Downmodulation of mitochondrial F0F1 ATP synthase by diazoxide in cardiac myoblasts:: a dual effect of the drug

Similar to ischemic preconditioning, diazoxide was documented to elicit beneficial bioenergetic consequences linked to cardioprotection. Inhibition of ATPase activity of mitochondrial F0F1 ATP synthase may have a role in such effect and may involve the natural inhibitor protein IF1. We recently documented, using purified enzyme and isolated mitochondrial membranes from beef heart, that diazoxide interacts with the F-1 sector of F0F1 ATP synthase by promoting IF1 binding and reversibly inhibiting ATP hydrolysis. Here we investigated the effects of diazoxide on the enzyme in cultured myoblasts. Specifically, embryonic heart-derived H9c2 cells were exposed to diazoxide and mitochondrial ATPase was assayed in conditions maintaining steady-state IF1 binding ( basal ATPase activity) or detaching bound IF1 at alkaline pH. Mitochondrial transmembrane potential and uncoupling were also investigated, as well as ATP synthesis flux and ATP content. Diazoxide at a cardioprotective concentration ( 40 mu M cell-associated concentration) transiently downmodulated basal ATPase activity, concomitant with mild mitochondria uncoupling and depolarization, without affecting ATP synthesis and ATP content. Alkaline stripping of IF1 from F0F1 ATP synthase was less in diazoxide-treated than in untreated cells. Pretreatment with glibenclamide prevented, together with mitochondria depolarization, inhibition of ATPase activity under basal but not under IF1-stripping conditions, indicating that diazoxide alters alkaline IF1 release. Diazoxide inhibition of ATPase activity in IF1-stripping conditions was observed even when mitochondrial transmembrane potential was reduced by FCCP. The results suggest that diazoxide in a model of normoxic intact cells directly promotes binding of inhibitor protein IF1 to F0F1 ATP synthase and enhances IF1 binding indirectly by mildly uncoupling and depolarizing mitochondria.