mice (WT)], with no change in muscle mass observed. IMCL content was significantly increased (+75.1 +/- 25%), whereas epididymal fat mass, although elevated, was not different in the KO mice compared with WT (+35.1 +/- 23%; P = 0.16). Fiber-type composition was unaltered, although type IIB fiber area was increased in KO mice (+25.5 +/- 6%). In situ muscle stimulation revealed lower peak tetanic forces in KO mice relative to WT (-47.5 +/- 6%), with no change in low-frequency fatigue rates. These data demonstrate that the absence of Ad expression causes contractile dysfunction and phenotypical changes in skeletal muscle. Furthermore, we demonstrate that Ad is expressed in skeletal muscle and that its intramyocellular localization is associated with elevated IMCL, particularly in type IIA/D fibers.     

Purinergic signaling regulates neural progenitor cell expansion and neurogenesis

Neural stem and progenitor cells typically exhibit a density-dependent survival and expansion, such that critical densities are required below which clonogenic progenitors are lost. This suggests that short-range autocrine factors may be critical for progenitor cell maintenance. We report here that purines drive the expansion of ventricular zone neural stem and progenitor cells, and that purine receptor activation is required for progenitor cells to be maintained as such. Neural progenitors expressed P2Y purinergic receptors and mobilized intracellular calcium in response to agonist. Receptor antagonists suppressed proliferation and permitted differentiation into neurons and glia in vitro, while subsequent removal of purinergic inhibition restored progenitor cell expansion. Real-time bioluminescence imaging of extracellular ATP revealed that the source of extracellular nucleotides are the progenitor cells themselves, which appear to release ATP in episodic burst events. Enzyme histochemistry of the adult rat brain for ectonucleotidase activity revealed that NTDPase, which acts to degrade active ATP and thereby clears it from areas of active purinergic transmission, was selectively localized to the subventricular zone and the dentate gyrus, regions in which neuronal differentiation proceeds from the progenitor cell pool. These data suggest that purine nucleotides act as proliferation signals for neural progenitor cells, and thereby serve as negative regulators of terminal neuronal differentiation. As a result, progenitor cell-derived neurogenesis is thus associated with regions of both active purinergic signaling and modulation thereof.     

PUMA regulates intestinal progenitor cell radiosensitivity and gastrointestinal syndrome

Radiation is one of the most effective cancer treatments. However, gastrointestinal (GI) syndrome is a major limiting factor in abdominal and pelvic radiotherapy. The loss of crypt stem cells or villus endothelial cells has been suggested to be responsible for radiation-induced intestinal damage. We report here a critical role of the BH3-only protein p53 upregulated modulator of apoptosis (PUMA) in the radiosensitivity of intestinal epithelium and pathogenesis of GI syndrome. PUMA was induced in a p53-dependent manner and mediated radiation-induced apoptosis via the mitochondrial pathway in the intestinal mucosa. PUMA-deficient mice exhibited blocked apoptosis in the intestinal progenitor and stem cells, enhanced crypt proliferation and regeneration, and prolonged survival following lethal doses of radiation. Unexpectedly, PUMA deficiency had little effect on radiation-induced intestinal endothelial apoptosis. Suppressing PUMA expression by antisense oligonucleotides provided significant intestinal radioprotection. Therefore, PUMA-mediated apoptosis in the progenitor and stem cell compartments is crucial for radiation-induced intestinal damage.