Antiretroviral protease inhibitors prevent L6 muscle cell fusion by reducing calpain activity

The antiretroviral protease inhibitors indinavir (IDV) and ritonavir (RTV) are used in highly active antiretroviral therapies (HAART). Side effects from long-term HAART therapy include loss of muscle mass. Myoblasts when cultured in media low in growth factors withdraw from the cell cycle, express muscle-specific differentiation inducers and proteins, and fuse to form myotubes. The neutral protease, calpain, is required for myotube formation and RTV decreased calpain activity in vitro. We found lower calpain activity, but not protein, in homogenates of RTV-treated L6 cells than in control cultures. Importantly, L6 and C2C12 myoblasts did not form myotubes when cultured with 10 or 20 muM IDV or RTV. Control and drug-related L6 myoblasts showed identical decreases in proliferating cell nuclear antigens expression indicating proliferation arrest. Similarly, muscle differentiation inducers MyoD and myogenin and their downstream target, myosin heavy chain, were expressed at similar levels in control and drug-treated cells. Thus, whereas muscle differentiation was unaffected by protease inhibitors, calpain activity was reduced and myotube formation prevented. We conclude that RTV and IDV reduced myotube formation by reducing calpain activity. Our data suggest that protease inhibitors included in HAART might be directly involved in muscle wasting by reducing muscle remodeling.