Rotenone inhibits primary murine myotube formation via Raf-1 and ROCK2

Rotenone (ROT) is a widely used inhibitor of complex I (CI), the first complex of the mitochondrial oxidative phosphorylation (OXPHOS) system. However, particularly at high concentrations ROT was also described to display off-target effects. Here we studied how ROT affected in vitro primary murine myotube formation. We demonstrate that myotube formation is specifically inhibited by ROT (10-100 nM), but not by piericidin A (PA; 100 nM), another CI inhibitor. At 100 nM, both ROT and PA fully blocked myoblast oxygen consumption. Knock-down of Rho-associated, coiled-coil containing protein kinase 2 (ROCK2) and, to a lesser extent ROCK1, prevented the ROT-induced inhibition of myotube formation. Moreover, the latter was reversed by inhibiting Raf-1 activity. In contrast, ROT-induced inhibition of myotube formation was not prevented by knock-down of RhoA. Taken together, our results support a model in which ROT reduces primary myotube formation independent of its inhibitory effect on CI-driven mitochondrial ATP production, but via a mechanism primarily involving the Raf-1/ROCK2 pathway.