Role of protein kinase C in metabolic regulation of the cardiac Na + channel.

BACKGROUND: The reduced form of nicotinamide adenine dinucleotide (NADH) increases in cardiomyopathy, activates protein kinase C (PKC), up-regulates mitochondrial reactive oxygen species (mitoROS), and down-regulates the cardiac Na+ channel (NaV 1.5).

OBJECTIVE: The purpose of this study was to determine how NADH signals down-regulation of NaV 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. HEK293 cells stably expressing human NaV 1.5 were used for single channel recording, whole-cell patch-clamp recording, 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 PKCδ. Activated PKCδ translocated to mitochondria and up-regulated mitoROS (2.8- ± 0.3-fold, P <.01) by enhancing the activities of mitochondrial complexes I, II, and IV (1.1- to 1.5-fold, P <.01). PKCδ also interacted with NaV 1.5 to down-regulate Na+ current (INa ). Reduction in INa by activated PKCδ 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 PKCδ by enhancing PLD activity. PKCδ modulated both mitoROS and NaV 1.5. PKCδ elevated mitoROS by enhancing mitochondrial oxidative phosphorylation complex activities. PKCδ-mediated channel phosphorylation and mitoROS were both required to down-regulate NaV 1.5 and alter single channel conductance.