Mitochondrial Reactive Oxygen Species Regulate the Cardiac Na + Channel

Abstract

Background: Pyridine nucleotides regulate the cardiac Na+ current (INa) through PKC activation and reactive oxygen species (ROS), which is inhibited by NAD+ through PKA activation. Here, we investigated the source of ROS induced by elevated NADH and how it affected the cardiac Na+ channel (Nav1.5). Methods: HEK cells stably expressing Nav1.5 and rat neonatal ventricular myocytes were utilized. Effects on INa were assessed by whole-cell patch clamp recording. Also monitored were ROS generation, mitochondrial membrane potential (ΔΨm), and myocytes action potential (AP). Results: Decrease of INa (52±9%; P<0.01) induced by NADH (100 μM) in HEK cells was reversed by mitoTEMPO, rotenone, malonate, DIDS, PK11195 and 4’-chlorodiazepam. Antimycin A (20 μM) also decreased INa (51±4%, P<0.01), which was blocked by NAD+, forskolin, or rotenone. Inhibitors for complex IV, nitric oxide synthase, NADPH oxidases, xanthine oxidases, mitochondrial permeability transition pore, and mitochondrial ATP-sensitive K+ channel did not alter NADH effect on INa. Analogous results were observed in myocytes. Rotenone, mitoTEMPO, and 4’-chlorodiazepam also blocked the effect of a mutant A280V glycerol-3-phosphate dehydrogenase 1-like protein on reducing INa, indicating a mitochondrial role in Brugada syndrome. Fluorescent microscopy revealed that elevated NADH led to mitochondrial ROS generation but did not affect the mitochondrial ΔΨm. NADH treatment did not affect the resting potential and the AP duration of myocytes. Nevertheless, a decrease of the maximum upstroke velocity of action potential (68±12%, P<0.05) was observed. Conclusions: Altering the oxidized to reduced NAD(H) balance can activate mitochondrial ROS production from complex I/III and release from the mitochondrial inner membrane anion channel leading to reduced INa. This signaling cascade may help explain the link between altered metabolism, conduction block, and arrhythmic risk.