Abstract 16241: Impairment and Down-Regulation of the Flavin Subcomplex is Associated With the Injury of Complex I in the Mitochondria of Ischemic Heart
Mitochondrial Complex I (CxI) plays a seminal role in both energy transduction via electron transport and as a source of oxygen free radical production following ischemia-reperfusion (IR) injury. Because flavoprotein subcomplex (Fp) controls NADH binding, electron transfer, and superoxide generation by CxI, we hypothesized altered Fp activity is central to IR injury. Accordingly, we determined the role of Fp in IR injury in isolated rat hearts (30 min ischemia, 1 hr reperfusion). The electron transfer activity (ETA) of CxI in mitochondria was analyzed by ubiquinone-1-stimulated NADH oxidation. The ETA of CxI was decreased to 52±5 % (p<0.05, n=6) of normal heart after ischemia, but increased to 72±3 % (p<0.05, n=6) of control upon reperfusion. The ETA of Fp was decreased to 76±4% (p<0.05, n=6) of control after 30-min ischemia as assayed by NADH-ferricyanide reductase (NFR) activity. The results indicated that altered function of Fp accounts for 50% decline of intact CxI activity following IR injury. No significant change of NFR activity was detected in the post-ischemic heart with 1 hr reperfusion. Western immunoblots of the FMN-binding subunit of Fp revealed expression of Fp in the ischemic heart was decreased to 68±7 % of non-ischemic controls (p<0.05, n=6), suggesting ischemic degradation of Fp contributes to diminishing its ETA and subsequent injury of intact CxI. Protein expression of Fp was restored to normal levels (100.2±6.5%) upon reperfusion, indicating rapid biosynthesis of Fp at the elevated oxygen tension. No significant difference in the mRNA level of Fp was observed between ischemic and post-ischemic hearts. However, the reperfusion-induced Fp biosynthesis in myocardium was inhibited by cycloheximide (1 mM), suggesting the involvement of translational control for reperfusion-induced Fp biosynthesis. Western analysis revealed protein expression of cytosolic PGC-1α was reduced to 41±7% (p<0.01, n=6) after 30 min of ischemia, but returned to normal levels during reperfusion, suggesting the essential role of PGC-1α as an upstream regulator in controlling Fp biosynthesis during myocardial ischemia and reperfusion. We conclude that impairment and down-regulation of Fp contribute to the injury of CxI in the mitochondria of ischemic heart.
- © 2010 by American Heart Association, Inc.