Overexpression of Follistatin in Human Myoblasts Increases Their Proliferation and Differentiation, and Improves the Graft Success in SCID Mice

Abstract

Duchenne muscular dystrophy is caused by the absence of functional dystrophin, leading to the myofiber membrane instability and progressive muscle atrophy. Myoblast transplantation in dystrophic muscles is a potential therapy, as it permits the long-term restoration of dystrophin expression in transplanted muscles. However, the success of this approach is limited by the short period of muscle repair following myoblast transplantation. Myostatin, a powerful inhibitor of muscle growth, is involved in terminating the period of muscle repair following injury by reducing myoblast proliferation and differentiation. Follistatin forms a complex with myostatin, preventing its interaction with its receptor and thus blocking the myostatin signal. Here, we used a lentivirus to overexpress the follistatin protein in normal myoblasts to block the myostatin signaling. We measured the potential of transduced myoblasts to proliferate and to form multinucleated myotubes in vitro. And finally, we considered the engraftment success of those transduced myoblasts in comparison with control cells in vivo within SCID mice TA muscle. Our results first confirmed the overexpression of follistatin into lentivirus transduced myoblasts, and second showed that the overexpression of the follistatin in normal human myoblasts improved in vitro their proliferation rate by about 1.5-fold after 96 h and also their differentiation rate by about 1.6- and 1.8-fold, respectively, in the absence and in the presence of recombinant myostatin. Finally, our data demonstrated that the engraftment of human normal myoblasts overexpressing the follistatin protein into SCID mouse muscles was enhanced by twofold.

The Role of Stem Cells in Muscular Dystrophies

“…These observations confirmed those obtained in animal models showing that skeletal muscle provokes a strong immune response, even against minor antigens [33]. Different attempts were made to improve the ability of migration of myoblasts and to ameliorate their proliferative capacity [34,35]. Several problems arose so that the interest in myoblasts to treat muscular dystrophies diminished [24,36].…”

Human myostatin negatively regulates human myoblast growth and differentiation

“…In studies of the effect of exogenous addition of myostatin on the growth of human myoblast cultures, Shishkin et al (28) demonstrated that treatment with increasing concentrations of recombinant myostatin protein results in a dose-dependent decrease in the proliferation rate of cultured human myoblasts. Similarly, Benabdallah et al (5) showed that inhibition of myostatin signaling with follistatin increases proliferation and differentiation rates in human myoblasts, and that overexpression of follistatin partially rescued recombinant myostatin protein-mediated inhibition of human myogenic differentiation.…”

Identification of the crucial molecular events during the large-scale myoblast fusion in sheep

“…In mouse and chick, FST functions as a positive regulator of muscle development and growth by inhibiting TGF-␤ family members including myostatin (2,23,34,43,64). As to cellular behavior, FST regulates the proliferation (4,7,23,35), differentiation (4,32), and fusion (29) of muscle cells in myogenesis in vivo and in vitro of mouse and duck. The present evidence (Fig.…”

“…In skeletal myogenesis, myocyte proliferation, differentiation, or apoptosis are coordinated at the level of E2F (25,59), and downregulation of E2F1 mRNA is required for myogenic differentiation in C2 and C2C12 myoblasts (56,61). PIK3R1 (phosphoinositide-3-kinase, regulatory subunit 1) and FST (follistatin) are not only target genes of E2F1 (8), but also important members of signal pathways that regulate myoblast proliferation, differentiation, and fusion (4,21,23,29,36,52,53). During differentiation, MEF2A, a member of MEF2 family, plays a determining role in regulating muscle cell differentiation and inducing muscle specific gene transcription in conjunction with bHLH (basic helix-loophelix) transcription factors (5,6,42,44,60).…”