Abstract
IntroductionStimulating the commitment of implanted dystrophin+ muscle-derived stem cells (MDSCs) into myogenic, as opposed to lipofibrogenic lineages, is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD).MethodsTo examine whether counteracting myostatin, a negative regulator of muscle mass and a pro-lipofibrotic factor, would help this process, we compared the in vitro myogenic and fibrogenic capacity of MDSCs from wild-type (WT) and myostatin knockout (Mst KO) mice under various modulators, the expression of key stem cell and myogenic genes, and the capacity of these MDSCs to repair the injured gastrocnemius in aged dystrophic mdx mice with exacerbated lipofibrosis.ResultsSurprisingly, the potent in vitro myotube formation by WT MDSCs was refractory to modulators of myostatin expression or activity, and the Mst KO MDSCs failed to form myotubes under various conditions, despite both MDSC expressing Oct 4 and various stem cell genes and differentiating into nonmyogenic lineages. The genetic inactivation of myostatin in MDSCs was associated with silencing of critical genes for early myogenesis (Actc1, Acta1, and MyoD). WT MDSCs implanted into the injured gastrocnemius of aged mdx mice significantly improved myofiber repair and reduced fat deposition and, to a lesser extent, fibrosis. In contrast to their in vitro behavior, Mst KO MDSCs in vivo also significantly improved myofiber repair, but had few effects on lipofibrotic degeneration.ConclusionsAlthough WT MDSCs are very myogenic in culture and stimulate muscle repair after injury in the aged mdx mouse, myostatin genetic inactivation blocks myotube formation in vitro, but the myogenic capacity is recovered in vivo under the influence of the myostatin+ host-tissue environment, presumably by reactivation of key genes originally silenced in the Mst KO MDSCs.
Highlights
Stimulating the commitment of implanted dystrophin+ muscle-derived stem cells (MDSCs) into myogenic, as opposed to lipofibrogenic lineages, is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD)
MDSC cultures from the myostatin knockout (Mst KO) resemble their counterparts from WT mice in morphology, replication, cell markers, and multipotent differentiation WT MDSCs formed in vitro the most robust skeletal myotubes at about passage 13, and WT MDSCs and Mst KO MDSCs were compared from passages 10 through 28
The WT MDSC culture was previously shown to be Sca1+ [28]; Sca1 selection was used for both cultures, and flow cytometry confirmed its expression in subconfluent cultures in DM-10 of both the WT and Mst KO MDSCs (Figure 1A), with negligible isotype reaction
Summary
Stimulating the commitment of implanted dystrophin+ muscle-derived stem cells (MDSCs) into myogenic, as opposed to lipofibrogenic lineages, is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The lipofibrotic degeneration of skeletal muscle (that is, excessive deposition of endomysial collagen, other extracellular matrix, and fat), characterizes muscle dystrophy, and in particular Duchenne muscular dystrophy (DMD) [1,2], as seen in its animal model, the mdx mouse [3,4,5]. This process, associated with inflammation and oxidative stress [6], is partially responsible for the severe muscle contractile dysfunction in DMD and the mdx mouse, caused mainly by the bouts of myofiber necrosis due to dystrophin genetic inactivation. The same effects are generated in response to genetic deletion of myostatin in the myostatin knockout (MST KO) mouse, in which myofiber hypertrophy is associated with less fat and reduced fibrosis [19,20,21,22,23]
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