Abstract 13103: Increased Mitochondrial Oxidative Stress and Decreased Mitochondrial Respiration in an in vitro Model of Ischemia Reperfusion Injury

Abstract

Introduction: Peripheral artery disease (PAD) patients experience ischemia during activity and reperfusion of ischemic skeletal muscle during rest. Cycles of ischemia and reperfusion (I/R) result in a compromised metabolic myopathy that adds to the pathophysiology of PAD. We have recently developed an I/R in vitro model that can mimic the skeletal muscle damage seen in the PAD gastrocnemius. We hypothesized that cycles of I/R will increase mitochondrial oxidative stress and mitochondrial respiration in skeletal muscle myotubes. Methods: Primary human skeletal muscle cells derived from PAD patients and non-PAD-control gastrocnemius biopsy samples were differentiated in myotubes and exposed to constant normoxia, cycles of normoxia-hypoxia, or cycles of normoxia-hypoxia-hyperoxia, to mimic I/R, with the OxyCyler C42. A substrate inhibitor protocol was used to measure mitochondrial hydrogen peroxide and respiration using an Oroboros fluorespirometer. Mitochondrial gene and protein expressions and transmitted electron microscopy were used to identify changes in mitochondrial function and morphology. Results: The oxygen consumption rate was significantly reduced in the PAD myotubes compared to control myotubes followed by significant increase of H 2 O 2 production. The mRNA and protein expression of several mitochondrial energy metabolism components were significantly reduced in the PAD myotubes. Cycling of normoxia-hypoxia and normoxia-hypoxia-hyperoxia significantly altered the mitochondrial function and morphology in both control and PAD myotubes. Conclusions: Our in vitro model of I/R injury can alter oxidative stress, mitochondrial function and morphology in control and PAD myotubes. Therefore, the pathophysiology of PAD can be modeled in vitro , which allows for a method to test novel therapeutics to treat PAD.