Fasudil blocks muscle atrophy and C2C12 myoblasts respiratory dysfunction triggered by ROCK1 / 第二军医大学学报

ABSTRACT

Objective To confirm whether fasudil can block C2C12 myoblasts respiration dysfunction triggered by Rho-associated coiled-coil containing protein kinase 1 (ROCK1), and whether it can block the occurrence of muscle atrophy. Methods C2C12 myoblasts were cultured in vitro, and 2% horse serum was used to induce cell differentiation and maturation. The obtained mature muscle tubule cells were divided into four groups according to the different stimuli Ad-GFP group, only transfected GPT-adenovirus vector (Adv) in C2C12 myoblasts; Ad-ROCKl group, transfected ROCKl-Adv in C2C12 myoblasts to induce ROCK1 overexpression; Ad-GFPF group, transfected GFP-Adv and given 10 μmol/L fasudil in C2C12 myoblasts; and Ad-ROCKIF group, transfected ROCKl-Adv and given 10 μmol/L fasudil in C2C12 myoblasts. The oxygen consumption rate (OCR) and extracelluar acidification rate (ECAR) of C2C12 myoblasts under different stimulation conditions were evaluated by cell energy metabolism analyzer (Seahorse), so as to determine the effect of ROCK1 overexpression and fasudil stimulation on the respiratory function of C2C12 myoblasts. Mitochondrial fission was measured by MitoTracker® red fluorescent probes. The expressions of ROCK1, mitochondrial related protein 1 (Drpl) and phosphorylated p-Drpl, E3 ubiquitin ligase muscle RING finger-1 protein (MuRFl) and muscle atrophy F-box (MAFbx, Atrogin 1) was measured by Western blotting analysis. Results Seahorse analysis showed that the OCR, ECAR, basal respiration, maximal respiration and respiration required for coupling ATP of C2C12 myoblasts in the Ad ROCK1 group were significantly increased compared with those in the Ad-GFP group (P<0.01); Meanwhile, MitoTracker® staining showed that the mitochondrial fission was increased and the mitochondrial size frequency distribution shifted left in the Ad-ROCK1 group. After exposed to fasudil. the OCR and EACR of C2C12 myoblasts in the Ad-ROCKIF group were significantly decreased versus the Ad-ROCK1 group, and the basal respiration and maximal respiration were significantly increased (P<0.05). Western blotting analysis showed that p-Drpl/Drpl ratio, and the expressions of ROCK1, MuRFl and Atroginl in Ad-ROCKIF group were significantly reduced compared with Ad-ROCKl group (P<0.05). Conclusion Fasudil, an inhibitor of ROCK1, can block the abnormal cell respiration of C2C12 myoblasts caused by overexpressed ROCK 1 in vitro, and can reduce the activity of mitochondrial kinetic protein and the expression of muscle atrophy-related proteins.