Abstract 381: Cardioprotection By Yeast Complex I Homolog, Internal Rotenone-insensitive NADH-Q Oxidoreductase (ndi1), In Ischemia Reperfusion Injury
One critical complication of ischemia reperfusion (IR) injury is mitochondrial respiratory dysfunction leading to energetic deficits and severe cellular toxicity from reactive oxygen species (ROS). ROS are produced by, and toxic to, mitochondrial electron transfer complex I. Mammalian complex I, a NADH-quinone oxidoreductase enzyme, is a multiple subunit enzyme that oxidizes NADH and pumps protons across the inner membrane to generate a gradient that drives ATP synthesis. Defects in complex I impair ATP synthesis and result in generation of superoxide radicals causing protein unfolding, lipid peroxidation and mitochondrial DNA mutations. Internal rotenone insensitive NADH-quinone oxidoreductase (Ndi1) is one of two enzymes in S. cerevisiae mitochondria that function analogously to mammalian complex I as the entry site of electrons to the respiratory chain. Studies have shown Ndi1 is able to co-express with complex I at the inner mitochondrial membrane of mammalian cells. In complex I deficient cells, Ndi1 restores NADH-oxidation activity and reduces superoxide production. We expressed the yeast Ndi1 gene in the cardiomyocyte-derived HL-1 cell line and in neonatal rat ventricular cardiac myocytes via transient transfection. Following simulated ischemia-reperfusion (sIR), we found a 17% and 18% decrease in cell death of HL-1 and neonatal myocytes, respectively, as measured by YoPro1 staining. We also demonstrate reduced ROS production and preserved ATP levels following sIR in cells expressing Ndi1. Linking the 11- amino acid protein transduction domain (PTD) of the HIV TAT protein to Ndi1 generates a TAT-Ndi1 fusion protein capable of crossing cell membranes. Langendorff perfusion of TAT-Ndi1 in rat hearts followed by 30min no-flow ischemia and 2hr reperfusion reduced infarct size from 40% to 18% (+/−2.1%, p<0.05, n=4), demonstrating a cardioprotective capacity for Ndi1. TAT-Ndi1 expression in perfused rat hearts was confirmed by western analysis and immunohistochemistry. This is the first time a TAT-fusion protein has been successfully targeted to mitochondrial membranes and demonstrates the potential for TAT-protein transduction as a therapeutic tool in the delivery of cross-species proteins for treatment of mitochondrial disorders.
This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada & Utah).