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

ABSTRACT: 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 (NaV1.5).The purpose of this study was to determine how NADH signals down-regulation of NaV1.5.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 NaV1.5 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.NADH enhanced PLD activity (1.6- ± 0.1-fold, P <.01) and activated PKC?. Activated PKC? 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). PKC? also interacted with NaV1.5 to down-regulate Na+ current (INa). Reduction in INa by activated PKC? 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.NADH activated PKC? by enhancing PLD activity. PKC? modulated both mitoROS and NaV1.5. PKC? elevated mitoROS by enhancing mitochondrial oxidative phosphorylation complex activities. PKC?-mediated channel phosphorylation and mitoROS were both required to down-regulate NaV1.5 and alter single channel conductance.

SUBMITTER: Liu M

PROVIDER: S-EPMC5316355 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Role of protein kinase C in metabolic regulation of the cardiac Na<sup>+</sup> channel.

Liu Man M Shi Guangbin G Yang Kai-Chien KC Gu Lianzhi L Kanthasamy Anumantha G AG Anantharam Vellareddy V Dudley Samuel C SC

Heart rhythm 20161215 3

<h4>Background</h4>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<sup>+</sup> channel (Na<sub>V</sub>1.5).<h4>Objective</h4>The purpose of this study was to determine how NADH signals down-regulation of Na<sub>V</sub>1.5.<h4>Methods</h4>Isolated mouse cardiomyocytes were used for patch-clamp recording and for monitoring mitoR ...[more]

PMID: 27989687

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Project description:Human gene variants affecting ion channel biophysical activity and/or membrane localization are linked to potentially fatal cardiac arrhythmias. However, the mechanism for many human arrhythmia variants remains undefined despite more than a decade of investigation. Posttranslational modulation of membrane proteins is essential for normal cardiac function. Importantly, aberrant myocyte signaling has been linked to defects in cardiac ion channel posttranslational modifications and disease. We recently identified a novel pathway for posttranslational regulation of the primary cardiac voltage-gated Na(+) channel (Na(v)1.5) by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). However, a role for this pathway in cardiac disease has not been evaluated.We evaluated the role of CaMKII-dependent phosphorylation in human genetic and acquired disease. We report an unexpected link between a short motif in the Na(v)1.5 DI-DII loop, recently shown to be critical for CaMKII-dependent phosphorylation, and Na(v)1.5 function in monogenic arrhythmia and common heart disease. Experiments in heterologous cells and primary ventricular cardiomyocytes demonstrate that the human arrhythmia susceptibility variants (A572D and Q573E) alter CaMKII-dependent regulation of Na(v)1.5, resulting in abnormal channel activity and cell excitability. In silico analysis reveals that these variants functionally mimic the phosphorylated channel, resulting in increased susceptibility to arrhythmia-triggering afterdepolarizations. Finally, we report that this same motif is aberrantly regulated in a large-animal model of acquired heart disease and in failing human myocardium.We identify the mechanism for 2 human arrhythmia variants that affect Na(v)1.5 channel activity through direct effects on channel posttranslational modification. We propose that the CaMKII phosphorylation motif in the Na(v)1.5 DI-DII cytoplasmic loop is a critical nodal point for proarrhythmic changes to Na(v)1.5 in congenital and acquired cardiac disease.

Dataset Information

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

Publications

Role of protein kinase C in metabolic regulation of the cardiac Na<sup>+</sup> channel.

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