Extracellular matrix factors influence myoblast activation, differentiation and fusion.
Satellite cells are muscle stem cells that reside in a niche between the basal lamina and sarcolemma of mature muscle fibers. Upon muscle injury, these cells are activated to myoblasts that subsequently proliferate, migrate and differentiate into myotubes in order to facilitate repair. Extracellular matrix (ECM) and growth factors are known to regulate certain aspects of myogenesis however, a comprehensive study of the direct effects of niche ECM factors on C2C12 myogenesis has not previously been conducted and forms the core of this study. We first examined the role of hepatocyte growth factor (HGF) on C2C12 myogenesis. HGF is known to initiate activation of quiescent satellite cells in the niche and regulate various aspects of differentiation. In this study, we determined that HGF has a dose-dependent dual role in C2C12 myogenesis. HGF (2 ng/ml) significantly promoted cell division, but reduced myogenic commitment and fusion. Conversely, 10 ng/ml HGF reduced proliferative capability, but increased differentiation. This is potentially regulated by changes in c-Met expression; analysis revealed significantly decreased c-Met expression in differentiating cells cultured with 2 ng/ml HGF, but increased expression in proliferating cells with 10 ng/ml HGF. Furthermore, investigation into the mechanisms by which HGF affects myogenesis, revealed that mitogen-activated protein kinase (MAPKs: ERK, JNK or p38K) and phosphatidylinositol-3-kinase (PI3K) inhibition abrogated the HGF-stimulated increase in cell number. Interestingly, PI3K and p38 kinase facilitated the negative effect of HGF on proliferation, while ERK inhibition abrogated the HGF-mediated decrease in differentiation. Next, we analyzed the effect of the satellite cell niche ECM on C2C12 myogenesis. Collagen IV and laminin, the major components of the basal lamina, bind to and interact with satellite cells via integrins and other cell surface proteins such as the tetraspanin, CD9. Matrigel significantly increased terminal fusion but had no effect on Pax7+ and MyoD+ cell numbers. Collagen IV, the second largest constituent of Matrigel, was observed to significantly increase MyoD+ cell numbers and terminal fusion without effecting percentage Pax7+ cell numbers. Furthermore collagen IV stimulated an increase in CD9 expression on differentiating cells, such that cells cultured on collagen IV required higher levels of neutralizing anti-CD9 monoclonal antibodies to reduce fusion. These results indicate, for the first time, that the interaction of collagen IV with CD9 is a critical mediator of skeletal muscle fusion and that the observed pro-myogenic effect is accompanied, on a molecular level, by an increase in the number of committed MyoD positive cells. Extracellular matrix (ECM) and growth factors are known to have complex interactions that may modulate their activity in vivo. Lastly, in an attempt to more closely mimic in vivo conditions, murine C2C12 myoblasts were cultured on collagen IV in HGF-supplemented media followed by assessment of differentiation and proliferation. Collagen IV was not able to negate the negative effect of HGF (2 ng/ml) on fusion but was able to restore normal MyHC expression. Due to a collagen IV stimulated increase in CD9 expression in differentiating myoblasts, cells cultured on collagen IV required higher levels of neutralizing anti-CD9 monoclonal antibodies to reduce fusion; an effect not observed when cells were differentiated in the presence of HGF alone. HGF (10 ng/ml) treated samples we unable to demonstrate any fusion when CD9 was completely blocked suggesting that CD9 is a crucial co-factor in HGF (10 ng/ml)-induced fusion. These results show, for the first time, that collagen IV is able to modulate certain aspects of the dual role of HGF on myogenesis. In summary, we identified a novel dose-dependent dual role of HGF in myogenesis and uncovered that these effects are mediated by changes in c-Met expression and downstream MAPK and PI3K signalling. We showed, for the first time, that collagen IV is able to positively mediate C2C12 differentiation via a CD9-dependent pathway. Lastly, we revealed that collagen IV is able to mediate the dose-dependent effects of HGF on C2C12 myogenesis.