title: Rotenone inhibits primary murine myotube formation via Raf-1 and ROCK2.
abstract: 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 (10-100nM), but not by piericidin A (PA; 100nM), another CI inhibitor. At 100nM, 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.
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Sander
authors.initials: S
authors.lastname: Grefte
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Jori A L
authors.initials: JA
authors.lastname: Wagenaars
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Renate
authors.initials: R
authors.lastname: Jansen
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Peter H G M
authors.initials: PH
authors.lastname: Willems
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands. Electronic address: Werner.Koopman@radboudumc.nl.
authors.forename: Werner J H
authors.initials: WJ
authors.lastname: Koopman
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D002459:Cell Fusion; D002478:Cells, Cultured; D005260:Female; D005455:Fluorescent Antibody Technique; D055785:Gene Knockdown Techniques; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D024510:Muscle Development / Q000187:drug effects*; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism*; D010101:Oxygen Consumption / Q000187:drug effects; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D012402:Rotenone / Q000494:pharmacology*; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: D012402:Rotenone; D019908:Proto-Oncogene Proteins c-raf; D054460:rho-Associated Kinases
keywords: Fusion index; GW5074; Piericidin A; Rho-GTPase; Rotenone; U0126