Gating of the Late Na+ Channel in Normal and Failing Human Myocardium

doi:10.1006/jmcc.2002.2100

Journal of Molecular and Cellular Cardiology

Volume 34, Issue 11, November 2002, Pages 1477-1489

https://doi.org/10.1006/jmcc.2002.2100 Get rights and content

Abstract

A. I. Undrovinas, V. A. Maltsev, J., W. Kyle, N. S ilverman and H. N. Sabbah. Gating of the Late Na⁺ Channel in Normal and Failing Human Myocardium. Journal of Molecular and Cellular Cardiology (2002) 34, 1477–1489. We previously reported an ultraslow inactivating late Na⁺ current (I_NaL) in left ventricular cardiomyocytes (VC) isolated from normal (NVC) and failing (FVC) human hearts. This current could play a role in heart failure-induced repolarization abnormalities. To identify properties of NaCh contributing to I_NaL, we examined early and late openings in cell-attached patches of HEK293 cells expressing human cardiac NaCh α-subunit (α-HEK) and in VC of one normal and three failing human hearts. Two types of the late NaCh openings underlay I_NaL in all three preparations: scattered late (SLO) and bursts (BO). Amplitude analysis revealed that slope conductance for both SLO and BO was the same compared to the main level of early openings (EO) in both VC (21 vs 22.7 pS, NVC; 22.7 vs 22.6 pS, FVC) and α-HEK (23.2 vs 23 pS), respectively. Analysis of SLO latencies revealed voltage-independent ultraslow inactivation in all preparations with tendency to be slower in FVC compared to NCV. EO and SLO render one open voltage-independent state (τ∼0.4 ms) for NVC and FVC. One open (voltage-dependent) and two closed states (one voltage-dependent and another voltage-independent) were found in BO of both specimens. Burst duration tend to be longer in FVC (∼50 ms) than in NVC (∼30 ms). In FVC we found both modes SLO and BO at membrane potential of −10 mV that is attribute for take-off voltages (from −18 to −2 mV) for early afterdepolarizations (EAD's) in FVC. In conclusions, we found a novel gating mode SLO that manifest slow (hundreds of ms), voltage-independent inactivation in both NVC and FVC. We were unable to reliably demonstrate any differences in the properties of the late NaCh in failing vs a normal human heart. Accordingly, the late current appears to be generated by a single population of channels in normal and failing human ventricular myocardium. Both SLO and BO could be implicated in EADs in HF.

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In myocardial pathology such as heart failure a late sodium current (I_NaL) augmentation is known to be involved in conditions of arrhythmogenesis. However, the underlying mechanisms of the I_NaL generation are not entirely understood. By now evidence is growing that non-cardiac sodium channel isoforms could also be involved in the I_NaL generation. The present study investigates the contribution of the neuronal sodium channel isoform Na_V1.8 to arrhythmogenesis in a clearly-defined setting of enhanced I_NaL by using anemone toxin II (ATX-II) in the absence of structural heart disease.
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In WT cardiomyocytes exposure to ATX-II augmented I_NaL, prolonged APD, increased SR-Ca²⁺-leak and induced proarrhythmic triggers such as early afterdepolarizations (EADs) and Ca²⁺-waves. All of them could be significantly reduced by applying Na_V1.8 blockers PF-01247324 and A-803467. Both blockers had no relevant effects on cellular electrophysiology of SCN10A^−/− cardiomyocytes. Moreover, in SCN10A^−/−-cardiomyocytes, the ATX-II-dependent increase in I_NaL, SR-Ca²⁺-leak and APD prolongation was less than in WT and comparable to the results which were obtained with WT cardiomyocytes being exposed to ATX-II and Na_V1.8 inhibitors in parallel. Moreover, we found a decrease in reverse mode NCX current and reduced CaMKII-dependent RyR2-phosphorylation after application of PF-01247324 as an underlying explanation for the Na⁺-mediated Ca²⁺-dependent proarrhythmic triggers.
The current findings demonstrate that Na_V1.8 is a significant contributor for I_NaL-induced arrhythmic triggers. Therefore, Na_V1.8 inhibition under conditions of an enhanced I_NaL constitutes a promising antiarrhythmic strategy which merits further investigation.
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Although late sodium current (I_Na-late) has long been known to contribute to plateau formation of mammalian cardiac action potentials, lately it was considered as possible target for antiarrhythmic drugs. However, many aspects of this current are still poorly understood. The present work was designed to study the true profile of I_Na-late in canine and guinea pig ventricular cells and compare them to I_Na-late recorded in undiseased human hearts. I_Na-late was defined as a tetrodotoxin-sensitive current, recorded under action potential voltage clamp conditions using either canonic- or self-action potentials as command signals. Under action potential voltage clamp conditions the amplitude of canine and human I_Na-late monotonically decreased during the plateau (decrescendo-profile), in contrast to guinea pig, where its amplitude increased during the plateau (crescendo profile). The decrescendo-profile of canine I_Na-late could not be converted to a crescendo-morphology by application of ramp-like command voltages or command action potentials recorded from guinea pig cells. Conventional voltage clamp experiments revealed that the crescendo I_Na-late profile in guinea pig was due to the slower decay of I_Na-late in this species. When action potentials were recorded from multicellular ventricular preparations with sharp microelectrode, action potentials were shortened by tetrodotoxin, which effect was the largest in human, while smaller in canine, and the smallest in guinea pig preparations. It is concluded that important interspecies differences exist in the behavior of I_Na-late. At present canine myocytes seem to represent the best model of human ventricular cells regarding the properties of I_Na-late. These results should be taken into account when pharmacological studies with I_Na-late are interpreted and extrapolated to human. Accordingly, canine ventricular tissues or myocytes are suggested for pharmacological studies with I_Na-late inhibitors or modifiers. Incorporation of present data to human action potential models may yield a better understanding of the role of I_Na-late in action potential morphology, arrhythmogenesis, and intracellular calcium dynamics.
β-adrenergic regulation of late Na<sup>+</sup> current during cardiac action potential is mediated by both PKA and CaMKII
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Late Na⁺ current (I_NaL) significantly contributes to shaping cardiac action potentials (APs) and increased I_NaL is associated with cardiac arrhythmias. β-adrenergic receptor (βAR) stimulation and its downstream signaling via protein kinase A (PKA) and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) pathways are known to regulate I_NaL. However, it remains unclear how each of these pathways regulates I_NaL during the AP under physiological conditions. Here we performed AP-clamp experiments in rabbit ventricular myocytes to delineate the impact of each signaling pathway on I_NaL at different AP phases to understand the arrhythmogenic potential. During the physiological AP (2 Hz, 37 °C) we found that I_NaL had a basal level current independent of PKA, but partially dependent on CaMKII. βAR activation (10 nM isoproterenol, ISO) further enhanced I_NaL via both PKA and CaMKII pathways. However, PKA predominantly increased I_NaL early during the AP plateau, whereas CaMKII mainly increased I_NaL later in the plateau and during rapid repolarization. We also tested the role of key signaling pathways through exchange protein activated by cAMP (Epac), nitric oxide synthase (NOS) and reactive oxygen species (ROS). Direct Epac stimulation enhanced I_NaL similar to the βAR-induced CaMKII effect, while NOS inhibition prevented the βAR-induced CaMKII-dependent I_NaL enhancement. ROS generated by NADPH oxidase 2 (NOX2) also contributed to the ISO-induced I_NaL activation early in the AP. Taken together, our data reveal differential modulations of I_NaL by PKA and CaMKII signaling pathways at different AP phases. This nuanced and comprehensive view on the changes in I_NaL during AP deepens our understanding of the important role of I_NaL in reshaping the cardiac AP and arrhythmogenic potential under elevated sympathetic stimulation, which is relevant for designing therapeutic treatment of arrhythmias under pathological conditions.
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Action potential (AP) prolongation and resulting QT prolongation are hallmark arrhythmogenic changes in the failing myocardium [4–7]. While increased late sodium current has been demonstrated in association with AP prolongation in HF [8–10], voltage-dependent potassium currents are critical determinants of cardiac AP duration (APD) [11]. In humans, the rapid component of the delayed rectifier potassium current (IKr) is largely responsible for ventricular repolarization.
Heart failure (HF) claims 250,000 lives per year in the US, and nearly half of these deaths are sudden and presumably due to ventricular tachyarrhythmias. QT interval and action potential (AP) prolongation are hallmark proarrhythmic changes in the failing myocardium, which potentially result from alterations in repolarizing potassium currents. Thus, we aimed to examine whether decreased expression of the rapid delayed rectifier potassium current, I_Kr, contributes to repolarization abnormalities in human HF. To map functional I_Kr expression across the left ventricle (LV), we optically imaged coronary-perfused LV free wall from donor and end-stage failing human hearts. The LV wedge preparation was used to examine transmural AP durations at 80% repolarization (APD80), and treatment with the I_Kr-blocking drug, E-4031, was utilized to interrogate functional expression. We assessed the percent change in APD80 post-I_Kr blockade relative to baseline APD80 (∆APD80) and found that ∆APD80s are reduced in failing versus donor hearts in each transmural region, with 0.35-, 0.43-, and 0.41-fold reductions in endo-, mid-, and epicardium, respectively (p = 0.008, 0.037, and 0.022). We then assessed hERG1 isoform gene and protein expression levels using qPCR and Western blot. While we did not observe differences in hERG1a or hERG1b gene expression between donor and failing hearts, we found a shift in the hERG1a:hERG1b isoform stoichiometry at the protein level. Computer simulations were then conducted to assess I_Kr block under E-4031 influence in failing and nonfailing conditions. Our results confirmed the experimental observations and E-4031-induced relative APD80 prolongation was greater in normal conditions than in failing conditions, provided that the cellular model of HF included a significant downregulation of I_Kr. In human HF, the response to I_Kr blockade is reduced, suggesting decreased functional I_Kr expression. This attenuated functional response is associated with altered hERG1a:hERG1b protein stoichiometry in the failing human LV, and failing cardiomyoctye simulations support the experimental findings. Thus, of I_Kr protein and functional expression may be important determinants of repolarization remodeling in the failing human LV.
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^f1: Please address all correspondence to: Albertas I. Undrovinas, PhD, Henry Ford Hospital, Cardiovascular Research, Education & Research Bldg. Room 4015, 2799 West Grand Boulevard, Detroit, MI 48202-2689, USA. Tel: (313)-916-1321; Fax: (313)-916-3001; E-mail: [email protected]

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Regular ArticleGating of the Late Na+ Channel in Normal and Failing Human Myocardium

Abstract

Biophys J

J Mol Cell Cardiol

J Mol Cell Cardiol

Biophys J

Biophys J

Effect of tetrodotoxin on action potentials of the conducting system in the dog heart

Am J Physiol

The intracellular sodium activity of sheep heart Purkinje fibres: effects of local anaesthetics and tetrodotoxin

J Physiol (London)

Factors related to the low resting membrane potentials of diseased human atrial muscles

Japan J Physiol

Late sodium current and its contribution to action potential configuration in guinea pig ventricular myocytes

Circ Res

Background inward current in ventricular and atrial cells of the guinea-pig

Proc R Soc Lond B Biol Sci

The effects of sodium substitution on currents determining the resting potential in guinea-pig ventricular cells

Exp Physiol

Long QT syndrome patients with mutations of the SCN5A and HERG genes have differential responses to Na+ channel blockade and to increases in heart rate. Implications for gene-specific therapy

Circulation

Novel, ultraslow inactivating sodium current in human ventricular cardiomyocytes

Circulation

Late sodium current in heart failure

Circulation

Repolarization abnormalities in cardiomyocytes of dogs with chronic heart failure: Role of sustained inward current

Cell Mol Life Sci

Distribution of a persistent sodium current across the ventricular wall in guinea pigs [In Process Citation]

Circ Res

Larger late sodium conductance in M cells contributes to electrical heterogeneity in canine ventricle

Am J Physiol

Cardiac excitation-contraction coupling

Nature

A persistent sodium current in rat ventricular myocytes

J Physiol (London)

Hypoxia increases persistent sodium current in rat ventricular myocytes

J Physiol(London)

Late Na channels in cardiac cells: the physiological role of background Na channels

Biophys J

Effects of III–IV linker mutations on human heart Na+ channel inactivation gating

Circ Res

Optimization of a mammalian expression system for the measurement of sodium channel gating currents

Am J Physiol

Cytoskeleton modulates gating of voltage-dependent sodium channel in heart

Am J Physiol

Regular Article
Gating of the Late Na⁺ Channel in Normal and Failing Human Myocardium

Long QT syndrome patients with mutations of the SCN5A and HERG genes have differential responses to Na⁺ channel blockade and to increases in heart rate. Implications for gene-specific therapy

Effects of III–IV linker mutations on human heart Na⁺ channel inactivation gating