Background— Oxidative stress is causally linked to the progression of heart failure, and mitochondria are critical sources of reactive oxygen species in failing myocardium. We previously observed that in heart failure, elevated cytosolic Na⁺ ([Na⁺]_i) reduces mitochondrial Ca²⁺ ([Ca²⁺]_m) by accelerating Ca²⁺ efflux via the mitochondrial Na⁺/Ca²⁺ exchanger. Because the regeneration of antioxidative enzymes requires NADPH, which is indirectly regenerated by the Krebs cycle, and Krebs cycle dehydrogenases are activated by [Ca²⁺]_m, we speculated that in failing myocytes, elevated [Na⁺]_i promotes oxidative stress.

Methods and Results— We used a patch-clamp–based approach to simultaneously monitor cytosolic and mitochondrial Ca²⁺ and, alternatively, mitochondrial H₂O₂ together with NAD(P)H in guinea pig cardiac myocytes. Cells were depolarized in a voltage-clamp mode (3 Hz), and a transition of workload was induced by β-adrenergic stimulation. During this transition, NAD(P)H initially oxidized but recovered when [Ca²⁺]_m increased. The transient oxidation of NAD(P)H was closely associated with an increase in mitochondrial H₂O₂ formation. This reactive oxygen species formation was potentiated when mitochondrial Ca²⁺ uptake was blocked (by Ru360) or Ca²⁺ efflux was accelerated (by elevation of [Na⁺]_i). In failing myocytes, H₂O₂ formation was increased, which was prevented by reducing mitochondrial Ca²⁺ efflux via the mitochondrial Na⁺/Ca²⁺ exchanger.

Conclusions— Besides matching energy supply and demand, mitochondrial Ca²⁺ uptake critically regulates mitochondrial reactive oxygen species production. In heart failure, elevated [Na⁺]_i promotes reactive oxygen species formation by reducing mitochondrial Ca²⁺ uptake. This novel mechanism, by which defects in ion homeostasis induce oxidative stress, represents a potential drug target to reduce reactive oxygen species production in the failing heart.