Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. Here we show that myostatin, a TGF-β member, signals satellite cell quiescence and also negatively regulates satellite cell self-renewal. BrdU labeling in vivo revealed that, among the Myostatin-deficient satellite cells, higher numbers of satellite cells are activated as compared with wild type. In contrast, addition of Myostatin to myofiber explant cultures inhibits satellite cell activation. Cell cycle analysis confirms that Myostatin up-regulated p21, a Cdk inhibitor, and decreased the levels and activity of Cdk2 protein in satellite cells. Hence, Myostatin negatively regulates the G1 to S progression and thus maintains the quiescent status of satellite cells. Immunohistochemical analysis with CD34 antibodies indicates that there is an increased number of satellite cells per unit length of freshly isolated Mstn−/− muscle fibers. Determination of proliferation rate suggests that this elevation in satellite cell number could be due to increased self-renewal and delayed expression of the differentiation gene (myogenin) in Mstn−/− adult myoblasts. Taken together, these results suggest that Myostatin is a potent negative regulator of satellite cell activation and thus signals the quiescence of satellite cells.

Numerous stimulatory growth factors that can influence muscle regeneration are known. Recently, it has been demonstrated that neutralization of muscle growth inhibitory factors, such as myostatin (Mstn; also known as growth differentiation factor 8, Gdf8), also leads to increased muscle regeneration in mdx mice that are known to have cycles of degeneration. However, the precise mechanism by which Mstn regulates muscle regeneration has not yet been fully determined. To investigate the role of Mstn in adult skeletal muscle regeneration, wild-type and myostatin-null (Mstn-/-) mice were injured with notexin. Forty-eight hours after injury, accelerated migration and enhanced accretion of myogenic cells (MyoD1+) and macrophages (Mac-1+) was observed at the site of regeneration in Mstn-/- muscle as compared with wild-type muscle. Inflammatory cell numbers decreased more rapidly in the Mstn-/- muscle, indicating that the whole process of inflammatory cell response is accelerated in Mstn-/- mice. Consistent with this result, the addition of recombinant Mstn reduced the activation of satellite cells (SCs) and chemotactic movements of both myoblasts and macrophages ex vivo. Examination of regenerated muscle (28 days after injury) also revealed that Mstn-/- mice showed increased expression of decorin mRNA, reduced fibrosis and improved healing as compared with wild-type mice. On the basis of these results, we propose that Mstn negatively regulates muscle regeneration not only by controlling SC activation but also by regulating the migration of myoblasts and macrophages to the site of injury. Thus, antagonists of Mstn could potentially be useful as pharmacological agents for the treatment of disorders of overt degeneration and regeneration.

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We present a simple and efficient method for expressing cDNAs in Purkinje neurons (PNs) present in heterogeneous mouse cerebellar cultures. The method combines the transfection of freshly-dissociated cerebellar cells via nucleofection with the use of novel expression plasmids containing a fragment of the L7 (Pcp2) gene that, within the cerebellum, drives PN-specific expression. The efficiency of PN transfection (determined 13 days post nucleofection) is approximately 70%. Double and triple transfections are routinely achieved at slightly lower efficiencies. Expression in PNs is obvious after one week in culture and still strong after three weeks, by which time these neurons are well-developed. Moreover, high-level expression is restricted almost exclusively to the PNs present in these mixed cultures, which greatly facilitates the characterization of PN-specific functions. As proof of principle, we used this method to visualize (1) the morphology of living PNs expressing mGFP, (2) the localization and dynamics of the dendritic spine proteins PSD-93 and Homer-3a tagged with mGFP and (3) the interaction of live PNs expressing mGFP with other cerebellar neurons expressing mCherry from a β-Actin promoter plasmid. Finally, we created a series of L7-plasmids containing different fluorescent protein cDNAs that are suited for the expression of cDNAs of interest as N- and C-terminally tagged fluorescent fusion proteins. In summary, this procedure allows for the highly efficient, long-term, and specific expression of multiple cDNAs in differentiated PNs, and provides a favorable alternative to two procedures (viral transduction, ballistic gene delivery) used previously to express genes in cultured PNs.