Modulation of mitochondrial permeability transition pore by the F 1 Fo ATP synthase O subunit

Abstract

Inhibition of glycogen synthase kinase (GSK)-3 has been shown to reduce myocardial ischemia/reperfusion (IR) injury, but the underlying mechanisms are not well understood. Administration of a GSK-3 inhibitor (SB 216763) in isolated rat hearts 15 minutes prior to ischemia reduced myocardial cell death and improved post-ischemic contractile dysfunction. To identify signaling pathways involved in cardioprotection, we used proteomic methods to identify proteins that might undergo post-translational modifications in SB 216763-treated hearts. We found that SB 216763 treatment with resulted in F1Fo ATP synthase O subunit (ATPO) phosphorylation at serine 102. We hypothesize that phosphorylation of this residue plays an important role for cardioprotection. We mutated the serine residue 102 of ATPO to an alanine residue (S102A) to determine its biological effect on mitochondrial function. We first confirmed a 45-fold increase in overexpression of mutated ATPO in HepG2 cells. Next, we assessed mitochondrial permeability transition pore (mPTP) opening using the calcein AM-cobalt chloride quenching method. HepG2 cells transfected with empty vector showed a 86 ± 3.4% loss of mitochondrial calcein fluorescence following ionomycin treatment, which induces mPTP opening. HepG2 cells with mutated ATPO showed a similar 88 ± 5.0% decrease in fluorescence with mPTP opening. Interestingly, SB 216763 treatment reduced the ionomycin-mediated loss of mitochondrial calcein fluorescence in HepG2 cells with empty vector to 63 ± 7.4% (n=5, p<0.05), but had no effect on the loss of fluorescence in the cells with mutated ATPO. These data suggest that a GSK-3 inhibitor-mediated increase in ATPO phosphorylation plays a role in reducing mPTP opening and that serine 102 is the functional residue orchestrating this effect. The research is supported by the Intramural Research Program of NHLBI.