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An interplay between Ca 2+ /calmodulin-dependent protein kinase IIδc (CaMKIIδc) and late Na + current (I NaL ) is known to induce arrhythmias in the failing heart. Here, we elucidate the role of the sodium channel isoform Na V 1.8 for CaMKIIδc-dependent proarrhythmia. In a CRISPR-Cas9-generated human iPSC-cardiomyocyte homozygous knock-out of Na V 1.8, we demonstrate that Na V 1.8 contributes to I NaL formation. In addition, we reveal a direct interaction between Na V 1.8 and CaMKIIδc in cardiomyocytes isolated from patients with heart failure (HF). Using specific blockers of Na V 1.8 and CaMKIIδc, we show that Na V 1.8-driven I NaL is CaMKIIδc-dependent and that Na V 1.8-inhibtion reduces diastolic SR-Ca 2+ leak in human failing cardiomyocytes. Moreover, increased mortality of CaMKIIδc-overexpressing HF mice is reduced when a Na V 1.8 knock-out is introduced. Cellular and in vivo experiments reveal reduced ventricular arrhythmias without changes in HF progression. Our work therefore identifies a proarrhythmic CaMKIIδc downstream target which may constitute a prognostic and antiarrhythmic strategy. In heart failure, increased CaMKII activity is decisively involved in arrhythmia formation. Here, the authors introduce the neuronal sodium channel Na V 1.8 as a CaMKII downstream target as its specific knock-out reduces arrhythmias and improves survival in a CaMKII-overexpressing mouse model.

Pharmacologic approaches for the treatment of atrial arrhythmias are limited due to side effects and low efficacy. Thus, the identification of new antiarrhythmic targets is of clinical interest. Recent genome studies suggested an involvement of SCN10A sodium channels (NaV1.8) in atrial electrophysiology. This study investigated the role and involvement of NaV1.8 (SCN10A) in arrhythmia generation in the human atria and in mice lacking NaV1.8. NaV1.8 mRNA and protein were detected in human atrial myocardium at a significant higher level compared to ventricular myocardium. Expression of NaV1.8 and NaV1.5 did not differ between myocardium from patients with atrial fibrillation and sinus rhythm. To determine the electrophysiological role of NaV1.8, we investigated isolated human atrial cardiomyocytes from patients with sinus rhythm stimulated with isoproterenol. Inhibition of NaV1.8 by A-803467 or PF-01247324 showed no effects on the human atrial action potential. However, we found that NaV1.8 significantly contributes to late Na+ current and consequently to an increased proarrhythmogenic diastolic sarcoplasmic reticulum Ca2+ leak in human atrial cardiomyocytes. Selective pharmacological inhibition of NaV1.8 potently reduced late Na+ current, proarrhythmic diastolic Ca2+ release, delayed afterdepolarizations as well as spontaneous action potentials. These findings could be confirmed in murine atrial cardiomyocytes from wild-type mice and also compared to SCN10A−/− mice (genetic ablation of NaV1.8). Pharmacological NaV1.8 inhibition showed no effects in SCN10A−/− mice. Importantly, in vivo experiments in SCN10A−/− mice showed that genetic ablation of NaV1.8 protects against atrial fibrillation induction. This study demonstrates that NaV1.8 is expressed in the murine and human atria and contributes to late Na+ current generation and cellular arrhythmogenesis. Blocking NaV1.8 selectively counteracts this pathomechanism and protects against atrial arrhythmias. Thus, our translational study reveals a new selective therapeutic target for treating atrial arrhythmias.

The SGLT2 inhibitor empagliflozin improved cardiovascular outcomes in patients with diabetes. As the cardiac mechanisms remain elusive, we investigated the long-term effects (up to 2 months) of empagliflozin on excitation-contraction (EC)-coupling in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) in a blinded manner. IPSC from 3 donors, differentiated into pure iPSC-CM (4 differentiations), were treated with a clinically relevant concentration of empagliflozin (0.5 μmol/l) or vehicle control. Treatment, data acquisition, and analysis were conducted externally blinded. Epifluorescence microscopy measurements in iPSC-CM showed that empagliflozin has neutral effects on Ca2+ transient amplitude, diastolic Ca2+ levels, Ca2+ transient kinetics, or sarcoplasmic Ca2+ load after 2 weeks or 8 weeks of treatment. Confocal microscopy determining possible effects on proarrhythmogenic diastolic Ca2+ release events showed that in iPSC-CM, Ca2+ spark frequency and leak was not altered after chronic treatment with empagliflozin. Finally, in patch-clamp experiments, empagliflozin did not change action potential duration, amplitude, or resting membrane potential compared with vehicle control after long-term treatment. Next-generation RNA sequencing (NGS) and mapped transcriptome profiles of iPSC-CMs untreated and treated with empagliflozin for 8 weeks showed no differentially expressed EC-coupling genes. In line with NGS data, Western blots indicate that empagliflozin has negligible effects on key EC-coupling proteins. In this blinded study, direct treatment of iPSC-CM with empagliflozin for a clinically relevant duration of 2 months did not influence cardiomyocyte EC-coupling and electrophysiology. Therefore, it is likely that other mechanisms independent of cardiomyocyte EC-coupling are responsible for the beneficial treatment effect of empagliflozin.Key messagesThis blinded study investigated the clinically relevant long-term effects (up to 2 months) of empagliflozin on cardiomyocyte excitation-contraction (EC)-coupling.Human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) were used to study a human model including a high repetition number of experiments.Empagliflozin has neutral effects on cardiomyocyte Ca2+ transients, sarcoplasmic Ca2+ load, and diastolic sarcoplasmic Ca2+ leak.In patch-clamp experiments, empagliflozin did not change the action potential.Next-generation RNA sequencing, mapped transcriptome profiles, and Western blots of iPSC-CM untreated and treated with empagliflozin showed no differentially expressed EC-coupling candidates.

In heart failure and atrial fibrillation, a persistent Na[sup.+] current (I[sub.NaL]) exerts detrimental effects on cellular electrophysiology and can induce arrhythmias. We have recently shown that Na[sub.V]1.8 contributes to arrhythmogenesis by inducing a I[sub.NaL]. Genome-wide association studies indicate that mutations in the SCN10A gene (Na[sub.V]1.8) are associated with increased risk for arrhythmias, Brugada syndrome, and sudden cardiac death. However, the mediation of these Na[sub.V]1.8-related effects, whether through cardiac ganglia or cardiomyocytes, is still a subject of controversial discussion. We used CRISPR/Cas9 technology to generate homozygous atrial SCN10A-KO-iPSC-CMs. Ruptured-patch whole-cell patch-clamp was used to measure the I[sub.NaL] and action potential duration. Ca[sup.2+] measurements (Fluo 4-AM) were performed to analyze proarrhythmogenic diastolic SR Ca[sup.2+] leak. The I[sub.NaL] was significantly reduced in atrial SCN10A KO CMs as well as after specific pharmacological inhibition of Na[sub.V]1.8. No effects on atrial APD[sub.90] were detected in any groups. Both SCN10A KO and specific blockers of Na[sub.V]1.8 led to decreased Ca[sup.2+] spark frequency and a significant reduction of arrhythmogenic Ca[sup.2+] waves. Our experiments demonstrate that Na[sub.V]1.8 contributes to I[sub.NaL] formation in human atrial CMs and that Na[sub.V]1.8 inhibition modulates proarrhythmogenic triggers in human atrial CMs and therefore Na[sub.V]1.8 could be a new target for antiarrhythmic strategies.

In heart failure and atrial fibrillation, a persistent Na+ current (INaL) exerts detrimental effects on cellular electrophysiology and can induce arrhythmias. We have recently shown that NaV1.8 contributes to arrhythmogenesis by inducing a INaL. Genome-wide association studies indicate that mutations in the SCN10A gene (NaV1.8) are associated with increased risk for arrhythmias, Brugada syndrome, and sudden cardiac death. However, the mediation of these NaV1.8-related effects, whether through cardiac ganglia or cardiomyocytes, is still a subject of controversial discussion. We used CRISPR/Cas9 technology to generate homozygous atrial SCN10A-KO-iPSC-CMs. Ruptured-patch whole-cell patch-clamp was used to measure the INaL and action potential duration. Ca2+ measurements (Fluo 4-AM) were performed to analyze proarrhythmogenic diastolic SR Ca2+ leak. The INaL was significantly reduced in atrial SCN10A KO CMs as well as after specific pharmacological inhibition of NaV1.8. No effects on atrial APD90 were detected in any groups. Both SCN10A KO and specific blockers of NaV1.8 led to decreased Ca2+ spark frequency and a significant reduction of arrhythmogenic Ca2+ waves. Our experiments demonstrate that NaV1.8 contributes to INaL formation in human atrial CMs and that NaV1.8 inhibition modulates proarrhythmogenic triggers in human atrial CMs and therefore NaV1.8 could be a new target for antiarrhythmic strategies.

The sodium channel Na[sub.V]1.8, encoded by the SCN10A gene, has recently emerged as a potential regulator of cardiac electrophysiology. We have previously shown that Na[sub.V]1.8 contributes to arrhythmogenesis by inducing a persistent Na[sup.+] current (late Na[sup.+] current, I[sub.NaL]) in human atrial and ventricular cardiomyocytes (CM). We now aim to further investigate the contribution of Na[sub.V]1.8 to human ventricular arrhythmogenesis at the CM-specific level using pharmacological inhibition as well as a genetic knockout (KO) of SCN10A in induced pluripotent stem cell CM (iPSC-CM). In functional voltage-clamp experiments, we demonstrate that I[sub.NaL] was significantly reduced in ventricular SCN10A-KO iPSC-CM and in control CM after a specific pharmacological inhibition of Na[sub.V]1.8. In contrast, we did not find any effects on ventricular APD[sub.90]. The frequency of spontaneous sarcoplasmic reticulum Ca[sup.2+] sparks and waves were reduced in SCN10A-KO iPSC-CM and control cells following the pharmacological inhibition of Na[sub.V]1.8. We further analyzed potential triggers of arrhythmias and found reduced delayed afterdepolarizations (DAD) in SCN10A-KO iPSC-CM and after the specific inhibition of Na[sub.V]1.8 in control cells. In conclusion, we show that Na[sub.V]1.8-induced I[sub.NaL] primarily impacts arrhythmogenesis at a subcellular level, with minimal effects on systolic cellular Ca[sup.2+] release. The inhibition or knockout of Na[sub.V]1.8 diminishes proarrhythmic triggers in ventricular CM. In conjunction with our previously published results, this work confirms Na[sub.V]1.8 as a proarrhythmic target that may be useful in an anti-arrhythmic therapeutic strategy.

Sarcoplasmic reticulum (SR) Ca 2+ leak induced by Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is centrally involved in atrial and ventricular arrhythmogenesis as well as heart failure remodeling. Consequently, treating SR Ca 2+ leak has been proposed as a novel therapeutic paradigm, but compounds for use in humans are lacking. SMP-114 (“Rimacalib”) is a novel, orally available CaMKII inhibitor developed for human use that has already entered clinical phase II trials to treat rheumatoid arthritis. We speculated that SMP-114 might also be useful to treat cardiac SR Ca 2+ leak. SMP-114 significantly reduces SR Ca 2+ leak (as assessed by Ca 2+ sparks) in human atrial (0.72 ± 0.33 sparks/100 µm/s vs. control 3.02 ± 0.91 sparks/100 µm/s) and failing left ventricular (0.78 ± 0.23 vs. 1.69 ± 0.27 sparks/100 µm/s) as well as in murine ventricular cardiomyocytes (0.30 ± 0.07 vs. 1.50 ± 0.28 sparks/100 µm/s). Associated with lower SR Ca 2+ leak, we found that SMP-114 suppressed the occurrence of spontaneous arrhythmogenic spontaneous Ca 2+ release (0.356 ± 0.109 vs. 0.927 ± 0.216 events per 30 s stimulation cessation). In consequence, post-rest potentiation of Ca 2+ -transient amplitude (measured using Fura-2) during the 30 s pause was improved by SMP-114 (52 ± 5 vs. 37 ± 4%). Noteworthy, SMP-114 has these beneficial effects without negatively impairing global excitation–contraction coupling: neither systolic Ca 2+ release nor single cell contractility was compromised, and also SR Ca 2+ reuptake, in line with resulting cardiomyocyte relaxation, was not impaired by SMP-114 in our assays. SMP-114 demonstrated potential to treat SR Ca 2+ leak and consequently proarrhythmogenic events in rodent as well as in human atrial cardiomyocytes and cardiomyocytes from patients with heart failure. Further research is necessary towards clinical use in cardiac disease.

The sodium channel NaV1.8, encoded by the SCN10A gene, has recently emerged as a potential regulator of cardiac electrophysiology. We have previously shown that NaV1.8 contributes to arrhythmogenesis by inducing a persistent Na+ current (late Na+ current, INaL) in human atrial and ventricular cardiomyocytes (CM). We now aim to further investigate the contribution of NaV1.8 to human ventricular arrhythmogenesis at the CM-specific level using pharmacological inhibition as well as a genetic knockout (KO) of SCN10A in induced pluripotent stem cell CM (iPSC-CM). In functional voltage-clamp experiments, we demonstrate that INaL was significantly reduced in ventricular SCN10A-KO iPSC-CM and in control CM after a specific pharmacological inhibition of NaV1.8. In contrast, we did not find any effects on ventricular APD90. The frequency of spontaneous sarcoplasmic reticulum Ca2+ sparks and waves were reduced in SCN10A-KO iPSC-CM and control cells following the pharmacological inhibition of NaV1.8. We further analyzed potential triggers of arrhythmias and found reduced delayed afterdepolarizations (DAD) in SCN10A-KO iPSC-CM and after the specific inhibition of NaV1.8 in control cells. In conclusion, we show that NaV1.8-induced INaL primarily impacts arrhythmogenesis at a subcellular level, with minimal effects on systolic cellular Ca2+ release. The inhibition or knockout of NaV1.8 diminishes proarrhythmic triggers in ventricular CM. In conjunction with our previously published results, this work confirms NaV1.8 as a proarrhythmic target that may be useful in an anti-arrhythmic therapeutic strategy.

In heart failure (HF), Ca(2+)/calmodulin kinase II (CaMKII) expression is increased. Altered Na(+) channel gating is linked to and may promote ventricular tachyarrhythmias (VTs) in HF. Calmodulin regulates Na(+) channel gating, in part perhaps via CaMKII. We investigated effects of adenovirus-mediated (acute) and Tg (chronic) overexpression of cytosolic CaMKIIdelta(C) on Na(+) current (I(Na)) in rabbit and mouse ventricular myocytes, respectively (in whole-cell patch clamp). Both acute and chronic CaMKIIdelta(C) overexpression shifted voltage dependence of Na(+) channel availability by -6 mV (P < 0.05), and the shift was Ca(2+) dependent. CaMKII also enhanced intermediate inactivation and slowed recovery from inactivation (prevented by CaMKII inhibitors autocamtide 2-related inhibitory peptide [AIP] or KN93). CaMKIIdelta(C) markedly increased persistent (late) inward I(Na) and intracellular Na(+) concentration (as measured by the Na(+) indicator sodium-binding benzofuran isophthalate [SBFI]), which was prevented by CaMKII inhibition in the case of acute CaMKIIdelta(C) overexpression. CaMKII coimmunoprecipitates with and phosphorylates Na(+) channels. In vivo, transgenic CaMKIIdelta(C) overexpression prolonged QRS duration and repolarization (QT intervals), decreased effective refractory periods, and increased the propensity to develop VT. We conclude that CaMKII associates with and phosphorylates cardiac Na(+) channels. This alters I(Na) gating to reduce availability at high heart rate, while enhancing late I(Na) (which could prolong action potential duration). In mice, enhanced CaMKIIdelta(C) activity predisposed to VT. Thus, CaMKII-dependent regulation of Na(+) channel function may contribute to arrhythmogenesis in HF.

Background: Phosphodiesterases (PDE) critically regulate myocardial cAMP and cGMP levels. PDE2 is stimulated by cGMP to hydrolyze cAMP, mediating a negative crosstalk between both pathways. PDE2 upregulation in heart failure contributes to desensitization to β-adrenergic overstimulation. After isoprenaline (ISO) injections, PDE2 overexpressing mice (PDE2 OE) were protected against ventricular arrhythmia. Here, we investigate the mechanisms underlying the effects of PDE2 OE on susceptibility to arrhythmias. Methods: Cellular arrhythmia, ion currents, and Ca2+-sparks were assessed in ventricular cardiomyocytes from PDE2 OE and WT littermates. Results: Under basal conditions, action potential (AP) morphology were similar in PDE2 OE and WT. ISO stimulation significantly increased the incidence of afterdepolarizations and spontaneous APs in WT, which was markedly reduced in PDE2 OE. The ISO-induced increase in ICaL seen in WT was prevented in PDE2 OE. Moreover, the ISO-induced, Epac- and CaMKII-dependent increase in INaL and Ca2+-spark frequency was blunted in PDE2 OE, while the effect of direct Epac activation was similar in both groups. Finally, PDE2 inhibition facilitated arrhythmic events in ex vivo perfused WT hearts after reperfusion injury. Conclusion: Higher PDE2 abundance protects against ISO-induced cardiac arrhythmia by preventing the Epac- and CaMKII-mediated increases of cellular triggers. Thus, activating myocardial PDE2 may represent a novel intracellular anti-arrhythmic therapeutic strategy in HF.