Dystrophin is expressed in differentiated myofibers where it is required for sarcolemmal integrity, and loss-of-function mutations in its gene result in Duchenne Muscular Dystrophy (DMD), a disease characterized by progressive and severe skeletal muscle degeneration. Here we found that dystrophin is also highly expressed in activated muscle stem cells (also known as satellite cells) where it associates with the Ser/Thr kinase Mark2 (also known as Par1b), an important regulator of cell polarity. In the absence of dystrophin, expression of Mark2 protein is downregulated, resulting in the inability to polarize Pard3 to the opposite side of the cell. Consequently, the number of asymmetric divisions is strikingly reduced in dystrophin-deficient satellite cells, while also displaying a loss of polarity, abnormal division patterns including centrosome amplification, impaired mitotic spindle orientation, and prolonged cell divisions. Altogether, these intrinsic defects strongly reduce the generation of myogenic progenitors needed for proper muscle regeneration. Therefore, we conclude that dystrophin has an essential role in the regulation of satellite cell polarity and asymmetric division. Our findings indicate that muscle wasting in DMD is not only caused by myofiber fragility, but is also exacerbated by impaired regeneration due to intrinsic satellite cell dysfunction.
SUMMARY Brown adipose tissue (BAT) is an energy-dispensing thermogenic tissue that plays an important role in balancing energy metabolism. Lineage-tracing experiments indicate that brown adipocytes are derived from myogenic progenitors during embryonic development. However, adult skeletal muscle stem cells (satellite cells) have long been considered uniformly determined toward the myogenic lineage. Here, we report that adult satellite cells give rise to brown adipocytes and that microRNA-133 regulates the choice between myogenic and brown adipose determination by targeting the 3′UTR of Prdm16. Antagonism of microRNA-133 during muscle regeneration increases uncoupled respiration, glucose uptake, and thermogenesis in local treated muscle and augments whole-body energy expenditure, improves glucose tolerance, and impedes the development of diet-induced obesity. Finally, we demonstrate that miR-133 levels are downregulated in mice exposed to cold, resulting in de novo generation of satellite cell-derived brown adipocytes. Therefore, microRNA-133 represents an important therapeutic target for the treatment of obesity.
Satellite cells (SCs) sustain muscle growth and empower adult skeletal muscle with vigorous regenerative abilities. Here, we report that EZH2, the enzymatic subunit of the Polycomb-repressive complex 2 (PRC2), is expressed in both Pax7 + /Myf5 À stem cells and Pax7 + /Myf5 + committed myogenic precursors and is required for homeostasis of the adult SC pool. Mice with conditional ablation of Ezh2 in SCs have fewer muscle postnatal Pax7 + cells and reduced muscle mass and fail to appropriately regenerate. These defects are associated with impaired SC proliferation and derepression of genes expressed in nonmuscle cell lineages. Thus, EZH2 controls self-renewal and proliferation, and maintains an appropriate transcriptional program in SCs.
Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1 . In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2,3 . The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions [3][4][5][6] . The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7 . Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium [1][2][3] . This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process [1][2][3]
The in vitro development of human myotubes carrying genetic diseases, such as Duchenne Muscular Dystrophy, will open new perspectives in the identification of innovative therapeutic strategies. Through the proper design of the substrate, we guided the differentiation of human healthy and dystrophic myoblasts into myotubes exhibiting marked functional differentiation and highly defined sarcomeric organization. A thin film of photo cross-linkable elastic poly-acrylamide hydrogel with physiological-like and tunable mechanical properties (elastic moduli, E: 12, 15, 18 and 21 kPa) was used as substrate. The functionalization of its surface by micro-patterning in parallel lanes (75 microm wide, 100 microm spaced) of three adhesion proteins (laminin, fibronectin and matrigel) was meant to maximize human myoblasts fusion. Myotubes formed onto the hydrogel showed a remarkable sarcomere formation, with the highest percentage (60.0% +/- 3.8) of myotubes exhibiting sarcomeric organization, of myosin heavy chain II and alpha-actinin, after 7 days of culture onto an elastic (15 kPa) hydrogel and a matrigel patterning. In addition, healthy myotubes cultured in these conditions showed a significant membrane-localized dystrophin expression. In this study, the culture substrate has been adapted to human myoblasts differentiation, through an easy and rapid methodology, and has led to the development of in vitro human functional skeletal muscle myotubes useful for clinical purposes and in vitro physiological study, where to carry out a broad range of studies on human muscle physiopathology.
Satellite cells are a heterogeneous population of muscle progenitors with stem cell properties responsible for the regeneration of adult skeletal muscle. Increasing interest in the therapeutic potential of satellite cells has challenged researchers with the need to purify a homogenous population of muscle progenitors. Here we provide a detailed protocol for the isolation of a pure population of satellite cells using fluorescence activated cell sorting. We give specific guidelines to ameliorate the reproducibility of the satellite cell isolation protocol with the goal to standardize procedures across labs. This protocol identifies satellite cells within adult skeletal muscle as an enriched population of Integrin α7(+)/CD34(+) double positive cells and CD45, CD31, CD11b, and Sca1 negative (Lin(-)) cells (Integrin α7(+)/CD34(+)/Lin(-)). Functional assay shows that Integrin α7(+)/CD34(+)/Lin(-) satellite cells possess high myogenic potential and ability to regenerate muscle depleted satellite cells upon transplantation.
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