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

Abstract

Background NADH increases in cardiomyopathy, activates protein kinase C (PKC), upregulates mitochondrial reactive oxygen species (mitoROS), and downregulates the cardiac Na+ channel (Nav1.5). Objective The objective was to determine how NADH signals downregulation of Nav1.5. Methods Isolated mouse cardiomyocytes were used for patch clamp recording and to monitor mitoROS with MitoSOX™ Red. HEK293 cells were used for transient transfections. HEK293 cells stably expressing human Nav1.5 were utilized for single channel recording, whole-cell patch clamp recording, activity measurements of phospholipase C and 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<0.01) and activated PKCδ. Activated PKCδ translocated to mitochondria and upregulated mitoROS (2.8±0.3-fold, P<0.01) by enhancing the activities of mitochondrial complexes I, II and IV (1.1- to 1.5-fold, P<0.01). PKCδ also interacted with Nav1.5 to downregulate 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 PKA was a change in single channel conductance. Conclusion NADH activated PKCδ by enhancing PLD activity. PKCδ modulated both mitoROS and Nav1.5. PKCδ elevated mitoROS via enhancing the mitochondrial oxidative phosphorylation complex activities. PKCδ-mediated channel phosphorylation and mitoROS were both required to downregulate Nav1.5 and altered single channel conductance.

Emerging potential benefits of modulating NAD+metabolism in cardiovascular disease

“…Other studies, however, have demonstrated that NADH-mediated PKC phosphorylation at S1503 modulates single channel conductance (58). In addition to NADH directly activating PKC by increasing phospholipase D activity and diacylglycerol levels, NAD ϩ has been shown to increase Na v 1.5 activity through PKA-mediated phosphorylation (57,58). Interestingly, in hearts isolated from mice that are haploinsufficient for Na v 1.5, perfusion of NAD ϩ for 20 min was protective against arrhythmogenesis assessed by programmed electrical stimulation.…”

“…PKC, in turn, phosphorylates Na v 1.5 at S1503 to decrease channel surface expression (98). Other studies, however, have demonstrated that NADH-mediated PKC phosphorylation at S1503 modulates single channel conductance (58). In addition to NADH directly activating PKC by increasing phospholipase D activity and diacylglycerol levels, NAD ϩ has been shown to increase Na v 1.5 activity through PKA-mediated phosphorylation (57,58).…”

Phosphorylation of cardiac voltage‐gated sodium channel: Potential players with multiple dimensions

“…Recently a metabolic pathway for PKC activation has been described where the elevated levels of NADH activate PKCδ, resulting in a decrease in I Na . Interestingly, this decrease in I Na was not because of any change in surface expression but rather channel conductance was decreased directly by phosphorylation of serine 1503 in Na V 1.5 channels and indirectly by increasing ROS production in mitochondria .…”

“…Recently a metabolic pathway for PKC activation has been described where the elevated levels of NADH activate PKCδ, resulting in a decrease in I Na . Interestingly, this decrease in I Na was not because of any change in surface expression but rather channel conductance was decreased directly by phosphorylation of serine 1503 in Na V 1.5 channels and indirectly by increasing ROS production in mitochondria . The PKCδ antagonism completely reversed both decrease in I Na and ROS production from the mitochondria while specific inhibition of PKCα could partially recover I Na without any effect on ROS production, thus indicating the involvement of more than one PKC isozyme.…”

“…The PKCδ antagonism completely reversed both decrease in I Na and ROS production from the mitochondria while specific inhibition of PKCα could partially recover I Na without any effect on ROS production, thus indicating the involvement of more than one PKC isozyme. By mutational analysis it was described that both phosphorylation of serine 1503 in Na V 1.5 channels and ROS production in mitochondria are required for PKCδ‐mediated modulation of the Na V 1.5 channel . Alteration in sodium current is detrimental in underlying disease conditions and may trigger arrhythmias leading to sudden cardiac death .…”