Subcellular dynamics and functional activity of the cleaved Na+ channel β1 subunit intracellular domain

The voltage-gated Na+ channel β1 subunit, encoded by SCN1B, regulates cell surface expression and gating of α subunits, and participates in cell adhesion. β1 is cleaved by α/β and γ-secretases, releasing an extracellular domain and intracellular domain (ICD) respectively. Abnormal SCN1B expression/function is linked to pathologies including epilepsy, cardiac arrhythmia, and cancer. In this study, we sought to determine the effect of secretase cleavage on β1 function in breast cancer cells. Using a series of GFP-tagged β1 constructs, we show that β1-GFP is mainly retained intracellularly, particularly in the endoplasmic reticulum and endolysosomal pathway, and accumulates in the nucleus. Reduction in endosomal β1-GFP levels occurred following γ-secretase inhibition, implicating endosomes, and/or the preceding plasma membrane, as important sites for secretase processing. Using live-cell imaging, we report β1-ICD-GFP accumulation in the nucleus. Furthermore, β1-GFP and β1ICD-GFP both increased Na+ current, whereas β1STOP-GFP, which lacks the ICD, did not, thus highlighting that the β1-ICD was necessary and sufficient to increase Na+ current measured at the plasma membrane. Importantly, although the endogenous Na+ current expressed in MDA-MB-231 cells is TTX-resistant (carried by Nav1.5), the Na+ current increased by β1-GFP or β1ICD-GFP was TTX-sensitive. Taken together, this work suggests that the β1-ICD is a critical regulator of β subunit function. Our data further support the notion that γ-secretase may play a key role in regulating β1 function in breast cancer cells. This work thus highlights proteolytic processing of β1 by secretase cleavage to be a relevant mechanism in diseases associated with abnormal β1 function.

Increased NaV1.8 expression in patients with sleep-disordered breathing induces pro-arrhythmic activity

Background Sleep-disordered breathing (SDB) is often associated with atrial fibrillation, but detailed mechanisms remain elusive. Interestingly, late Na current (late INa) has been shown to be increased in patients with SDB, while expression of cardiac Na channel NaV1.5 and peak Na current were decreased. Indeed, recent data demonstrated that enhanced NaV1.8-dependent late INa may also induce pro-arrhythmic activity. Purpose We tested whether Na-V1.8 expression and subsequent NaV1.8-dependent pro-arrhythmic activity are increased in patients with SDB. Methods We prospectively analysed 29 right atrial appendage biopsies of patients undergoing elective coronary artery bypass grafting. SDB was assessed using polygraphy in the preoperative night and an apnoea-hypopnea index (AHI) ≥15/h defined SDB. Micro-dissected atrial trabeculae were electrically field stimulated (at 1 Hz, 5 V for 50 ms, at 37°C) to elicit regular contractions. Trabecular arrhythmias were induced using 100 nM isoproterenol at [Ca]o of 3.5 mmol/L and pro-arrhythmic activity was scored from 0 (no arrhythmias) to 5 (salve). Sarcoplasmic reticulum Ca leak was estimated by the contractility after paused stimulation (at 2 Hz, normalized to before pause). To correlate functional and expression data for each individual patient, NaV1.8 mRNA expression was quantified in each trabeculum using qPCR. Results NaV1.8 mRNA expression was increased in patients with SDB, leading to a significant positive correlation with the severity of SDB (i.e. AHI, p=0.02, r2=0.22, Fig. 1A). Multivariate regression analysis revealed that this association was independent from age, sex, atrial fibrillation, heart failure, diabetes mellitus, and renal function (p=0.03, r2=0.35). Accordingly, selective NaV1.8 blockade with PF-01247324 (PF, 1 μM, 30 min) significantly improved post-pause contractility of isolated trabeculae from 1.69±0.31 to 2.95±0.54 in patients with SDB (p=0.001), whereas no significant improvement was observed in patients without SDB. This resulted in significant positive correlations between the PF-dependent improvement of post-pause contractility and both AHI (p=0.047, r2=0.19) and NaV1.8 mRNA expression (p=0.03, r2=0.17). Most importantly, we also observed a significant increase in arrhythmia severity in patients with SDB of 2.21±0.52 (vs. 1.00±0.49, p=0.03) that could be significantly reduced by selective NaV1.8 inhibition with PF to 0.25±0.18 (p=0.0008, Fig. 1B). In accordance, there was a significant positive correlation between arrhythmia severity and AHI (p=0.01, r2=0.28) that was abolished in the presence of PF (interaction analysis: p=0.ehab724.33141, r2=0.46). Conclusion In patients with SDB, enhanced NaV1.8 expression contribute to atrial pro-arrhythmic activity independent from comorbidities. Selective NaV1.8 inhibition may have therapeutic implications for patients with SDB. FUNDunding Acknowledgement Type of funding sources: Other. Main funding source(s): Part of the study was supported by grants from Philips Respironics (Murrysville, PA 15668) and the Medical Faculty at the University of Regensburg. Figure 1

Calcium Signaling Silencing in Atrial Fibrillation: Implications for Atrial Sodium Homeostasis

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia, affecting more than 33 million people worldwide. Despite important advances in therapy, AF’s incidence remains high, and treatment often results in recurrence of the arrhythmia. A better understanding of the cellular and molecular changes that (1) trigger AF and (2) occur after the onset of AF will help to identify novel therapeutic targets. Over the past 20 years, a large body of research has shown that intracellular Ca2+ handling is dramatically altered in AF. While some of these changes are arrhythmogenic, other changes counteract cellular arrhythmogenic mechanisms (Calcium Signaling Silencing). The intracellular Na+ concentration ([Na+])i is a key regulator of intracellular Ca2+ handling in cardiac myocytes. Despite its importance in the regulation of intracellular Ca2+ handling, little is known about [Na+]i, its regulation, and how it might be changed in AF. Previous work suggests that there might be increases in the late component of the atrial Na+ current (INa,L) in AF, suggesting that [Na+]i levels might be high in AF. Indeed, a pharmacological blockade of INa,L has been suggested as a treatment for AF. Here, we review calcium signaling silencing and changes in intracellular Na+ homeostasis during AF. We summarize the proposed arrhythmogenic mechanisms associated with increases in INa,L during AF and discuss the evidence from clinical trials that have tested the pharmacological INa,L blocker ranolazine in the treatment of AF.

Dual Mechanisms of Cardiac Action Potential Prolongation by 4-Oxo-Nonenal Increasing the Risk of Arrhythmia; Late Na+ Current Induction and hERG K+ Channel Inhibition

4-Oxo-nonenal (4-ONE) is an endogenous lipid peroxidation product that is more reactive than 4-hydroxy-nonenal (4-HNE). We previously reported the arrhythmic potential of 4-HNE by suppression of cardiac human Ether-a-go-go Related Gene (hERG) K+ channels with prolonged action potential duration (APD) in cardiomyocytes. Here, we illustrate the higher arrhythmic risk of 4-ONE by modulating the cardiac hNaV1.5 channel currents (INaV). Although the peak amplitude of INaV was not significantly changed by 4-ONE up to 10 μM, the rate of INaV inactivation was slowed, and the late Na+ current (INaL) became larger by 10 μM 4-ONE. The chemical modification of specific residues in hNaV1.5 by 4-ONE was identified using MS-fingerprinting analysis. In addition to the changes in INaV, 4-ONE decreased the delayed rectifier K+ channel currents including the hERG current. The L-type Ca2+ channel current was decreased, whereas its inactivation was slowed by 4-ONE. The APD prolongation by 10 μM of 4-ONE was more prominent than that by 100 μM of 4-HNE. In the computational in silico cardiomyocyte simulation analysis, the changes of INaL by 4-ONE significantly exacerbated the risk of arrhythmia exhibited by the TdP marker, qNet. Our study suggests an arrhythmogenic effect of 4-ONE on cardiac ion channels, especially hNaV1.5.

Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergent arrhythmogenic phenotypes

In cardiomyocytes, NaV1.5 channels mediate initiation and fast propagation of action potentials. The Ca2+-binding protein calmodulin (CaM) serves as a de facto subunit of NaV1.5. Genetic studies and atomic structures suggest that this interaction is pathophysiologically critical, as human mutations within the NaV1.5 carboxy-terminus that disrupt CaM binding are linked to distinct forms of life-threatening arrhythmias, including long QT syndrome 3, a “gain-of-function” defect, and Brugada syndrome, a “loss-of-function” phenotype. Yet, how a common disruption in CaM binding engenders divergent effects on NaV1.5 gating is not fully understood, though vital for elucidating arrhythmogenic mechanisms and for developing new therapies. Here, using extensive single-channel analysis, we find that the disruption of Ca2+-free CaM preassociation with NaV1.5 exerts two disparate effects: 1) a decrease in the peak open probability and 2) an increase in persistent NaV openings. Mechanistically, these effects arise from a CaM-dependent switch in the NaV inactivation mechanism. Specifically, CaM-bound channels preferentially inactivate from the open state, while those devoid of CaM exhibit enhanced closed-state inactivation. Further enriching this scheme, for certain mutant NaV1.5, local Ca2+ fluctuations elicit a rapid recruitment of CaM that reverses the increase in persistent Na current, a factor that may promote beat-to-beat variability in late Na current. In all, these findings identify the elementary mechanism of CaM regulation of NaV1.5 and, in so doing, unravel a noncanonical role for CaM in tuning ion channel gating. Furthermore, our results furnish an in-depth molecular framework for understanding complex arrhythmogenic phenotypes of NaV1.5 channelopathies.

The SCN5A point mutation M1875T, associated with familial atrial fibrillation, causes a gain-of-function effect of the cardiac Nav1.5 channel in atrial cardiomyocytes

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Leducq Foundation Background The point mutation M1875T in the SCN5A gene, which encodes the pore-forming α-subunit of the cardiac voltage-gated Na+ channel Nav1.5, has been associated with familial atrial fibrillation (AF), but its effects on atrial cardiomyocyte electrophysiology is unclear. Aim To investigate the effect of the point mutation M1875T on atrial electrophysiological parameters. Methods In a novel heterozygous knock-in murine model (Scn5a-M1875T+/-), whole-cell patch clamp electrophysiology was used to investigate Na+ currents in left atrial (LA) cardiomyocytes isolated from hearts of young adult mice (10-16 weeks). LA microelectrode and optical mapping recordings were used to study action potential (AP) characteristics. Cardiac size and function were measured by transthoracic echocardiography. Atrial Scn5a gene and Nav1.5 protein expression were assessed by Rt-PCR and Western blot. Results The Na+ current was increased in cardiomyocytes isolated from Scn5a-M1875T+/- LA (wildtype (WT) -22.7 ± 0.9 pA/pF (N = 14, n = 115); Scn5a-M1875T+/- -28.3 ± 1.1 pA/pF (N = 15, n = 117)). Scn5a-M1875T+/- intact isolated superfused LA had an elevated AP amplitude (100 ms pacing cycle length (PCL): WT 86.4 ± 0.9 mV (N = 8, n = 24); Scn5a-M1875T+/- 91.2 ± 0.7 mV (N = 8, n = 25)) and a faster peak upstroke velocity (100 ms PCL: WT 127.98 ± 3.28 mV/ms; Scn5a-M1875T+/- 142.80 ± 3.98 mV/ms). AP duration (APD) was not different apart from a small APD shortening at slow rates. Echocardiography revealed no difference in size and function at the age of investigation. Atrial Scn5a gene and Nav1.5 protein expression were comparable. When challenged with flecainide (1 µM), Scn5a-M1875T+/- LA showed less conduction slowing than WT (100 ms PCL: WT -10.43 ± 1.27 cm/s (N = 12); Scn5a-M1875T+/- -6.10 ± 1.34 cm/s (N = 12)). 5 µM flecainide caused significant increase in WT refractoriness (7/12 atria lost 1:1 capture at PCL ≤ 120 ms) compared to Scn5a-M1875T+/- (1/12). Conclusion(s): SCN5A point mutation M1875T increases the Na+ current in atrial cardiomyocytes and intact atria, leading to a faster AP upstroke and an attenuated response to flecainide. Abstract Figure 1: Current-Voltage relationship

Is selective late sodium current inhibition different from class I/B antiarrhythmic action? Comparison of the effects of GS967 to mexiletine in canine ventricular myocardium

Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): National Research, Development and Innovation Office New National Excellence Programme Enhancement of the late Na+ current (INa,late) increases arrhythmia propensity in the heart, while suppression of the current is antiarrhythmic. GS-458967 (GS) is an agent considered to be a selective blocker of INa,late. In the present study, effects of GS967 on INa,late, on L-type calcium current (ICaL), and on action potential (AP) morphology were studied in canine ventricular myocytes by using conventional voltage clamp, action potential voltage clamp and sharp microelectrode techniques. These effects of GS were compared to tetrodotoxin (TTX) and to the class I/B antiarrhythmic compound mexiletine. GS (1 μM), mexiletine (40 μM) and TTX (10 μM) dissected largely similarly shaped inward currents under action potential voltage clamp conditions. In case of GS and mexiletine, the amplitude and integral of this inward current was significantly smaller when measured in the presence of 1 μM nisoldipine, while no difference was observed in case of TTX. Under conventional voltage clamp conditions, INa,late was significantly reduced by 1 μM GS and 40 μM mexiletine (about 79% and 63% reduction of current integrals, respectively). The integral of ICa,L was moderately but significantly decreased by both drugs (reduction of 9% and 14%, respectively). These changes were associated with a faster inactivation of ICa,L. Drug effects on early Na+ current (INa,early) were assessed by analyzing the maximal rate of depolarization (V + max) in multicellular preparations. Both GS and mexiletine showed fast onset and offset kinetics: 110 ms and 289 ms offset time constants, respectively, as determined from V + max measurements in right ventricular papillary muscles, while the onset kinetics was characterized by 5.3 AP and 2.6 AP lengths, respectively, at 2.5 Hz. Effects on beat-to-beat variability of AP duration (APD) was studied in isolated myocytes. Short-term variability was significantly decreased by both GS and mexiletine (average reduction of 42% and 24%, respectively) while they caused similar shortening of the APD. The electrophysiological effects of GS are similar to those of mexiletine, but with a somewhat faster offset kinetics of V + max block. However, since GS reduced V+ max and INa,late in the same concentration, the currently accepted view that GS that selectively blocks INa,late has to be questioned and it is suggested that GS should be classified as a class I/B (or I/B + IV) antiarrhythmic agent.

Deletion of Trpm4 Alters the Function of the Nav1.5 Channel in Murine Cardiac Myocytes

Transient receptor potential melastatin member 4 (TRPM4) encodes a Ca2+-activated, non-selective cation channel that is functionally expressed in several tissues, including the heart. Pathogenic mutants in TRPM4 have been reported in patients with inherited cardiac diseases, including conduction blockage and Brugada syndrome. Heterologous expression of mutant channels in cell lines indicates that these mutations can lead to an increase or decrease in TRPM4 expression and function at the cell surface. While the expression and clinical variant studies further stress the importance of TRPM4 in cardiac function, the cardiac electrophysiological phenotypes in Trpm4 knockdown mouse models remain incompletely characterized. To study the functional consequences of Trpm4 deletion on cardiac electrical activity in mice, we performed perforated-patch clamp and immunoblotting studies on isolated atrial and ventricular cardiac myocytes and surfaces, as well as on pseudo- and intracardiac ECGs, either in vivo or in Langendorff-perfused explanted mouse hearts. We observed that TRPM4 is expressed in atrial and ventricular cardiac myocytes and that deletion of Trpm4 unexpectedly reduces the peak Na+ currents in myocytes. Hearts from Trpm4−/− mice presented increased sensitivity towards mexiletine, a Na+ channel blocker, and slower intraventricular conduction, consistent with the reduction of the peak Na+ current observed in the isolated cardiac myocytes. This study suggests that TRPM4 expression impacts the Na+ current in murine cardiac myocytes and points towards a novel function of TRPM4 regulating the Nav1.5 function in murine cardiac myocytes.

CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells

Tumor necrosis factor (TNF) is known to activate the epithelial Na+ channel (ENaC) in A549 cells. A549 cells are widely used model for ENaC research. The role of δ-ENaC subunit in TNF-induced activation has not been studied. In this study we hypothesized that δ-ENaC plays a major role in TNF-induced activation of ENaC channel in A549 cells which are widely used model for ENaC research. We used CRISPR/Cas 9 approach to knock down (KD) the δ-ENaC in A549 cells. Western blot and immunofluorescence assays were performed to analyze efficacy of δ-ENaC protein KD. Whole-cell patch clamp technique was used to analyze the TNF-induced activation of ENaC. Overexpression of wild type δ-ENaC in the δ-ENaC KD of A549 cells restored the TNF-induced activation of whole-cell Na+ current. Neither N-linked glycosylation sites nor carboxyl terminus domain of δ-ENaC was necessary for the TNF-induced activation of whole-cell Na+ current in δ-ENaC KD of A549 cells. Our data demonstrated that in A549 cells the δ-ENaC plays a major role in TNF-induced activation of ENaC.