Modulation of Cardiac Na+ Channels by PKC and Mitochondria

Abstract

<h3>Background</h3> Elevated NADH downregulates cardiac Na⁺ channel (Na_v1.5) current (I_Na) acutely to a magnitude seen in Brugada syndrome by activating protein kinase C (PKC) and inducing mitochondrial ROS overproduction. Here, we studied the mechanism whereby this kinase alters I_Na. <h3>Methods</h3> HEK293 cells transfected with human cardiac Na_v1.5 wild type (WT), S1503A, and S1503D were utilized for whole-cell patch-clamp recording. Isolated mouse cardiomyocytes were used to monitor mitochondrial reactive oxygen species (mitoROS) with MitoSOX^TM Red. <h3>Results</h3> The peak I_Na of mutants was similar to that of the WT channel. Application of NADH reduced I_Na in PKC site mutant S1503D (42 ± 2% of untreated S1503D, <i>P</i> <.01) but not S1503A (81 ± 6% of untreated S1503A, <i>P</i> >.05). Similar results were seen with phorbol 12-myristate 13-acetate on S1503D (37 ± 6% of untreated S1503D, <i>P</i> <.01) and S1503A (87 ± 11% of untreated S1503A, <i>P</i> >.05). Inducing mitochondrial ROS with antimycin A decreased I_Na of WT SCN5A but not of S1503A. Antimycin A's effect on I_Na was not blocked chelerythrine, implying PKC activation alone is insufficient to reduce I_Na. Because δV1-1 (a specific inhibitor for PKCδ) but not PKCα or ε inhibitors blocked mitochondrial ROS overproduction and reduction of I_Na induced by elevated NADH, it would appear that PKCδ activation is upstream of mitochondrial ROS and that, in addition to PKC phosphorylation of the channel, mitochondrial ROS is required for current reduction with NADH. <h3>Conclusions</h3> PKC regulates Na_v1.5 in two ways: indirectly through modification of mitochondrial ROS production and directly through channel phosphorylation at S1503. PKCδ activation induces mitochondrial ROS overproduction. The I_Na of S1503D alone was equivalent to wild type, suggesting PKC phosphorylation of the Na_v1.5 channel is essential for channel downregulation and I_Na reduction but not sufficient for the NADH effect.