Pyridine nucleotides regulate the cardiac Na⁺ current (I_Na) through generation of reactive oxygen species (ROS).

We investigated the source of ROS induced by elevated NADH.

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 μmol/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 μmol/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⁺.

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.