Reactive oxygen species originating from mitochondria regulate the cardiac sodium channel

Abstract

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 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(+).

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.

Figure 1

The source of ROS induced by NADH is the mitochondria. (A) Representative traces of I_Na demonstrate the decrease in current in the presence of [NADH]_i (100 μmol/L) was blocked by mitoTEMPO (5 μmol/L). (B) The downregulation of peak I_Na by [NADH]_i at 100 μmol/L (**P<0.01 versus SCN5A group) is not reversed by L-NAME, apocynin, or allopurinol (P>0.05 versus NADH group), but is reversed by mitoTEMPO at 5 μmol/L (P>0.05 versus SCN5A group, P<0.01 versus NADH group). All these compounds have no effect on I_Na when applied alone (P>0.05 versus SCN5A group). Numbers in parentheses indicate the number of experiments.

Figure 2

Mitochondrial ROS production in response to [NADH]_i was monitored by MitoSOX^™ Red with SCN5A cells and myocytes. The control groups were untreated, the PL groups were treated with 1 and 10 mmol/L pyruvate/lactate for 10 min, and the NAD-PL groups were incubated with 500 μmol/L NAD⁺ for ~6 hours and then treated with pyruvate/lactate buffer for 10 min. The pictures in the upper panel are representative images of myocytes of three groups. The scale bar indicates 10 μm. The lower panel shows the relative MitoSOX^™ Red fluorescent intensity, ***P<0.001 versus the untreated cells or NAD-PL groups. For each group, 9–16 samples were averaged.

Figure 3

PKC, the electron transport chain and the IMAC are involved in downregulation of I_Na by [NADH]_i. (A) Downregulation of I_Na by [NADH]_i (**P<0.01 versus SCN5A) is reversed by rotenone (1 μmol/L), but not by 5-HD. Diazoxide does not affect I_Na (P>0.05 versus SCN5A). (B) Malonate (1 mmol/L) blocks the NADH effect on reducing I_Na, and antimycin A (20 μmol/L) reproduces the [NADH]_i effect (**P<0.01 versus SCN5A group). The antimycin A-induced reduction in I_Na is prevented by [NAD⁺]_o, forskolin, or rotenone. Azide failed to block the NADH effect. (C) Chelerythrine failed to block the antimycin A effect on reducing I_Na, confirming that PKC activation is required for ROS generation. (D) Downregulation of I_Na by [NADH]_i is reversed by DIDS, PK11195 and 4′-CD, but not by CsA (**P<0.01 versus SCN5A groups). Numbers in parentheses indicate the number of experiments.

Figure 4

Neonatal ventricular myocytes show analogous downregulation of I_Na by [NADH]_i. Downregulation can be blocked by rotenone and 4′-CD, but not L-NAME. Antimycin A decreases I_Na similarly to that of [NADH]_i (**P<0.01 and ***P<0.001 versus control myocytes). Numbers in parentheses indicate the number of experiments.

Figure 5

Downregulation of I_Na by A280V GPD1-L is reversed by mitoTEMPO, rotenone, and 4′-CD (**P<0.01 versus all other groups). ). Peak currents at −20 mV were normalized to cell capacitance and divided by the current obtained with SCN5A cells transfected with WT GPD1-L. Numbers in parentheses indicate the number of experiments.

Figure 6

A proposed signaling pathway by which the mutant GPD1-L and NADH downregulate cardiac Na⁺ channel by causing PKC activation and ROS overproduction from the complex III of mitochondrial electron transport chain. Reactive oxygen species (ROS) are released from the mitochondria by the IMAC that is modulated by the mBzR. NAD⁺ upregulates the cardiac Na⁺ channel through PKA activation and inhibition of ROS overproduction.