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

¹Department of Biomedical Sciences and Technologies, ²Microgravity, Ageing, Training and Immobility Center of Excellence, and ³Interdepartmental Center for Regenerative Medicine, University of Udine, Udine, Italy

Submitted 7 April 2006 ; accepted in final form 22 September 2006

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 F₁ 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 µM 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.

energy metabolism; H9c2 myoblasts; inhibitor protein IF₁

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