Peptidyl Inhibition of Nox2 Enhances Stress Response and Mitigates Muscle Fiber Atrophy with Simulated Microgravity

Abstract

Skeletal muscle is a dynamic and pliable tissue, which contains up to 75% of all proteins in the body. The regulation of proteins synthesis and degradation determines the muscle responsive adaptations to alterations in mechanical loading, nutrition availability, exercise intensity, etc. The pathway by which skeletal muscle cells are equipped with the ability of converting changes in mechanical force to biochemical signals is entitled “mechanotransduction”. The translocation of mu-splice variant of neuronal Nitric Oxide Synthase (nNOSµ) from sarcolemma to sarcoplasm is ascertained a causal player in mechanotransduction in unloaded skeletal muscle. In addition, Reactive Oxygen Species (ROS) are demonstrated to be causal in the translocation of nNOSμ, suppression of anabolic signaling, and muscle fiber atrophy. Mitochondria is known to be one of the sources of ROS production during unloading. Moreover, the sarcolemmal NADPH oxidase-2 (Nox2), a mechano-sensitive source of ROS, is also revealed to be increased in unloaded skeletal muscles. The upregulated Nox2 would further contribute directly to nNOSμ translocation, perturbation in heat shock proteins (e.g., HSP70) and antioxidant enzyme (e.g., MnSOD), and muscle atrophy during unloading. Upstream mechanisms and downstream effects involved in the regulation of Nox2 activity during exposure to weightlessness are partially understood due to their complex nature. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor reported to protect cells in response to stressors. We postulate that peptidyl inhibition of Nox2 would alleviate the disruption of HSP70, MnSOD, and Nrf2, and the translocation of sarcolemmal nNOS during unloading and, therefore, muscle fiber atrophy. 4-month-old F344 rats were partitioned into 3 groups: ambulatory control (CON), hindlimb unloaded (HU), and hindlimb unloaded + 5 mg/kg/day peptidyl Nox2 inhibitor gp91dstat (HUG). At the end of the study (7 days), soleus muscles were removed and frozen in isopentane chilled in liquid N2. Samples were then fractionized for further analysis by western blot, immunofluorescence, etc. Our data revealed that exposure to mechanical unloading significantly decreases the protein level of HSP70, MnSOD, and NrF2. Although, no significant protective effect of gp91dstat treatment was observed in MnSOD protein abundance, Nrf2 and HSP70 levels were notably protected during unloading when Nox2 was inhibited, suggestive of the preservation of antioxidant protection. In addition, the nNOS sarcolemmal activity, which was reduced in the hindlimb unloading study group, was elevated with the use of peptidyl Nox2 inhibitor. In conclusion, this study provides direct evidence that upregulated Nox2 can act as a causal role in skeletal muscle mechanotransduction and cellular remodeling during mechanical unloading.