Reactive Oxygen Species Originating From Mitochondria Regulate the Cardiac Sodium Channel

Rationale: Pyridine nucleotides regulate the cardiac Na+ current (I-Na) through generation of reactive oxygen species (ROS). Objective: We investigated the source of ROS induced by elevated NADH. Methods and Results: In human embryonic kidney (HEK) cells stably expressing the cardiac Na+ channel, the decrease of I-Na (52 +/- 9%; P<0.01) induced by cytosolic NADH application (100 mu mol/L) was reversed by mitoTEMPO, rotenone, malonate, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), PK11195, and 4'-chlorodiazepam, a specific scavenger of mitochondrial superoxide and inhibitors of the mitochondrial complex I, complex II, voltage-dependent anion channels, and benzodiazepine receptor, respectively. Anti-mycin A (20 mu mol/L), a complex III inhibitor known to generate ROS, decreased I-Na (51 +/- 4%, P<0.01). This effect was blocked by NAD(+), forskolin, or rotenone. Inhibitors of complex IV, nitric oxide synthase, the NAD(P)H oxidases, xanthine oxidases, the mitochondrial permeability transition pore, and the mitochondrial ATP-sensitive K+ channel did not change the NADH effect on I-Na. Analogous results were observed in cardiomyocytes. Rotenone, mitoTEMPO, and 4'-chlorodiazepam also blocked the mutant A280V GPD1-L (glycerol-3-phosphate dehydrogenase 1-like) effect on reducing I-Na, indicating a role for mitochondria in the Brugada syndrome caused by this mutation. Fluorescent microscopy confirmed mitochondrial ROS generation with elevated NADH and ROS inhibition by NAD(+). Conclusions: Altering the oxidized to reduced NAD(H) balance can activate mitochondrial ROS production, leading to reduced I-Na. This signaling cascade may help explain the link between altered metabolism, conduction block, and arrhythmic risk. (Circ Res. 2010;107:967-974.)