Catabolic Response Of C2C12 Myotubes Following Doxorubicin Exposure

Doxorubicin, a commonly prescribed chemotherapeutic agent, causes skeletal muscle wasting in cancer patients undergoing chemotherapy. We have previously shown doxorubicin causes loss of skeletal muscle mass and increases oxidants in vivo. PURPOSE: The purpose of our study was to investigate the cellular response of direct doxorubicin exposure in vitro. We hypothesized that doxorubicin causes a catabolic response in C2C12 myotubes, increasing ROS-sensitive signaling and promoting atrophy. METHODS: Cultured C2C12 myotubes were exposed to doxorubicin (0.2 μM) at various time points (2-48 hrs). Cytosolic oxidant activity was measured using the fluorochrome probe dichlorofluorescin (DCFH). The loss of myofibrillar proteins actin and myosin were measured using western blot analysis. We measured mRNA and protein levels of the E3 ubiquitin ligases MAFbx/atrogin-1 and MuRF1, and protein levels of the protease caspase-3. RESULTS: Oxidant activity was elevated 13 9 % (2 hrs, n=18, p<0.05) compared to control. Following doxorubicin exposure (48 hrs) actin (mean SE; -49 4 %, n=3, p<0.01) and myosin (-40 9 %, n=11, p<0.05) proteins were decreased compared to control. Doxorubicin increased MAFbx/atrogin-1 mRNA 16 hrs (74 8%, n=3, p<0.01) and 24 hrs (132 8 %, n=3, p<0.01) following exposure, and elevated protein at 24 hrs (15 4 %, n=13, p<0.05). Doxorubicin did not alter MuRF1 mRNA or protein at any time point (data not shown). Caspase-3, both precursor and active form, were elevated 6 hrs (precursor: 25 7 %, n=3, p<0.05; active: 125 35 %, n=3) and 24 hrs (precursor: 36 6, n=3, p<0.01; active: 87 12 %, n=3, p<0.01) following doxorubicin. CONCLUSIONS: Our data suggests that doxorubicin increases oxidants, leading to downstream catabolic signaling. Supported by: T32 HL086341-02(EWP) and AHA predoctoral fellowship (LAAG)