Basal lamina remodeling at the skeletal muscle stem cell niche mediates stem cell self-renewal

A central question in stem cell biology is the relationship between stem cells and their niche. Although previous reports have uncovered how signaling molecules released by niche cells support stem cell function, the role of the extra-cellular matrix (ECM) within the niche is unclear. Here, we show that upon activation, skeletal muscle stem cells (satellite cells) induce local remodeling of the ECM and the deposition of laminin-α1 and laminin-α5 into the basal lamina of the satellite cell niche. Genetic ablation of laminin-α1, disruption of integrin-α6 signaling or blocking matrix metalloproteinase activity impairs satellite cell expansion and self-renewal. Collectively, our findings establish that remodeling of the ECM is an integral process of stem cell activity to support propagation and self-renewal, and may explain the effect laminin-α1-containing supports have on embryonic and adult stem cells, as well as the regenerative activity of exogenous laminin-111 therapy

Prelamin A mediates myocardial inflammation in dilated and HIV-associated cardiomyopathies

Cardiomyopathies are complex heart muscle diseases that can be inherited or acquired. Dilated cardiomyopathy can result from mutations in LMNA, encoding the nuclear intermediate filament proteins lamin A/C. Some LMNA mutations lead to accumulation of the lamin A precursor, prelamin A, which is disease causing in a number of tissues, yet its impact upon the heart is unknown. Here, we discovered myocardial prelamin A accumulation occurred in a case of dilated cardiomyopathy, and we show that a potentially novel mouse model of cardiac-specific prelamin A accumulation exhibited a phenotype consistent with inflammatory cardiomyopathy, which we observed to be similar to HIV-associated cardiomyopathy, an acquired disease state. Numerous HIV protease therapies are known to inhibit ZMPSTE24, the enzyme responsible for prelamin A processing, and we confirmed that accumulation of prelamin A occurred in HIV+ patient cardiac biopsies. These findings (a) confirm a unifying pathological role for prelamin A common to genetic and acquired cardiomyopathies; (b) have implications for the management of HIV patients with cardiac disease, suggesting protease inhibitors should be replaced with alternative therapies (i.e., nonnucleoside reverse transcriptase inhibitors); and (c) suggest that targeting inflammation may be a useful treatment strategy for certain forms of inherited cardiomyopathy

VGLL3 operates via TEAD1, TEAD3 and TEAD4 to influence myogenesis in skeletal muscle.

VGLL proteins are transcriptional co-factors that bind TEAD family transcription factors to regulate events ranging from wing development in fly, to muscle fibre composition and immune function in mice. Here, we characterise Vgll3 in skeletal muscle. We found that mouse Vgll3 was expressed at low levels in healthy muscle but that its levels increased during hypertrophy or regeneration; in humans, VGLL3 was highly expressed in tissues from patients with various muscle diseases, such as in dystrophic muscle and alveolar rhabdomyosarcoma. Interaction proteomics revealed that VGLL3 bound TEAD1, TEAD3 and TEAD4 in myoblasts and/or myotubes. However, there was no interaction with proteins from major regulatory systems such as the Hippo kinase cascade, unlike what is found for the TEAD co-factors YAP (encoded by YAP1) and TAZ (encoded by WWTR1). Vgll3 overexpression reduced the activity of the Hippo negative-feedback loop, affecting expression of muscle-regulating genes including Myf5, Pitx2 and Pitx3, and genes encoding certain Wnts and IGFBPs. VGLL3 mainly repressed gene expression, regulating similar genes to those regulated by YAP and TAZ. siRNA-mediated Vgll3 knockdown suppressed myoblast proliferation, whereas Vgll3 overexpression strongly promoted myogenic differentiation. However, skeletal muscle was overtly normal in Vgll3-null mice, presumably due to feedback signalling and/or redundancy. This work identifies VGLL3 as a transcriptional co-factor operating with the Hippo signal transduction network to control myogenesis

Not all activated satellite cell progeny commit to differentiation

Satellite cells provide skeletal muscle with a remarkable capacity for repeated repair and regeneration. Quiescent satellite cells are MyoD-ve, with MyoD being induced in >98% of satellite cells within 24hrs of activation (Zammit, Exp. Cell Res. 281, 39, 2002). Here, we explore the subsequent fate of these satellite cells, as they proliferate and begin to differentiate. Satellite cells associated with myofibres, cultured without exposure to serum/CEE, still activated MyoD but the majority failed to divide, demonstrating an uncoupling of the two events. In contrast, myofibres cultured with serum/CEE had clones of satellite cells. MyoD-ve cells were detected in these clones, in which the rest of the cells were MyoD+ve, implying they had arisen after division but were not adopting the same fate. Three possible explanations for MyoD-ve cells were explored: phosphorylated histone markers and BrdU labelling showed that the lack of MyoD was not cell cycle dependent; myogenin immunostaining demonstrated that satellite cells committing to differentiation initially contained MyoD; Pax7 expression was generally down-regulated in the majority of satellite cells by 72hrs, but some remained strongly Pax7+ve. Together, these data suggest that MyoD-ve cells are returning to a quiescent state

Maintaining the regenerative compartment of adult skeletal muscle

Skeletal muscle is predominantly composed of highly specialised contractile myofibres, each maintained by hundreds of postmitotic myonuclei. Throughout adult life, new myonuclei are required to meet the persistent demands of myofibre turnover, growth and repair. These are provided by the differentiation of myoblasts, generated from the satellite cells that reside between the plasmalemma and surrounding basal lamina of mature myofibres. Although the progression from satellite cell to myonucleus is well established, the mechanisms by which an effective regenerative compartment is maintained remain controversial. Indeed, it has been suggested that satellite cells are “arrested” myogenic precursors that are replenished from other stem cells located within the muscle interstitium and/or from outside the tissue. Using an isolated myofibre culture system to investigate early events of satellite cell activation, proliferation and differentiation, we have shown that in the context of the sublaminal niche, satellite cell progeny can adopt divergent fates. In this model system, quiescent Pax7+ve/MyoD-ve satellite cells are synchronously activated to produce a homogenous Pax7+ve/MyoD+ve population. These cells proliferate and generate clusters of myoblasts, most of which become Pax7-ve/myogenin+ve and are fated to undergo terminal differentiation. Significantly, some cells within the clusters maintain expression of Pax7 and down regulate MyoD, apparently returning to a state of quiescence comparable to that of the initial Pax7+ve/MyoD-ve population. Our observations demonstrate that satellite cells have the potential to generate both myonuclei and new satellite cells and suggest that the alternate fate decisions may be extrinsically imposed on a homogeneous population. Most importantly, these findings suggest that the satellite cell pool may be maintained by self-renewal and does not necessarily require a contribution from elsewhere

Integration of embryonic and fetal skeletal myogenic programs at the myosin light chain 1f/3f locus.

AbstractThe genetic control of skeletal muscle differentiation at the onset of myogenesis in the embryo is relatively well understood compared to the formation of muscle during the fetal period giving rise to the bulk of skeletal muscle fibers at birth. The Mlc1f/3f (Myl1) locus encodes two alkali myosin light chains, Mlc1f and Mlc3f, from two promoters that are differentially regulated during development. The Mlc1f promoter is active in embryonic, fetal and adult fast skeletal muscle whereas the Mlc3f promoter is upregulated during fetal development and remains on in adult fast skeletal muscle. Two enhancer elements have been identified at the mammalian Mlc1f/3f locus, a 3′ element active at all developmental stages and an intronic enhancer activated during fetal development. Here, using transgenesis, we demonstrate that these enhancers act combinatorially to confer the spatial, temporal and quantitative expression profile of the endogenous Mlc3f promoter. Using double reporter transgenes we demonstrate that each enhancer can activate both Mlc1f and Mlc3f promoters in vivo, revealing enhancer sharing rather than exclusive enhancer–promoter interactions. Finally, we demonstrate that the fetal activated enhancer contains critical E-box myogenic regulatory factor binding sites and that enhancer activation is impaired in vivo in the absence of myogenin but not in the absence of innervation. Together our observations provide insights into the regulation of fetal myogenesis and the mechanisms by which temporally distinct genetic programs are integrated at a single locus

The Hippo effector TAZ (WWTR1) transforms myoblasts and TAZ abundance is associated with reduced survival in embryonal rhabdomyosarcoma.

The Hippo effector YAP has recently been identified as a potent driver of embryonal rhabdomyosarcoma (ERMS). Most reports suggest that the YAP paralogue TAZ (gene symbol WWTR1) functions as YAP but, in skeletal muscle, TAZ has been reported to promote myogenic differentiation, whereas YAP inhibits it. Here, we investigated whether TAZ is also a rhabdomyosarcoma oncogene or whether TAZ acts as a YAP antagonist. Immunostaining of rhabdomyosarcoma tissue microarrays revealed that TAZ is significantly associated with poor survival in ERMS. In 12% of fusion gene-negative rhabdomyosarcomas, the TAZ locus is gained, which is correlated with increased expression. Constitutively active TAZ S89A significantly increased proliferation of C2C12 myoblasts and, importantly, colony formation on soft agar, suggesting transformation. However, TAZ then switches to enhance myogenic differentiation in C2C12 myoblasts, unlike YAP. Conversely, lentiviral shRNA-mediated TAZ knockdown in human ERMS cells reduced proliferation and anchorage-independent growth. While TAZ S89A or YAP1 S127A similarly activated the 8XGTIIC-Luc Hippo reporter, only YAP1 S127A activated the Brachyury (T-box) reporter. Consistent with its oncogene function, TAZ S89A induced expression of the ERMS cancer stem cell gene Myf5 and the serine biosynthesis pathway (Phgdh, Psat1, Psph) in C2C12 myoblasts. Thus, TAZ is associated with poor survival in ERMS and could act as an oncogene in rhabdomyosarcoma. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland