Iman S. Gurung

Cardiac Na+ Current Regulation by Pyridine Nucleotides

Rationale: Mutations in glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) protein reduce cardiac Na+ current (INa) and cause Brugada Syndrome (BrS). GPD1-L has >80% amino acid homology with glycerol-3-phosphate dehydrogenase, which is involved in NAD-dependent energy metabolism. Objective: Therefore, we tested whether NAD(H) could regulate human cardiac sodium channels (Nav1.5). Methods and Results: HEK293 cells stably expressing Nav1.5 and rat neonatal cardiomyocytes were used. The influence of NADH/NAD+ on arrhythmic risk was evaluated in wild-type or SCN5A+/− mouse heart. A280V GPD1-L caused a 2.48±0.17-fold increase in intracellular NADH level (P<0.001). NADH application or cotransfection with A280V GPD1-L resulted in decreased INa (0.48±0.09 or 0.19±0.04 of control group, respectively; P<0.01), which was reversed by NAD+, chelerythrine, or superoxide dismutase. NAD+ antagonism of the Na+ channel downregulation by A280V GPD1-L or NADH was prevented by a protein kinase (PK)A inhibitor, PKAI6–22. The effects of NADH and NAD+ were mimicked by a phorbol ester and forskolin, respectively. Increasing intracellular NADH was associated with an increased risk of ventricular tachycardia in wild-type mouse hearts. Extracellular application of NAD+ to SCN5A+/− mouse hearts ameliorated the risk of ventricular tachycardia. Conclusions: Our results show that Nav1.5 is regulated by pyridine nucleotides, suggesting a link between metabolism and INa. This effect required protein kinase C activation and was mediated by oxidative stress. NAD+ could prevent this effect by activating PKA. Mutations of GPD1-L may downregulate Nav1.5 by altering the oxidized to reduced NAD(H) balance.

A role for membrane potential in regulating GPCRs

G-protein-coupled receptors (GPCRs) have ubiquitous roles in transducing extracellular signals into cellular responses. Therefore, the concept that members of this superfamily of surface proteins are directly modulated by changes in membrane voltage could have widespread consequences for cell signalling. Although several studies have indicated that GPCRs can be voltage dependent, particularly P2Y(1) receptors in the non-excitable megakaryocyte, the evidence has been mostly indirect. Recent work on muscarinic receptors has stimulated substantial interest in this field by reporting the first voltage-dependent charge movements for a GPCR. An underlying mechanism is proposed whereby a voltage-induced conformational change in the receptor alters its ability to couple to the G protein and thereby influences its affinity for an agonist. We discuss the strength of the evidence behind this hypothesis and include suggestions for future work. We also describe other examples in which direct voltage control of GPCRs can account for effects of membrane potential on downstream signals and highlight the possible physiological consequences of this phenomenon.

Effects of L‐type Ca2+ channel antagonism on ventricular arrhythmogenesis in murine hearts containing a modification in the Scn5a gene modelling human long QT syndrome 3

Ventricular arrhythmogenesis in long QT 3 syndrome (LQT3) involves both triggered activity and re‐entrant excitation arising from delayed ventricular repolarization. Effects of specific L‐type Ca2+ channel antagonism were explored in a gain‐of‐function murine LQT3 model produced by a ΔKPQ 1505–1507 deletion in the SCN5A gene. Monophasic action potentials (MAPs) were recorded from epicardial and endocardial surfaces of intact, Langendorff‐perfused Scn5a+/Δ hearts. In untreated Scn5a+/Δ hearts, epicardial action potential duration at 90% repolarization (APD90) was 60.0 ± 0.9 ms compared with 46.9 ± 1.6 ms in untreated wild‐type (WT) hearts (P < 0.05; n= 5). The corresponding endocardial APD90 values were 52.0 ± 0.7 ms and 53.7 ± 1.6 ms in Scn5a+/Δ and WT hearts, respectively (P > 0.05; n= 5). Epicardial early afterdepolarizations (EADs), often accompanied by spontaneous ventricular tachycardia (VT), occurred in 100% of MAPs from Scn5a+/Δ but not in any WT hearts (n= 10). However, EAD occurrence was reduced to 62 ± 7.1%, 44 ± 9.7%, 10 ± 10% and 0% of MAPs following perfusion with 10 nm, 100 nm, 300 nm and 1 μm nifedipine, respectively (P < 0.05; n= 5), giving an effective IC50 concentration of 79.3 nm. Programmed electrical stimulation (PES) induced VT in all five Scn5a+/Δ hearts (n= 5) but not in any WT hearts (n= 5). However, repeat PES induced VT in 3, 2, 2 and 0 out of 5 Scn5a+/Δ hearts following perfusion with 10 nm, 100 nm, 300 nm and 1 μm nifedipine, respectively. Patch clamp studies in isolated ventricular myocytes from Scn5a+/Δ and WT hearts confirmed that nifedipine (300 nm) completely suppressed the inward Ca2+ current but had no effect on inward Na+ currents. No significant effects were seen on epicardial APD90, endocardial APD90 or ventricular effective refractory period in Scn5a+/Δ and WT hearts following perfusion with nifedipine at 1 nm, 10 nm, 100 nm, 300 nm and 1 μm nifedipine concentrations. We conclude that L‐type Ca2+ channel antagonism thus exerts specific anti‐arrhythmic effects in Scn5a+/Δ hearts through suppression of EADs.

Scn3b knockout mice exhibit abnormal ventricular electrophysiological properties

We report for the first time abnormalities in cardiac ventricular electrophysiology in a genetically modified murine model lacking the Scn3b gene (Scn3b−/−). Scn3b−/− mice were created by homologous recombination in embryonic stem (ES) cells. RT-PCR analysis confirmed that Scn3b mRNA was expressed in the ventricles of wild-type (WT) hearts but was absent in the Scn3b−/− hearts. These hearts also showed increased expression levels of Scn1b mRNA in both ventricles and Scn5a mRNA in the right ventricles compared to findings in WT hearts. Scn1b and Scn5a mRNA was expressed at higher levels in the left than in the right ventricles of both Scn3b−/− and WT hearts. Bipolar electrogram and monophasic action potential recordings from the ventricles of Langendorff-perfused Scn3b−/− hearts demonstrated significantly shorter ventricular effective refractory periods (VERPs), larger ratios of electrogram duration obtained at the shortest and longest S1–S2 intervals, and ventricular tachycardias (VTs) induced by programmed electrical stimulation. Such arrhythmogenesis took the form of either monomorphic or polymorphic VT. Despite shorter action potential durations (APDs) in both the endocardium and epicardium, Scn3b−/− hearts showed ΔAPD90 values that remained similar to those shown in WT hearts. The whole-cell patch-clamp technique applied to ventricular myocytes isolated from Scn3b−/− hearts demonstrated reduced peak Na+ current densities and inactivation curves that were shifted in the negative direction, relative to those shown in WT myocytes. Together, these findings associate the lack of the Scn3b gene with arrhythmic tendencies in intact perfused hearts and electrophysiological features similar to those in Scn5a+/− hearts.

Direct Voltage Control of Signaling via P2Y1 and Other Gαq-coupled Receptors

Emerging evidence suggests that Ca2+ release evoked by certain G-protein-coupled receptors can be voltage-dependent; however, the relative contribution of different components of the signaling cascade to this response remains unclear. Using the electrically inexcitable megakaryocyte as a model system, we demonstrate that inositol 1,4,5-trisphosphate-dependent Ca2+ mobilization stimulated by several agonists acting via Gαq-coupled receptors is potentiated by depolarization and that this effect is most pronounced for ADP. Voltage-dependent Ca2+ release was not induced by direct elevation of inositol 1,4,5-trisphosphate, by agents mimicking diacylglycerol actions, or by activation of phospholipase Cγ-coupled receptors. The response to voltage did not require voltage-gated Ca2+ channels as it persisted in the presence of nifedipine and was only weakly affected by the holding potential. Strong predepolarizations failed to affect the voltage-dependent Ca2+ increase; thus, an alteration of G-protein βγ subunit binding is also not involved. Megakaryocytes from P2Y1-/- mice lacked voltage-dependent Ca2+ release during the application of ADP but retained this response after stimulation of other Gαq-coupled receptors. Although depolarization enhanced Ca2+ mobilization resulting from GTPγS dialysis and to a lesser extent during \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{AlF}_{4}^{-}\) \end{document} or thimerosal, these effects all required the presence of P2Y1 receptors. Taken together, the voltage dependence to Ca2+ release via Gαq-coupled receptors is not due to control of G-proteins or down-stream signals but, rather, can be explained by a voltage sensitivity at the level of the receptor itself. This effect, which is particularly robust for P2Y1 receptors, has wide-spread implications for cell signaling.

Comparison of regulated passive membrane conductance in action potential-firing fast- and slow-twitch muscle

In several pathological and experimental conditions, the passive membrane conductance of muscle fibers (Gm) and their excitability are inversely related. Despite this capacity of Gm to determine muscle excitability, its regulation in active muscle fibers is largely unexplored. In this issue, our previous study (Pedersen et al. 2009. J. Gen. Physiol. doi:10.1085/jgp.200910291) established a technique with which biphasic regulation of Gm in action potential (AP)-firing fast-twitch fibers of rat extensor digitorum longus muscles was identified and characterized with temporal resolution of seconds. This showed that AP firing initially reduced Gm via ClC-1 channel inhibition but after ∼1,800 APs, Gm rose substantially, causing AP excitation failure. This late increase of Gm reflected activation of ClC-1 and KATP channels. The present study has explored regulation of Gm in AP-firing slow-twitch fibers of soleus muscle and compared it to Gm dynamics in fast-twitch fibers. It further explored aspects of the cellular signaling that conveyed regulation of Gm in AP-firing fibers. Thus, in both fiber types, AP firing first triggered protein kinase C (PKC)-dependent ClC-1 channel inhibition that reduced Gm by ∼50%. Experiments with dantrolene showed that AP-triggered SR Ca2+ release activated this PKC-mediated ClC-1 channel inhibition that was associated with reduced rheobase current and improved function of depolarized muscles, indicating that the reduced Gm enhanced muscle fiber excitability. In fast-twitch fibers, the late rise in Gm was accelerated by glucose-free conditions, whereas it was postponed when intermittent resting periods were introduced during AP firing. Remarkably, elevation of Gm was never encountered in AP-firing slow-twitch fibers, even after 15,000 APs. These observations implicate metabolic depression in the elevation of Gm in AP-firing fast-twitch fibers. It is concluded that regulation of Gm is a general phenomenon in AP-firing muscle, and that differences in Gm regulation may contribute to the different phenotypes of fast- and slow-twitch muscle.

Mechanisms of ventricular arrhythmogenesis in mice following targeted disruption of KCNE1 modelling long QT syndrome 5

Mutations within KCNE1 encoding a transmembrane protein which coassembles with K+ channels mediating slow K+, IKs, currents are implicated in cardiac action potential prolongation and ventricular arrhythmogenicity in long QT syndrome 5. We demonstrate the following potentially arrhythmogenic features in simultaneously recorded, left ventricular, endocardial and epicardial monophasic action potentials from Langendorff‐perfused murine KCNE1−/− hearts for the first time. (1) Prolonged epicardial (57.1 ± 0.5 ms cf. 36.1 ± 0.07 ms in wild‐type (WT), P < 0.001; n= 5) and endocardial action potential duration at 90% repolarication (APD90) (54.4 ± 2.4 ms cf. 48.5 ± 0.3 ms, P < 0.05; n= 5). (2) Negative transmural repolarization gradients (ΔAPD90: endocardial minus epicardial APD90) (−2.5 ± 2.4 ms, compared with 12.4 ± 1.1 ms in WT, P < 0.001; n= 5). (3) Frequent epicardial early afterdepolarizations (EADs) and spontaneous ventricular tachycardia (VT) in 4 out of 5 KCNE1−/− hearts but not WT (n= 5). EADs were especially frequent following temporary cessations of ventricular pacing. (4) Monomorphic VT lasting 1.36 ± 0.2 s in 5 out of 5 KCNE1−/− hearts, following premature stimuli but not WT (n= 5). (5) Epicardial APD alternans. Perfusion of KCNE1−/− hearts with 1 μm nifedipine induced potentially anti‐arrhythmic changes including: (1) restored epicardial APD90 (from 57.1 ± 0.5 ms to 42.3 ± 0.4 ms, P < 0.001; n= 5); (2) altered ΔAPD90 to values (11.2 ± 2.6) close to WT (P > 0.05; n= 5); (3) EAD suppression during both spontaneous activity and following cessation of ventricular pacing (n= 5) to give similar features to WT controls (n= 5); (4) suppression of programmed electrical stimulation‐induced VT; and (5) suppression of APD alternans. These findings suggest arrhythmic effects of reduced outward currents expected in KCNE1−/− hearts and their abolition by antagonism of inward L‐type Ca2+ current.

Sensitivity limits for voltage control of P2y receptor-evoked Ca2+ mobilization in the rat megakaryocyte

G‐protein‐coupled receptor signalling has been suggested to be voltage dependent in a number of cell types; however, the limits of sensitivity of this potentially important phenomenon are unknown. Using the non‐excitable rat megakaryocyte as a model system, we now show that P2Y receptor‐evoked Ca2+ mobilization is controlled by membrane voltage in a graded and bipolar manner without evidence for a discrete threshold potential. Throughout the range of potentials studied, the peak increase in intracellular Ca2+ concentration ([Ca2+]i) in response to depolarization was always larger than the maximal reduction in [Ca2+]i following an equivalent amplitude hyperpolarization. Significant [Ca2+]i increases were observed in response to small amplitude (<5 mV, 5 s duration) or short duration (25 ms, 135 mV) depolarizations. Individual cardiac action potential waveforms were also able to repeatedly potentiate P2Y receptor‐evoked Ca2+ release and the response to trains of normally paced stimuli fused to generate prolonged [Ca2+]i increases. Furthermore, elevation of the temperature to physiological levels (36°C) resulted in a more sustained depolarization‐evoked Ca2+ increase compared with more transient or oscillatory responses at 20–24°C. The ability of signalling via a G‐protein‐coupled receptor to be potentiated by action potential waveforms and small amplitude depolarizations has broad implications in excitable and non‐excitable tissues.

Novel consequences of voltage‐dependence to G‐protein‐coupled P2Y1 receptors

Emerging evidence suggests that activation of G‐protein‐coupled receptors (GPCRs) can be directly regulated by membrane voltage. However, the physiological and pharmacological relevance of this effect remains unclear. We have further examined this phenomenon for P2Y1 receptors in the non‐excitable megakaryocyte using a range of agonists and antagonists.

Novel consequences of voltage-dependence to G-protein-coupled P2Y receptors

Emerging evidence suggests that activation of G‐protein‐coupled receptors (GPCRs) can be directly regulated by membrane voltage. However, the physiological and pharmacological relevance of this effect remains unclear. We have further examined this phenomenon for P2Y1 receptors in the non‐excitable megakaryocyte using a range of agonists and antagonists.