Investigating the effects of an endogenous and an exogenous oxidant stressor on protein synthesis in C2C12 myoblasts
Date
2015
Authors
Musci, Robert V., author
Miller, Benjamin, advisor
Hamilton, Karyn, advisor
Gentile, Chris, committee member
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Abstract
Reactive oxygen species (ROS) related signaling is important for stress adaptation. Specifically, ROS may alter protein turnover in a duration dependent manner with acute ROS exposure increasing protein synthesis for cellular adaptation. In vitro experimentation remains a valuable tool investigate the mechanisms underlying ROS-mediated protein synthesis. This study investigates paraquat (PQ), a mitochondrial complex I inhibitor that induces superoxide production, as a potential candidate to induce endogenous ROS production in vitro to simulate an oxidative stress similar to in vivo conditions. We hypothesized that acute PQ treatment would induce protein synthesis in cells similar to exogenously added hydrogen peroxide (H₂O₂). We treated C2C12 myoblasts with 0, 1, or 2mM PQ or 0, 50, 100, or 500uM H₂O₂ for 4, 8, or 12 h while using 4% deuterium oxide enriched media to measure protein synthesis of mixed, cytosolic, and mitochondrial (mito) fractions. 100uM H₂O₂ and 2mM PQ transiently increased mito protein synthesis at 4h compared to control (H₂O₂: 0mM: 1.89%/hr +/- 0.068, 100uM: 2.75%/hr +/- 0.098 p<0.05; PQ: 0mM: 4.30%/hr +/- 0.33, 2mM: 6.86%/hr +/- 0.552, p<0.05). We verified that PQ induced oxidative stress by demonstrating that 2mM PQ 2mM PQ augmented HO-1 content compared to control at 12h (0mM: 0.452a.u. +/- 0.133, 2mM: 1.434a.u. +/- 0.487 p<0.05). Thus, we have developed an in vitro model that induces endogenous production of ROS to induce oxidative stress and demonstrates a ROS-mediated increase in mito protein synthesis.