Abstract 1228: Decreased Mitochondrial Biogenesis and Increased Pro-oxidant Activity Are Associated with Oxidative Impairment of Complex II in the Postischemic Heart

Abstract

Increased O 2 ·− generated in mitochondria and NO is a key mechanism of cell death in the postischemic injury. Succinate ubiquinone reductase (SQR or Complex II) is a crucial segment of electron transport chain. Oxidative impairment of SQR has been observed in the postischemic myocardium. When rats are subjected to a 30 min coronary ligation, followed by a 24 h reperfusion, two polypeptides in tissue homogenate were found to react with the antibody against nuclear-encoded 29 kDa iron-sulfur protein (ISP) of SQR. One at 29 kDa corresponded to the mature form localized in mitochondria was present in normal amounts. The other with higher molecular weight 32 kDa corresponded to the precursor localized in cytosol was marked deficient. Similar results were observed when isolated heart was subjected to global ischemia (30min) and reperfusion (1h). Taken together, these results implicate mitochondria myopathy with an abnormality of biogenesis in the postischemic heart. The SQR mRNA level analyzed by quantitative real-time PCR was decreased 51%. Both mRNA and protein expressions of transcription coactivator PGC-1α were decreased in postischemic models 44% and 38% respectively, indicating a defect in the regulation and coordination of mitochondrial biogenesis. The mRNA levels of downstream transcription factors and antioxidant enzymes including NRF, TFAM, MnSOD, and UCP were similarly decreased; indicating that defect in mitochondrial biogenesis augments the oxidative stress in the postischemic heart. In addition, peroxynitrite formation was detected in the mitochondria of postischemic myocyte. The SQR isolated from myocardium was further used to study the interaction of NO with SQR in vitro . Under the conditions of enzyme turnover, SQR obtained after NO (25 fold excess) pretreatment gained catalytic function to generate hydroxyl radical detected by EPR spin trapping using DEPMPO. In the presence of catalase and iron chelator, the EPR signal of associated with DEPMPO/ · OH was completely abolished, suggesting the involvement of iron-H 2 O 2 dependent Fenton reaction. Direct EPR measurement at 77 °K indicated the formation of nonheme iron-NO complex, implying electron leakage to O 2 had occurred at the ISP of SQR and excess NO predisposed SQR to pro-oxidant activity.