Mitochondrial-targeted antioxidants protect skeletal muscle against immobilization-induced muscle atrophy

Abstract

Prolonged periods of muscular inactivity (e.g., limb immobilization) result in skeletal muscle atrophy. Although it is established that reactive oxygen species (ROS) play a role in inactivity-induced skeletal muscle atrophy, the cellular pathway(s) responsible for inactivity-induced ROS production remain(s) unclear. To investigate this important issue, we tested the hypothesis that elevated mitochondrial ROS production contributes to immobilization-induced increases in oxidative stress, protease activation, and myofiber atrophy in skeletal muscle. Cause-and-effect was determined by administration of a novel mitochondrial-targeted antioxidant (SS-31) to prevent immobilization-induced mitochondrial ROS production in skeletal muscle fibers. Compared with ambulatory controls, 14 days of muscle immobilization resulted in significant muscle atrophy, along with increased mitochondrial ROS production, muscle oxidative damage, and protease activation. Importantly, treatment with a mitochondrial-targeted antioxidant attenuated the inactivity-induced increase in mitochondrial ROS production and prevented oxidative stress, protease activation, and myofiber atrophy. These results support the hypothesis that redox disturbances contribute to immobilization-induced skeletal muscle atrophy and that mitochondria are an important source of ROS production in muscle fibers during prolonged periods of inactivity.

Fig. 1.

Muscle-to-body weight ratio in soleus (A) and plantaris (B) muscle of control group, immobilization (cast) group, and hindlimb immobilization group treated with SS-31 (Cast + SS) after 14 days of immobilization. Values are means ± SE (n = 7/group). *Significantly different (P < 0.05) from control.

Fig. 2.

Rates of H₂O₂ release from mitochondria in permeabilized skeletal muscle fibers prepared from soleus (A) and plantaris (B) muscles from control, cast, and cast + SS groups. Values are means ± SE. *Significantly different (P < 0.05) from control and cast + SS. #Significantly different from control.

Fig. 3.

Levels of β-unsaturated 4-hydroxynonenal (4-HNE)-conjugated cytosolic proteins in soleus (A) and plantaris (B) muscles from control, cast, and cast + SS groups. Data were analyzed as an indicator of lipid peroxidation via Western blot from the 3 experimental groups. Values are means ± SE (n = 7/group). *Significantly different (P < 0.05) from control and cast + SS. #Significantly different from control.

Fig. 4.

State 3 respiration, state 4 respiration, and respiratory control ratio (RCR) of mitochondria in permeabilized fibers prepared from soleus (A) and plantaris (B) muscle from control, cast, and cast + SS groups. Data were obtained using pyruvate/malate as substrate. Values are means ± SE. *Significantly different (P < 0.05) from control and cast + SS. #Significantly different from control.

Fig. 5.

Fiber cross-sectional area (CSA) of soleus and plantaris muscles from control, cast, and cast + SS groups. A: representative fluorescent staining of myosin heavy chain (MHC) I [4′,6-diamidino-2-phenylindole filter (blue)], MHC IIa [FITC filter (green)], and dystrophin [rhodamine filter (red)] proteins. B: type I, IIa, and IIx/IIb fiber CSA. Values are means ± SE (n = 7/group). *Significantly different (P < 0.05) from control and cast + SS.

Fig. 6.

Calpain activity in soleus (A) and plantaris (B) muscles from control, cast, and cast + SS groups. Data were analyzed via Western blot from the 3 experimental groups. Values are means ± SE (n = 7/group). *Significantly different (P < 0.05) from control and cast + SS. #Significantly different from control.

Fig. 7.

Caspase-3 activity in soleus (A) and plantaris (B) muscles from control, cast, and cast + SS groups. Data were analyzed via Western blot from the 3 experimental groups. Values are means ± SE (n = 7/group). *Significantly different (P < 0.05) from control and cast + SS.