Doxorubicin acts via mitochondrial ROS to stimulate catabolism in C2C12 myotubes

Abstract

Doxorubicin, a commonly prescribed chemotherapeutic agent, causes skeletal muscle wasting in cancer patients undergoing treatment and increases mitochondrial reactive oxygen species (ROS) production. ROS stimulate protein degradation in muscle by activating proteolytic systems that include caspase-3 and the ubiquitin-proteasome pathway. We hypothesized that doxorubicin causes skeletal muscle catabolism through ROS, causing upregulation of E3 ubiquitin ligases and caspase-3. We tested this hypothesis by exposing differentiated C2C12 myotubes to doxorubicin (0.2 μM). Doxorubicin decreased myotube width 48 h following exposure, along with a 40-50% reduction in myosin and sarcomeric actin. Cytosolic oxidant activity was elevated in myotubes 2 h following doxorubicin exposure. This increase in oxidants was followed by an increase in the E3 ubiquitin ligase atrogin-1/muscle atrophy F-box (MAFbx) and caspase-3. Treating myotubes with SS31 (opposes mitochondrial ROS) inhibited expression of ROS-sensitive atrogin-1/MAFbx and protected against doxorubicin-stimulated catabolism. These findings suggest doxorubicin acts via mitochondrial ROS to stimulate myotube atrophy.

Fig. 1.

Increasing concentrations of doxorubicin cause loss of cell integrity and total protein. A: averaged data for cell integrity (Sapphire700) 24 h following exposure to varying concentrations of doxorubicin (n = 3/group). B: averaged data for total protein 48 h following doxorubicin exposure (n = 3/group) and a representative gel showing total protein stain. Data are means ± SE. *P < 0.05 vs. control. Ctrl, control.

Fig. 2.

Doxorubicin exposure reduces myotube width and decreases myosin and actin protein levels. A: representative images of myotubes 24 and 48 h following exposure to doxorubicin (0.2 μM) or an equal volume of vehicle (control). B: averaged data for myotube width (n = 4/group). Averaged data for myosin (C; n = 6/group) and actin (D; n = 3/group) protein are shown relative to total protein. Data are means ± SE. *P < 0.05 vs. control. Representative Western blots may not be from contiguous lanes.

Fig. 3.

Doxorubicin simulates cytosolic oxidant activity in C2C12 myotubes. Images depict representative fluorescence (A) and averaged data for dichlorofluoroscein (DCF) fluorescence (B) in intact myotubes following 2 h of exposure to doxorubicin or an equal volume of vehicle (control; n = 18/group). Data are means ± SE. *P < 0.05 vs. control. Dox, doxorubicin.

Fig. 4.

Doxorubicin stimulates atrogin-1/MAFbx mRNA and protein. A: data depict atrogin-1/muscle atrophy F-box (MAFbx) mRNA in myotubes 6, 16, 24, and 48 h after doxorubicin exposure (n = 6/group at all time points). B: averaged data for atrogin-1/MAFbx protein 24 and 48 h following doxorubicin exposure (n = 13 for vehicle and 24 h; n = 4 for 48 h). Data are means ± SE. *P < 0.05 vs. control. Representative Western blots may not be from contiguous lanes. AU, arbitrary units.

Fig. 5.

Cleaved caspase-3 (19 kDa) and procaspase-3 (43 kDa) are elevated in myotubes following doxorubicin exposure. Averaged data for cleaved caspase-3 (A) and procaspase-3 (B) protein (n = 3/group) relative to total protein in myotubes exposed to doxorubicin. C: averaged data for actin fragment relative to total actin (n = 3/group). Data are means ± SE. *P < 0.05 vs. control. Representative Western blots may not be from contiguous lanes.

Fig. 6.

SS31 abolishes doxorubicin-induced cytosolic oxidant activity and atrogin-1/MAFbx. A: images depict representative fluorescence (left) and averaged data for DCF fluorescence (right) in intact myotubes following 2 h of exposure to doxorubicin and SS31 (n = 16/group). B: data depict atrogin-1/MAFbx mRNA levels in myotubes 24 h after doxorubicin and SS31 exposure (n = 3/group). Data are means ± SE. *P < 0.05 vs. control.

Fig. 7.

SS31 partially protects against doxorubicin-induced loss of myotube width and actin and myosin protein. A: representative images of myotubes 48 h following doxorubicin exposure with or without SS31. B: averaged data for myotube width (n = 6/group). Averaged data for myosin (C; n = 16/group) and actin (D; n = 19/group) protein are shown relative to total protein 48 h following doxorubicin and SS31 exposure. Data are means ± SE. *P < 0.05 vs. control. Representative Western blots may not be from contiguous lanes.

Fig. 8.

Summarized mechanism of doxorubicin-induced myotube atrophy. Model depicts intracellular pathways by which doxorubicin causes myotube atrophy based on data from the current findings. ROS, reactive oxygen species.