Dataset Information

Na+ channel regulation by Ca2+/calmodulin and Ca2+/calmodulin-dependent protein kinase II in guinea-pig ventricular myocytes.

ABSTRACT: Calmodulin (CaM) regulates Na+ channel gating through binding to an IQ-like motif in the C-terminus. Ca2+/CaM-dependent protein kinase II (CaMKII) regulates Ca2+ handling, and chronic overactivity of CaMKII is associated with left ventricular hypertrophy and dysfunction and lethal arrhythmias. However, the acute effects of Ca2+/CaM and CaMKII on cardiac Na+ channels are not fully understood.Purified Na(V)1.5-glutathione-S-transferase fusion peptides were phosphorylated in vitro by CaMKII predominantly on the I-II linker. Whole-cell voltage-clamp was used to measure Na+ current (I(Na)) in isolated guinea-pig ventricular myocytes in the absence or presence of CaM or CaMKII in the pipette solution. CaMKII shifted the voltage dependence of Na+ channel availability by approximately +5 mV, hastened recovery from inactivation, decreased entry into intermediate or slow inactivation, and increased persistent (late) current, but did not change I(Na) decay. These CaMKII-induced changes of Na+ channel gating were completely abolished by a specific CaMKII inhibitor, autocamtide-2-related inhibitory peptide (AIP). Ca2+/CaM alone reproduced the CaMKII-induced changes of I(Na) availability and the fraction of channels undergoing slow inactivation, but did not alter recovery from inactivation or the magnitude of the late current. Furthermore, the CaM-induced changes were also completely abolished by AIP. On the other hand, cAMP-dependent protein kinase A inhibitors did not abolish the CaM/CaMKII-induced alterations of I(Na) function.Ca2+/CaM and CaMKII have distinct effects on the inactivation phenotype of cardiac Na+ channels. The differences are consistent with CaM-independent effects of CaMKII on cardiac Na+ channel gating.

SUBMITTER: Aiba T

PROVIDER: S-EPMC2802203 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Publications

Na+ channel regulation by Ca2+/calmodulin and Ca2+/calmodulin-dependent protein kinase II in guinea-pig ventricular myocytes.

Aiba Takeshi T Hesketh Geoffrey G GG Liu Ting T Carlisle Rachael R Villa-Abrille Maria Celeste MC O'Rourke Brian B Akar Fadi G FG Tomaselli Gordon F GF

Cardiovascular research 20091001 3

<h4>Aims</h4>Calmodulin (CaM) regulates Na+ channel gating through binding to an IQ-like motif in the C-terminus. Ca2+/CaM-dependent protein kinase II (CaMKII) regulates Ca2+ handling, and chronic overactivity of CaMKII is associated with left ventricular hypertrophy and dysfunction and lethal arrhythmias. However, the acute effects of Ca2+/CaM and CaMKII on cardiac Na+ channels are not fully understood.<h4>Methods and results</h4>Purified Na(V)1.5-glutathione-S-transferase fusion peptides were ...[more]

PMID: 19797425

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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.

Project description:BACKGROUND:Catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited cardiac arrhythmia characterized by adrenergically triggered arrhythmias, is inadequately treated by current standard of care. Ca2+/calmodulin-dependent protein kinase II (CaMKII), an adrenergically activated kinase that contributes to arrhythmogenesis in heart disease models, is a candidate therapeutic target in CPVT. However, translation of CaMKII inhibition has been limited by the need for selective CaMKII inhibition in cardiomyocytes. Here, we tested the hypothesis that CaMKII inhibition with a cardiomyocyte-targeted gene therapy strategy would suppress arrhythmia in CPVT mouse models. METHODS:We developed AAV9-GFP-AIP, an adeno-associated viral vector in which a potent CaMKII inhibitory peptide, autocamtide-2-related inhibitory peptide [AIP], is fused to green fluorescent protein (GFP) and expressed from a cardiomyocyte selective promoter. The vector was delivered systemically. Arrhythmia burden was evaluated with invasive electrophysiology testing in adult mice. AIP was also tested on induced pluripotent stem cells derived from patients with CPVT with different disease-causing mutations to determine the effectiveness of our proposed therapy on human induced pluripotent stem cell-derived cardiomyocytes and different pathogenic genotypes. RESULTS:AAV9-GFP-AIP was robustly expressed in the heart without significant expression in extracardiac tissues, including the brain. Administration of AAV9-GFP-AIP to neonatal mice with a known CPVT mutation (RYR2R176Q/+) effectively suppressed ventricular arrhythmias induced by either ?-adrenergic stimulation or programmed ventricular pacing, without significant proarrhythmic effect. Intravascular delivery of AAV9-GFP-AIP to adolescent mice transduced ?50% of cardiomyocytes and was effective in suppressing arrhythmia in CPVT mice. Induced pluripotent stem cell-derived cardiomyocytes derived from 2 different patients with CPVT with different pathogenic mutations demonstrated increased frequency of abnormal calcium release events, which was suppressed by a cell-permeable form of AIP. CONCLUSIONS:This proof-of-concept study showed that AAV-mediated delivery of a CaMKII peptide inhibitor to the heart was effective in suppressing arrhythmias in a murine model of CPVT. CaMKII inhibition also reversed the arrhythmia phenotype in human CPVT induced pluripotent stem cell-derived cardiomyocyte models with different pathogenic mutations.

Project description:During the acquisition of memories, influx of Ca2+ into the postsynaptic spine through the pores of activated N-methyl-D-aspartate-type glutamate receptors triggers processes that change the strength of excitatory synapses. The pattern of Ca2+influx during the first few seconds of activity is interpreted within the Ca2+-dependent signaling network such that synaptic strength is eventually either potentiated or depressed. Many of the critical signaling enzymes that control synaptic plasticity,including Ca2+/calmodulin-dependent protein kinase II (CaMKII), are regulated by calmodulin, a small protein that can bindup to 4 Ca2+ ions. As a first step toward clarifying how the Ca2+-signaling network decides between potentiation or depression, we have created a kinetic model of the interactions of Ca2+, calmodulin, and CaMKII that represents our best understanding of the dynamics of these interactions under conditions that resemble those in a postsynaptic spine. We constrained parameters of the model from data in the literature, or from our own measurements, and then predicted time courses of activation and autophosphorylation of CaMKII under a variety of conditions. Simulations showed that species of calmodulin with fewer than four bound Ca2+ play a significant role in activation of CaMKII in the physiological regime,supporting the notion that processing of Ca2+ signals in a spine involves competition among target enzymes for binding to unsaturated species of CaM in an environment in which the concentration of Ca2+ is fluctuating rapidly. Indeed, we showed that dependence of activation on the frequency of Ca2+ transients arises from the kinetics of interaction of fluctuating Ca2+with calmodulin/CaMKII complexes. We used parameter sensitivity analysis to identify which parameters will be most beneficial to measure more carefully to improve the accuracy of predictions. This model provides a quantitative base from which to build more complex dynamic models of postsynaptic signal transduction during learning.

Dataset Information

Na+ channel regulation by Ca2+/calmodulin and Ca2+/calmodulin-dependent protein kinase II in guinea-pig ventricular myocytes.

Publications

Na+ channel regulation by Ca2+/calmodulin and Ca2+/calmodulin-dependent protein kinase II in guinea-pig ventricular myocytes.

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