NaV1.8 as Proarrhythmic Target in a Ventricular Cardiac Stem Cell Model.

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.

Molecular and Functional Relevance of NaV1.8-Induced Atrial Arrhythmogenic Triggers in a Human SCN10A Knock-Out Stem Cell Model.

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.

Electrophysiological and calcium-handling development during long-term culture of human-induced pluripotent stem cell-derived cardiomyocytes.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used for personalised medicine and preclinical cardiotoxicity testing. Reports on hiPSC-CM commonly describe heterogenous functional readouts and underdeveloped or immature phenotypical properties. Cost-effective, fully defined monolayer culture is approaching mainstream adoption; however, the optimal age at which to utilise hiPSC-CM is unknown. In this study, we identify, track and model the dynamic developmental behaviour of key ionic currents and Ca2+-handling properties in hiPSC-CM over long-term culture (30-80 days). hiPSC-CMs > 50 days post differentiation show significantly larger ICa,L density along with an increased ICa,L-triggered Ca2+-transient. INa and IK1 densities significantly increase in late-stage cells, contributing to increased upstroke velocity and reduced action potential duration, respectively. Importantly, our in silico model of hiPSC-CM electrophysiological age dependence confirmed IK1 as the key ionic determinant of action potential shortening in older cells. We have made this model available through an open source software interface that easily allows users to simulate hiPSC-CM electrophysiology and Ca2+-handling and select the appropriate age range for their parameter of interest. This tool, together with the insights from our comprehensive experimental characterisation, could be useful in future optimisation of the culture-to-characterisation pipeline in the field of hiPSC-CM research.

Different activation of MAPKs and Akt/GSK3ß after preload vs. afterload elevation.

AIMS: Pressure overload (PO) and volume overload (VO) lead to concentric or eccentric hypertrophy. Previously, we could show that activation of signalling cascades differ in in vivo mouse models. Activation of these signal cascades could either be induced by intrinsic load sensing or neuro-endocrine substances like catecholamines or the renin-angiotensin-aldosterone system. METHODS AND RESULTS: We therefore analysed the activation of classical cardiac signal pathways [mitogen-activated protein kinases (MAPKs) (ERK, p38, and JNK) and Akt-GSK3ß] in in vitro of mechanical overload (ejecting heart model, rabbit and human isolated muscle strips). Selective elevation of preload in vitro increased AKT and GSK3ß phosphorylation after 15 min in isolated rabbit muscles strips (AKT 49%, GSK3ß 26%, P < 0.05) and in mouse ejecting hearts (AKT 51%, GSK49%, P < 0.05), whereas phosphorylation of MAPKs was not influenced by increased preload. Selective elevation of afterload revealed an increase in ERK phosphorylation in the ejecting heart (43%, P < 0.05), but not in AKT, GSK3ß, and the other MAPKs. Elevation of preload and afterload in the ejecting heart induced a significant phosphorylation of ERK (95%, P < 0.001) and showed a moderate increased AKT (P = 0.14) and GSK3ß (P = 0.21) phosphorylation, which did not reach significance. Preload and afterload elevation in muscles strips from human failing hearts showed neither AKT nor ERK phosphorylation changes. CONCLUSIONS: Our data show that preload activates the AKT-GSK3ß and afterload the ERK pathway in vitro, indicating an intrinsic mechanism independent of endocrine signalling.

Generation of homozygous Nav1.8 knock-out iPSC lines by CRISPR Cas9 genome editing to investigate a potential new antiarrhythmic strategy.

The sodium channel Nav1.8, encoded by SCN10A, is reported to contribute to arrhythmogenesis by inducing the late INa and thereby enhanced persistent Na+ current. However, its exact electrophysiological role in cardiomyocytes remains unclear. Here, we generated induced pluripotent stem cells (iPSCs) with a homozygous SCN10A knock-out from a healthy iPSC line by CRISPR Cas9 genome editing. The edited iPSCs maintained full pluripotency, genomic integrity, and spontaneous in vitro differentiation capacity. The iPSCs are able to differentiate into iPSC-cardiomyocytes, hence making it possible to investigate the role of Nav1.8 in the heart.

Detrimental proarrhythmogenic interaction of Ca2+/calmodulin-dependent protein kinase II and NaV1.8 in heart failure.

An interplay between Ca2+/calmodulin-dependent protein kinase IIδc (CaMKIIδc) and late Na+ current (INaL) is known to induce arrhythmias in the failing heart. Here, we elucidate the role of the sodium channel isoform NaV1.8 for CaMKIIδc-dependent proarrhythmia. In a CRISPR-Cas9-generated human iPSC-cardiomyocyte homozygous knock-out of NaV1.8, we demonstrate that NaV1.8 contributes to INaL formation. In addition, we reveal a direct interaction between NaV1.8 and CaMKIIδc in cardiomyocytes isolated from patients with heart failure (HF). Using specific blockers of NaV1.8 and CaMKIIδc, we show that NaV1.8-driven INaL is CaMKIIδc-dependent and that NaV1.8-inhibtion reduces diastolic SR-Ca2+ leak in human failing cardiomyocytes. Moreover, increased mortality of CaMKIIδc-overexpressing HF mice is reduced when a NaV1.8 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.

The functional consequences of sodium channel NaV 1.8 in human left ventricular hypertrophy.

AIMS: In hypertrophy and heart failure, the proarrhythmic persistent Na+ current (INaL ) is enhanced. We aimed to investigate the electrophysiological role of neuronal sodium channel NaV 1.8 in human hypertrophied myocardium. METHODS AND RESULTS: Myocardial tissue of 24 patients suffering from symptomatic severe aortic stenosis and concomitant significant afterload-induced hypertrophy with preserved ejection fraction was used and compared with 12 healthy controls. We performed quantitative real-time PCR and western blot and detected a significant up-regulation of NaV 1.8 mRNA (2.34-fold) and protein expression (1.96-fold) in human hypertrophied myocardium compared with healthy hearts. Interestingly, NaV 1.5 protein expression was significantly reduced in parallel (0.60-fold). Using whole-cell patch-clamp technique, we found that the prominent INaL was significantly reduced after addition of novel NaV 1.8-specific blockers either A-803467 (30 nM) or PF-01247324 (1 µM) in human hypertrophic cardiomyocytes. This clearly demonstrates the relevant contribution of NaV 1.8 to this proarrhythmic current. We observed a significant action potential duration shortening and performed confocal microscopy, demonstrating a 50% decrease in proarrhythmic diastolic sarcoplasmic reticulum (SR)-Ca2+ leak and SR-Ca2+ spark frequency after exposure to both NaV 1.8 inhibitors. CONCLUSIONS: We show for the first time that the neuronal sodium channel NaV 1.8 is up-regulated on mRNA and protein level in the human hypertrophied myocardium. Furthermore, inhibition of NaV 1.8 reduced augmented INaL , abbreviated the action potential duration, and decreased the SR-Ca2+ leak. The findings of our study suggest that NaV 1.8 could be a promising antiarrhythmic therapeutic target and merits further investigation.

Sarcoplasmic reticulum calcium leak contributes to arrhythmia but not to heart failure progression.

Increased sarcoplasmic reticulum (SR) Ca2+ leak via the cardiac ryanodine receptor (RyR2) has been suggested to play a mechanistic role in the development of heart failure (HF) and cardiac arrhythmia. Mice treated with a selective RyR2 stabilizer, rycal S36, showed normalization of SR Ca2+ leak and improved survival in pressure overload (PO) and myocardial infarction (MI) models. The development of HF, measured by echocardiography and molecular markers, showed no difference in rycal S36- versus placebo-treated mice. Reduction of SR Ca2+ leak in the PO model by the rycal-unrelated RyR2 stabilizer dantrolene did not mitigate HF progression. Development of HF was not aggravated by increased SR Ca2+ leak due to RyR2 mutation (R2474S) in volume overload, an SR Ca2+ leak-independent HF model. Arrhythmia episodes were reduced by rycal S36 treatment in PO and MI mice in vivo and ex vivo in Langendorff-perfused hearts. Isolated cardiomyocytes from murine failing hearts and human ventricular failing and atrial nonfailing myocardium showed reductions in delayed afterdepolarizations, in spontaneous and induced Ca2+ waves, and in triggered activity in rycal S36 versus placebo cells, whereas the Ca2+ transient, SR Ca2+ load, SR Ca2+ adenosine triphosphatase function, and action potential duration were not affected. Rycal S36 treatment of human induced pluripotent stem cells isolated from a patient with catecholaminergic polymorphic ventricular tachycardia could rescue the leaky RyR2 receptor. These results suggest that SR Ca2+ leak does not primarily influence contractile HF progression, whereas rycal S36 treatment markedly reduces ventricular arrhythmias, thereby improving survival in mice.

Differential regulation of sodium channels as a novel proarrhythmic mechanism in the human failing heart.

Aims: In heart failure (HF), enhanced persistent Na+ current (INaL) exerts detrimental effects on cellular electrophysiology and can induce arrhythmias. However, the underlying regulatory mechanisms remain unclear. Our aim was to potentially investigate the regulation and electrophysiological contribution of neuronal sodium channel NaV1.8 in failing human heart and eventually to reveal a novel anti-arrhythmic therapy. Methods and results: By western blot, we found that NaV1.8 protein expression is significantly up-regulated, while of the predominant cardiac isoform NaV1.5 is inversely reduced in human HF. Furthermore, to investigate the relation of NaV1.8 regulation with the cellular proarrhythmic events, we performed comprehensive electrophysiology recordings and explore the effect of NaV1.8 on INaL, action potential duration (APD), Ca2+ spark frequency, and arrhythmia induction in human failing cardiomyocytes. NaV1.8 inhibition with the specific blockers A-803467 and PF-01247324 decreased INaL, abbreviated APD and reduced cellular-spontaneous Ca2+-release and proarrhythmic events in human failing cardiomyocytes. Consistently, in mouse cardiomyocytes stressed with isoproterenol, pharmacologic inhibition and genetically knockout of NaV1.8 (SCN10A-/-), were associated with reduced INaL and abbreviated APD. Conclusion: We provide first evidence of differential regulation of NaV1.8 and NaV1.5 in the failing human myocardium and their contribution to arrhythmogenesis due to generation of INaL. We propose inhibition of NaV1.8 thus constitutes a promising novel approach for selective anti-arrhythmic therapy in HF.

Antiarrhythmic effects of dantrolene in human diseased cardiomyocytes.

BACKGROUND: Cardiac type 2 ryanodine receptors (RyR2s) play a pivotal role in cellular electrophysiology and contractility. Increased RyR2-mediated diastolic sarcoplasmic reticulum (SR) Ca2+ release is linked to heart failure (HF) and arrhythmias. Dantrolene, a drug used for the treatment of malignant hyperthermia, is known to stabilize RyRs in skeletal muscle. OBJECTIVE: The purpose of this study was to investigate the effects of dantrolene on arrhythmogenic triggers and contractile function in human atrial fibrillation (AF) and HF cardiomyocytes (CM). METHODS: Human CM were isolated from either patients with HF (ventricular) or patients with AF (atrial), and Ca2+ imaging, patch-clamp, or muscle strip experiments were performed. RESULTS: After exposure to dantrolene, human atrial AF and left ventricular HF CM showed significant reductions in proarrhythmic SR Ca2+ spark frequency and diastolic SR Ca2+ leak. Moreover, dantrolene decreased the frequency of Ca2+ waves and spontaneous Ca2+ transients in HF CM. Patch-clamp experiments revealed that dantrolene significantly suppressed delayed afterdepolarizations in HF and AF CM. Importantly, dantrolene had no effect on action potential duration in AF or in HF CM. In addition, dantrolene had neutral effects on contractile force of human isometrically twitching ventricular HF trabeculae. CONCLUSION: Our study showed that dantrolene beneficially influenced disrupted SR Ca2+ homeostasis in human HF and AF CM. Cellular arrhythmogenic triggers were potently suppressed by dantrolene, whereas action potential duration and contractility were not affected. As a clinically approved drug for the treatment of malignant hyperthermia, dantrolene may be a potential antiarrhythmic drug for patients with rhythm disorders and merits further clinical investigation.

Enhanced cardiac TBC1D10C expression lowers heart rate and enhances exercise capacity and survival.

TBC1D10C is a protein previously demonstrated to bind and inhibit Ras and Calcineurin. In cardiomyocytes, also CaMKII is inhibited and all three targeted enzymes are known to promote maladaptive cardiomyocyte hypertrophy. Here, in accordance with lack of Calcineurin inhibition in vivo, we did not observe a relevant anti-hypertrophic effect despite inhibition of Ras and CaMKII. However, cardiomyocyte-specific TBC1D10C overexpressing transgenic mice exhibited enhanced longevity. Ejection fraction and exercise capacity were enhanced in transgenic mice, but shortening of isolated cardiomyocytes was not increased. This suggests longevity resulted from enhanced cardiac performance but independent of cardiomyocyte contractile force. In further search for mechanisms, a transcriptome-wide analysis revealed expressional changes in several genes pertinent to control of heart rate (HR) including Hcn4, Scn10a, Sema3a and Cacna2d2. Indeed, telemetric holter recordings demonstrated slower atrial conduction and significantly lower HR. Pharmacological reduction of HR was previously demonstrated to enhance survival in mice. Thus, in addition to inhibition of stress signaling, TBC1D10C economizes generation of cardiac output via HR reduction, enhancing exercise capacity and survival. TBC1D10C may be a new target for HR reduction and longevity.

The combined effects of ranolazine and dronedarone on human atrial and ventricular electrophysiology.

INTRODUCTION: Pharmacological rhythm control of atrial fibrillation (AF) in patients with structural heart disease is limited. Ranolazine in combination with low dose dronedarone remarkably reduced AF-burden in the phase II HARMONY trial. We thus aimed to investigate the possible mechanisms underlying these results. METHODS AND RESULTS: Patch clamp experiments revealed that ranolazine (5µM), low-dose dronedarone (0.3µM), and the combination significantly prolonged action potential duration (APD90) in atrial myocytes from patients in sinus rhythm (prolongation by 23.5±0.1%, 31.7±0.1% and 25.6±0.1% respectively). Most importantly, in atrial myocytes from patients with AF ranolazine alone, but more the combination with dronedarone, also prolonged the typically abbreviated APD90 (prolongation by 21.6±0.1% and 31.9±0.1% respectively). It was clearly observed that neither ranolazine, dronedarone nor the combination significantly changed the APD or contractility and twitch force in ventricular myocytes or trabeculae from patients with heart failure (HF). Interestingly ranolazine, and more so the combination, but not dronedarone alone, caused hyperpolarization of the resting membrane potential in cardiomyocytes from AF. As measured by confocal microscopy (Fluo-3), ranolazine, dronedarone and the combination significantly suppressed diastolic sarcoplasmic reticulum (SR) Ca(2+) leak in myocytes from sinus rhythm (reduction by ranolazine: 89.0±30.7%, dronedarone: 75.6±27.4% and combination: 78.0±27.2%), in myocytes from AF (reduction by ranolazine: 67.6±33.7%, dronedarone: 86.5±31.7% and combination: 81.0±33.3%), as well as in myocytes from HF (reduction by ranolazine: 64.8±26.5% and dronedarone: 65.9±29.3%). CONCLUSIONS: Electrophysiological measurements during exposure to ranolazine alone or in combination with low-dose dronedarone showed APD prolongation, cellular hyperpolarization and reduced SR Ca(2+) leak in human atrial myocytes. The combined inhibitory effects on various currents, in particular Na(+) and K(+) currents, may explain the anti-AF effects observed in the HARMONY trial. Therefore, the combination of ranolazine and dronedarone, but also ranolazine alone, may be promising new treatment options for AF, especially in patients with HF, and merit further clinical investigation.

Inhibition of CaMKII Attenuates Progressing Disruption of Ca(2+) Homeostasis Upon Left Ventricular Assist Device Implantation in Human Heart Failure.

In heart failure, left ventricular assist device (LVAD) implantation is performed to ensure sufficient cardiac output. Whereas some patients are subsequently weaned from LVAD support, other patients still need heart transplantation. To elucidate underlying mechanisms, we assessed the arrhythmogenic SR-Ca(2+) leak at the time of LVAD implantation (HF-Im) and heart transplantation (HF-Tx) and evaluated the effects of CaMKII-inhibition. Human left-ventricular cardiomyocytes were isolated, paced at 1 Hz for 10 beats to ensure SR-Ca(2+) loading and scanned for diastolic Ca(2+) sparks (confocal microscopy). In HF-Im, the high diastolic spark frequency (CaSpF) of 0.76 ± 0.12 × 100 µm(-1) × s(-1) could be reduced to 0.48 ± 0.10 × 100 µm(-1) × s(-1) by CaMKII inhibition (AIP, 1 µM). The amplitude of Ca(2+) sparks, width, and length was not significantly altered. In sum, CaMKII inhibition yielded a clear tendency toward a reduction of the SR-Ca(2+) leak (n cells/patients = 76/6 vs. 108/6, P = 0.08). In HF-Tx, we detected an even higher CaSpF of 1.00 ± 0.10 100 µm(-1) × s(-1) and a higher SR-Ca(2+) leak compared with HF-Im (increase by 81 ± 33%, n cells/patients = 156/7 vs. 130/7, P < 0.05), which fits to the further decreased LV function. Here, CaMKII inhibition likewise reduced CaSpF (0.35 ± 0.09 100 µm(-1) × s(-1,) P = 0.06) and significantly reduced spark duration (n sparks/patients = 58/3 vs. 159/3, P < 0.05). Conclusively, the SR-Ca(2+) leak was reduced by 69 ± 12% in HF-Tx upon CaMKII inhibition (n cells/patients = 53/3 vs. 91/3, P < 0.05). These data show that the SR-Ca(2+) leak correlates with the development of LV function after LVAD implantation and may represent an important pathomechanism. The fact that CaMKII inhibition reduces the SR-Ca(2+) leak in HF-Tx suggests that CaMKII inhibition may be a promising option to beneficially influence clinical course after LVAD implantation.

Sex-dependent alterations of Ca2+ cycling in human cardiac hypertrophy and heart failure.

AIMS: Clinical studies have shown differences in the propensity for malignant ventricular arrhythmias between women and men suffering from cardiomyopathies and heart failure (HF). This is clinically relevant as it impacts therapies like prophylactic implantable cardioverter-defibrillator implantation but the pathomechanisms are unknown. As an increased sarcoplasmic reticulum (SR) Ca(2+) leak is arrhythmogenic, it could represent a cellular basis for this paradox. METHODS/RESULTS: We evaluated the SR Ca(2+) leak with respect to sex differences in (i) afterload-induced cardiac hypertrophy (Hy) with preserved left ventricular (LV) function and (ii) end-stage HF. Cardiac function did not differ between sexes in both cardiac pathologies. Human cardiomyocytes isolated from female patients with Hy showed a significantly lower Ca(2+) spark frequency (CaSpF, confocal microscopy, Fluo3-AM) compared with men (P < 0.05). As Ca(2+) spark width and duration were similar in women and men, this difference in CaSpF did not yet translate into a significant difference of the calculated SR Ca(2+) leak between both sexes at this stage of disease (P = 0.14). Epifluorescence measurements (Fura2-AM) revealed comparable Ca(2+) cycling properties (diastolic Ca(2+) levels, amplitude of systolic Ca(2+) transients, SR Ca(2+) load) in patients of both sexes suffering from Hy. Additionally, the increased diastolic CaSpF in male patients with Hy did not yet translate into an elevated ratio of cells showing arrhythmic events (Ca(2+) waves, spontaneous Ca(2+) transients) (P = 0.77). In the transition to HF, both sexes showed an increase of the CaSpF (P < 0.05) and the sex dependence was even more pronounced. Female patients had a 69 ± 10% lower SR Ca(2+) leak (P < 0.05), which now even translated into a lower ratio of arrhythmic cells in female HF patients compared with men (P < 0.001). CONCLUSION: These data show that the SR Ca(2+) leak is lower in women than in men with comparable cardiac impairment. Since the SR Ca(2+) leak triggers delayed afterdepolarizations, our findings may explain why women are less prone to ventricular arrhythmias and confirm the rationale of therapeutic measures reducing the SR Ca(2+) leak.

Long term clinical outcome of dental implants placed in a patient with Singleton-Merten syndrome.

PATIENTS: Singleton-Merten syndrome is an extremely rare autosomal dominant condition with less than 10 reported cases in the literature. It is characterized by abnormal aortic calcifications and dental abnormalities. The goal of this case report is to discuss the abnormal oral clinical features and the modified treatment protocol that was used in order to achieve osseointegration of dental implants in a patient having abnormal bone density and bone turnover associated with Singleton-Merten Syndrome. DISCUSSION: Following extraction of the remaining teeth, titanium implants (Friadent GmbH, Mannheim, Germany and Straumann(®), Basel, Switzerland) were placed in the upper and lower jaw of the patient. The upper jaw which was treated with dental implants, received a bar supported implant retained prosthesis and the lower jaw an implant retained telescopic prosthesis. The patient was regularly followed up for the past 13 years during which, clinical and radiological evaluation of osseointegration was undertaken. All the loaded implants showed clinical and radiographic evidence of osseointegration. With a follow up of 13 years after insertion of the first implant, the patient reported functioning well with no complications. CONCLUSION: The treatment with dental implants in the extremely rare Singleton-Merten syndrome patients is a reasonable treatment option to rehabilitate maxillofacial aesthetics and establish normal function of the jaws.

Late INa increases diastolic SR-Ca2+-leak in atrial myocardium by activating PKA and CaMKII.

AIMS: Enhanced cardiac late Na current (late INa) and increased sarcoplasmic reticulum (SR)-Ca(2+)-leak are both highly arrhythmogenic. This study seeks to identify signalling pathways interconnecting late INa and SR-Ca(2+)-leak in atrial cardiomyocytes (CMs). METHODS AND RESULTS: In murine atrial CMs, SR-Ca(2+)-leak was increased by the late INa enhancer Anemonia sulcata toxin II (ATX-II). An inhibition of Ca(2+)/calmodulin-dependent protein kinase II (Autocamide-2-related inhibitory peptide), protein kinase A (H89), or late INa (Ranolazine or Tetrodotoxin) all prevented ATX-II-dependent SR-Ca(2+)-leak. The SR-Ca(2+)-leak induction by ATX-II was not detected when either the Na(+)/Ca(2+) exchanger was inhibited (KBR) or in CaMKIIδc-knockout mice. FRET measurements revealed increased cAMP levels upon ATX-II stimulation, which could be prevented by inhibition of adenylyl cyclases (ACs) 5 and 6 (NKY 80) but not by inhibition of phosphodiesterases (IBMX), suggesting PKA activation via an AC-dependent increase of cAMP levels. Western blots showed late INa-dependent hyperphosphorylation of CaMKII as well as PKA target sites at ryanodine receptor type-2 (-S2814 and -S2808) and phospholamban (-Thr17, -S16). Enhancement of late INa did not alter Ca(2+)-transient amplitude or SR-Ca(2+)-load. However, upon late INa activation and simultaneous CaMKII inhibition, Ca(2+)-transient amplitude and SR-Ca(2+)-load were increased, whereas PKA inhibition reduced Ca(2+)-transient amplitude and load and additionally slowed Ca(2+) elimination. In atrial CMs from patients with atrial fibrillation, inhibition of late INa, CaMKII, or PKA reduced the SR-Ca(2+)-leak. CONCLUSION: Late INa exerts distinct effects on Ca(2+) homeostasis in atrial myocardium through activation of CaMKII and PKA. Inhibition of late INa represents a potential approach to attenuate CaMKII activation and decreases SR-Ca(2+)-leak in atrial rhythm disorders. The interconnection with the cAMP/PKA system further increases the antiarrhythmic potential of late INa inhibition.

Enhanced late INa induces proarrhythmogenic SR Ca leak in a CaMKII-dependent manner.

OBJECTIVE: Enhanced late Na current (late INa) induces Na-dependent Ca overload as well as proarrhythmogenic events on the cellular level that include spatio-temporally uncoordinated diastolic Ca release from the sarcoplasmic reticulum (SR) and delayed afterdepolarizations (DADs). The Ca/calmodulin-dependent protein kinase II (CaMKII) gets activated upon increases in [Ca]i and mediates diastolic SR Ca leak as well as DADs. RATIONALE: We hypothesized that increased late INa (in disease-comparable ranges) exerts proarrhythmogenic events in isolated ventricular mouse myocytes in a manner depending on CaMKII-dependent SR Ca leak. We further tested whether inhibition of disease-related late INa may reduce proarrhythmogenic SR Ca leak in myocytes from failing human hearts. METHODS: Ventricular myocytes were isolated from healthy wildtype (WT), failing CaMKIIδC transgenic (TG) mouse, and failing human hearts. ATX-II (0.25-10 nmol/L) was used to enhance late INa. Spontaneous Ca loss from the SR during diastole (Ca sparks), DADs, non-triggered diastolic Ca transients in myocytes and premature beats of isometrically twitching papillary muscles were used as readouts for proarrhythmogenic events. CaMKII autophosphorylation was assessed by immunoblots. Late INa was inhibited using ranolazine (Ran, 10 µmol/L) or TTX (2 µmol/L), and CaMKII by KN-93 (1 µmol/L) or AIP (1 µmol/L). RESULTS: In WT myocytes, sub-nanomolar ATX-II exposure (0.5 nmol/L) enhanced late INa by ~60%, which resulted in increased diastolic SR Ca loss despite unaltered SR Ca content. In parallel, DADs and non-triggered diastolic Ca transients arose. Inhibition of enhanced late INa by RAN or TTX significantly attenuated diastolic SR Ca loss and suppressed DADs as well as mechanical alternans in mouse and diastolic SR Ca loss in failing human myocytes. ATX-II caused Ca-dependent CaMKII-activation without changes in protein expression, which was reversible by Ran or AIP. Conversely, CaMKII-inhibition decreased diastolic SR Ca loss, DADs and non-triggered diastolic Ca transients despite ATX-II-exposure. Finally, failing mouse myocytes with increased CaMKII activity (TG CaMKIIδC) showed an even aggravated diastolic SR Ca loss that was associated with an increased frequency of non-triggered diastolic Ca transients upon enhanced late INa. CONCLUSIONS: Increased late INa (in disease-comparable ranges) induces proarrhythmogenic events during diastole in healthy and failing mouse myocytes, which are mediated via CaMKII-dependent SR Ca loss. Inhibition of late INa not only attenuated these cellular arrhythmias in mouse myocytes but also in failing human myocytes indicating some antiarrhythmic potential for an inhibition of the elevated late INa/CaMKII signaling pathway in this setting.

Role of late sodium current as a potential arrhythmogenic mechanism in the progression of pressure-induced heart disease.

The aim of the study was to determine the characteristics of the late Na current (INaL) and its arrhythmogenic potential in the progression of pressure-induced heart disease. Transverse aortic constriction (TAC) was used to induce pressure overload in mice. After one week the hearts developed isolated hypertrophy with preserved systolic contractility. In patch-clamp experiments both, INaL and the action potential duration (APD90) were unchanged. In contrast, after five weeks animals developed heart failure with prolonged APDs and slowed INaL decay time which could be normalized by addition of the INaL inhibitor ranolazine (Ran) or by the Ca/calmodulin-dependent protein kinase II (CaMKII) inhibitor AIP. Accordingly the APD90 could be significantly abbreviated by Ran, tetrodotoxin and the CaMKII inhibitor AIP. Isoproterenol increased the number of delayed afterdepolarizations (DAD) in myocytes from failing but not sham hearts. Application of either Ran or AIP prevented the occurrence of DADs. Moreover, the incidence of triggered activity was significantly increased in TAC myocytes and was largely prevented by Ran and AIP. Western blot analyses indicate that increased CaMKII activity and a hyperphosphorylation of the Nav1.5 at the CaMKII phosphorylation site (Ser571) paralleled our functional observations five weeks after TAC surgery. In pressure overload-induced heart failure a CaMKII-dependent augmentation of INaL plays a crucial role in the AP prolongation and generation of cellular arrhythmogenic triggers, which cannot be found in early and still compensated hypertrophy. Inhibition of INaL and CaMKII exerts potent antiarrhythmic effects and might therefore be of potential therapeutic interest. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".

Diastolic dysfunction and arrhythmias caused by overexpression of CaMKIIδ(C) can be reversed by inhibition of late Na(+) current.

Transgenic (TG) Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) δ(C) mice develop systolic heart failure (HF). CaMKII regulates intracellular Ca(2+) handling proteins as well as sarcolemmal Na(+) channels. We hypothesized that CaMKII also contributes to diastolic dysfunction and arrhythmias via augmentation of the late Na(+) current (late I(Na)) in early HF (8-week-old TG mice). Echocardiography revealed severe diastolic dysfunction in addition to decreased systolic ejection fraction. Premature arrhythmogenic contractions (PACs) in isolated isometrically twitching papillary muscles only occurred in TG preparations (5 vs. 0, P < 0.05) which could be completely terminated when treated with the late I(Na) inhibitor ranolazine (Ran, 5 µmol/L). Force-frequency relationships revealed significantly reduced twitch force amplitudes in TG papillary muscles. Most importantly, diastolic tension increased with raising frequencies to a greater extent in TG papillary muscles compared to WT specimen (at 10 Hz: 3.7 ± 0.4 vs. 2.5 ± 0.3 mN/mm²; P < 0.05). Addition of Ran improved diastolic dysfunction to 2.1 ± 0.2 mN/mm² (at 10 Hz; P < 0.05) without negative inotropic effects. Mechanistically, the late I(Na) was markedly elevated in myocytes isolated from TG mice and could be completely reversed by Ran. In conclusion, our results show for the first time that TG CaMKIIδ(C) overexpression induces diastolic dysfunction and arrhythmogenic triggers possibly via an enhanced late I(Na). Inhibition of elevated late I(Na) had beneficial effects on arrhythmias as well as diastolic function in papillary muscles from CaMKIIδ(C) TG mice. Thus, late I(Na) inhibition appears to be a promising option for diastolic dysfunction and arrhythmias in HF where CaMKII is found to be increased.