Cellular microenvironments reveal defective mechanosensing responses and elevated YAP signaling in LMNA-mutated muscle precursors.

The mechanisms underlying the cell response to mechanical forces are crucial for muscle development and functionality. We aim to determine whether mutations of the LMNA gene (which encodes lamin A/C) causing congenital muscular dystrophy impair the ability of muscle precursors to sense tissue stiffness and to respond to mechanical challenge. We found that LMNA-mutated myoblasts embedded in soft matrix did not align along the gel axis, whereas control myoblasts did. LMNA-mutated myoblasts were unable to tune their cytoskeletal tension to the tissue stiffness as attested by inappropriate cell-matrix adhesion sites and cytoskeletal tension in soft versus rigid substrates or after mechanical challenge. Importantly, in soft two-dimensional (2D) and/or static three-dimensional (3D) conditions, LMNA-mutated myoblasts showed enhanced activation of the yes-associated protein (YAP) signaling pathway that was paradoxically reduced after cyclic stretch. siRNA-mediated downregulation of YAP reduced adhesion and actin stress fibers in LMNA myoblasts. This is the first demonstration that human myoblasts with LMNA mutations have mechanosensing defects through a YAP-dependent pathway. In addition, our data emphasize the crucial role of biophysical attributes of cellular microenvironment to the response of mechanosensing pathways in LMNA-mutated myoblasts.

Expression of mdr1 is required for efficient long term regeneration of dystrophic muscle.

The mouse mdr1a and mdr1b genes are expressed in skeletal muscle, though their precise role in muscle is unknown. Dystrophic muscle is characterized by repeated cycles of degeneration and regeneration. To explore the role of the mdr1 genes during muscle regeneration, we have created a triple knockout mouse lacking the mdr1a, mdr1b, and the dystrophin genes. The resulting ReX mice developed normally and were fertile. However, as adults, ReX had a higher proportion of degenerating muscle fibers and greater long-term loss of muscle mass than mdx. ReX muscles were also characterized by a reduced proportion of muscle side population (mSP) cells, of myogenic cells, and a reduced capacity for muscle regeneration. We found too that mSP cells derived from dystrophic muscle are more myogenic than those from normal muscle. Thus, in dystrophic muscle, the mdr1 gene plays an important role in the preservation of the mSP and of the myogenic regenerative potential. Moreover, our results suggest a hitherto unappreciated role of mdr1 in precursor cells of regenerating tissue; they therefore provide an important clue to the physiological significance of mdr1 expression in stem cells.

FGF6 mediated expansion of a resident subset of cells with SP phenotype in the C2C12 myogenic line.

Fibroblast growth factor 6 (FGF6) is selectively expressed during muscle development and regeneration. We examined its effect on muscle precursor cells (mpc) by forcing stable FGF6 expression in C2C12 cells in vitro. FGF6 produced in genetically engineered mpc was active, inducing strong morphological changes, altering cell adhesion and compromising their ability to differentiate into myotubes. Expression of MyoD and myogenin, but not of Myf5, was abrogated in FGF6 engineered mpc. These effects were reversed by FGF inhibitors. Ectopic expression of MyoD also restored fiber formation indicating that FGF6 interferes with the myogenic differentiation pathway upstream of MyoD. We also report that in the presence of FGF6, the minor (0.5-2%) subpopulation of cells actively excluding Hoechst 33342 in a verapamil-dependent manner (SP phenotype) was increased to 15-20% and the expression of the mdr1a gene (but not mdr1b) was upregulated by 400-fold. Our data establish a previously undescribed link between FGF6--a muscle specific growth factor--and a multidrug resistance gene expressed in stem cells, and suggest a role for FGF6 in the maintenance of a reserve pool of progenitor cells in the skeletal muscle.