Differentiation of activated satellite cells in denervated muscle following single fusions in situ and in cell culture

Abstract

Satellite cells represent a cellular source of regeneration in adult skeletal muscle. It remains unclear why a large pool of stem myoblasts in denervated muscle does not compensate for the loss of muscle mass during post-denervation atrophy. In this study, we present evidence that satellite cells in long-term denervated rat muscle are able to activate synthesis of contractile proteins after single fusions in situ. This process of early differentiation leads to formation of abnormally diminutive myotubes. The localization of such dwarf myotubes beneath the intact basal lamina on the surface of differentiated muscle fibers shows that they form by fusion of neighboring satellites or by the progeny of a single satellite cell following one or two mitotic divisions. We demonstrated single fusions of myoblasts using electron microscopy, immunocytochemical labeling and high resolution confocal digital imaging. Sequestration of nascent myotubes by the rapidly forming basal laminae creates a barrier that limits further fusions. The recruitment of satellite cells in the formation of new muscle fibers results in a progressive decrease in their local densities, spatial separation and ultimate exhaustion of the myogenic cell pool. To determine whether the accumulation of aberrant dwarf myotubes is explained by the intrinsic decline of myogenic properties of satellite cells, or depends on their spatial separation and the environment in the tissue, we studied the fusion of myoblasts isolated from normal and denervated muscle in cell culture. The experiments with a culture system demonstrated that the capacity of myoblasts to synthesize contractile proteins without serial fusions depended on cell density and the availability of partners for fusion. Satellite cells isolated from denervated muscle and plated at fusion-permissive densities progressed through the myogenic program and actively formed myotubes, which shows that their myogenic potential is not considerably impaired. The results of this study suggest that under conditions of denervation, progressive spatial separation and confinement of many satellite cells within the endomysial tubes of atrophic muscle fibers and progressive interstitial fibrosis are the important factors that prevent their normal differentiation. Our findings also provide an explanation of why denervated muscle partially and temporarily is able to restore its functional capacity following injury and regeneration: the release of satellite cells from their sublaminal location provides the necessary space for a more active regenerative process.

Expression of TGF-<i>β</i>1 and CTGF Is Associated with Fibrosis of Denervated Sternocleidomastoid Muscles in Mice

“…The negative effects of denervation in skeletal muscles, such as irreversible atrophy, extracellular matrix (ECM) formation, and connective tissue accumulation, may be among the major reasons for the poor prognosis in reinnervation (Borisov et al 2000(Borisov et al , 2005. Although protecting denervated skeletal muscles from fibrosis is key to successful reinnervation, including laryngeal reinnervation, the mechanisms of fibrosis remain unclear, and to date, there is no effective method.…”

“…According to available data, the regenerative potential of denervated skeletal muscles may be dependent on denervation-related changes in the affected muscles, such as progressive myofiber atrophy, as well as persistent fibrotic changes, and eventually, irreversible pathological change (Jergovic et al 2001;Sato et al 2003). This fibrosis can obstruct the recovery of muscle fibers and prevent full strength recovery, which may be a primary factor in the tendency for injury to recur after denervation (Borisov et al 2000(Borisov et al , 2005. However, very little is known regarding the molecular mechanisms of fibrosis in denervated skeletal muscle.…”

Age-Dependent Effect of Myostatin Blockade on Disease Severity in a Murine Model of Limb-Girdle Muscular Dystrophy

“…This impairment might be due to a progressive spatial separation and confinement of the satellite cells within the endomysial spaces of degenerated muscle fibers as well as by extreme interstitial fibrosis. 28 This defect in regenerative capacity might also be exacerbated by the advanced age of the muscle and accompanying decline of systemic factors (such as insulin-like growth factor-1 and hepatocyte growth factor). The net result of this impairment is continued degeneration and a progressive increase in fibrosis relative to control.…”

The satellite cell as a companion in skeletal muscle plasticity:currency, conveyance, clue, connector and colander

“…Schultz et al, 1978;Schultz et al, 1985;Carlson, 1995;Hansen-Smith and Carlson, 1979;Tank et al, 1977;Grounds and McGeachie, 1987;Grounds and McGeachie, 1989; J. E. Anderson McGeachie et al, 1993;McGeachie and Grounds, 1987), which showed that satellite cells must proliferate in this repair process. Repair capacity in denervated or tenotomized muscle is retained to a lesser extent, and in the longer term, is constrained by accumulation of interstitial collagen and reduced fusion (Borisov et al, 2005a;Borisov et al, 2005b;Dedkov et al, 2001;Dedkov et al, 2002;Lu et al, 1997;McGeachie, 1985;McGeachie, 1989).Satellite cell proliferation is not seen as the limiting factor in muscle regeneration or growth, although that capacity may be exhausted in severe conditions such as muscular dystrophy (Original micrograph at ϫ140.) (E) A myotube (arrow) forming through addition of myogenic cells, identified (by in situ hybridization) by their expression of myogenin transcripts, a muscle-specific regulatory gene.…”