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 (Na1.5).

OBJECTIVE

The purpose of this study was to determine how NADH signals down-regulation of Na1.5.

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 Na1.5 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 Na1.5 to down-regulate Na current (I). Reduction in I 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 Na1.5. PKCδ elevated mitoROS by enhancing mitochondrial oxidative phosphorylation complex activities. PKCδ-mediated channel phosphorylation and mitoROS were both required to down-regulate Na1.5 and alter single channel conductance.