Role of protein kinase C in metabolic regulation oft he cardiacNa+ channel

BACKGROUND The reduced form of nicotinamide adenine dinucleotide (NADH) increases in cardiomyopathy, activatesprotein kinase C (PKC), up-regulates mitochondrial reactive oxygen species (mitoROS), anddown-regulates the cardiac Na+ channel (Na(V)1.5). OBJECTIVE The purpose of this studywas to determine how NADH signals down-regulation of Na(V)1.5. METHODS Isolated mouse cardiomyocytes were used for patch-clamp recording and for monitoring mitoROS with MitoSOX Red. HEK293 cells were used for transient transfections. HEK293cells stably expressing human Na(V)1.5 were used for single channel recording, whole-cell patch-clamprecording, activity measurements of phospholipase C and phospholipase D(PLD), channel protein purification, and co-immunoprecipitation with PKC isoforms. HL-1 cells were used for mitochondria isolation. RESULTS NADH enhanced PLD activity(1.6- +/- 0.1-fold, P <. 01) and activated PKC delta. Activated PKC delta translocated to mitochondria and up-regulated mitoROS(2.8- +/- 0.3-fold, P <. 01) by enhancingthe activities of mitochondrial complexes I, II, and IV(1.1-to1.5fold, P <. 01). PKC delta also interacted with Na(V)1.5 to down-regulate Na+ current (I-Na). Reduction in I-Na by activated PKC delta was prevented by antioxidants and by mutating the known PKC phosphorylation site S1503. At the single channel level, the mechanism of current reduction by PKC and recovery by protein kinase A was a change in single channel conductance. CONCLUSION NADH activated PKC delta by enhancing PLD activity. PKCd modulated both mitoROS and Na(V)1.5. PKCd elevated mitoROS by enhancingmitochondrialoxidativephosphorylationcomplex activities. PKCd-mediated channel phosphorylation and mitoROS were both required to down-regulate Na(V)1.5 and alter single channel conductance.