Targeting late ICaL to close the window to ventricular arrhythmias

This commentary is on the paper by Angelini et al. Here, we set the original paper in the context of triggered arrhythmias, particularly early after depolarizations (EADs), emphasizing the importance of pharmacologically inhibiting late Ca2+ current to prevent EADs without affecting myocardial contractility.

CaMKII inhibition has dual effects on spontaneous Ca2+ release and Ca2+ alternans in ventricular cardiomyocytes from mice with a gain-of-function RyR2 mutation

In conditions with abnormally increased activity of the cardiac ryanodine receptor (RyR2), Ca2+/calmodulin-dependent protein kinase II (CaMKII) can contribute to a further destabilization of RyR2 that results in triggered arrhythmias. Therefore, inhibition of CaMKII in such conditions has been suggested as a strategy to suppress RyR2 activity and arrhythmias. However, suppression of RyR2 activity can lead to the development of arrhythmogenic Ca2+ alternans. Aim: To test whether suppression of RyR2 activity caused by inhibition of CaMKII increases propensity for Ca2+ alternans. Methods and results: We studied spontaneous Ca2+-release events and Ca2+ alternans in isolated left ventricular cardiomyocytes from mice carrying the gain-of-function RyR2 mutation RyR2-R2474S and from wild-type mice. CaMKII inhibition by KN-93 effectively decreased the frequency of spontaneous Ca2+-release events in RyR2‑R2474S cardiomyocytes exposed to the β‑adrenoceptor agonist isoprenaline. However, KN-93-treated RyR2-R2474S cardiomyocytes also showed increased propensity for Ca2+ alternans and increased Ca2+ alternans ratio compared with both an inactive analog of KN‑93 and with vehicle-treated controls. This increased propensity for Ca2+ alternans was explained by prolongation of Ca2+-release refractoriness. Importantly, the increased propensity for Ca2+ alternans in KN‑93-treated RyR2-R2474S cardiomyocytes did not surpass that of wild-type. Conclusions: Inhibition of CaMKII efficiently reduces spontaneous Ca2+-release, but promotes Ca2+ alternans in RyR2-R2474S cardiomyocytes with a gain-of-function RyR2 mutation. The dominant effect in RyR2-R2474S is to reduce spontaneous Ca2+-release, which supports this intervention as a therapeutic strategy in this specific condition. However, future studies on CaMKII inhibition in conditions with increased propensity for Ca2+ alternans should include investigation of both phenomena.

Nerol Attenuates Ouabain-Induced Arrhythmias

Nerol (C10H18O) is a monoterpene found in many essential oils, such as lemon balm and hop. In this study, we explored the contractile and electrophysiological properties of nerol and demonstrated its antiarrhythmic effects in guinea pig heart preparation. Nerol effects were evaluated on atrial and ventricular tissue contractility, electrocardiogram (ECG), voltage-dependent L-type Ca2+current (ICa,L), and ouabain-triggered arrhythmias. Overall our results revealed that by increasing concentrations of nerol (from 0.001 to 30 mM) there was a significant decrease in left atrium contractile force. This effect was completely and rapidly reversible after washing out (~ 2 min). Nerol (at 3 mM concentration) decreased the left atrium positive inotropic response evoked by adding up CaCl2in the extracellular medium. Interestingly, when using a lower concentration of nerol (30μM), it was not possible to clearly observe any significant ECG signal alterations but a small reduction of ventricular contractility was observed. In addition, 300μM nerol promoted a significant decrease on the cardiac rate and contractility. Important to note is the fact that in isolated cardiomyocytes, peak ICa,Lwas reduced by 58.9 ± 6.31% after perfusing 300μM nerol (n=7, p<0.05). Nerol, at 30 and 300μM, delayed the time of onset of ouabain-triggered arrhythmias and provoked a decrease in the diastolic tension induced by the presence of ouabain (50μM). Furthermore, nerol preincubation significantly attenuated arrhythmia severity index without changes in the positive inotropism elicited by ouabain exposure. Taken all together, we may be able to conclude that nerol primarily by reducing Ca2+influx through L-type Ca2+channel blockade lessened the severity of ouabain-triggered arrhythmias in mammalian heart.

Ablation of phospholamban rescues reperfusion arrhythmias but exacerbates myocardium infarction in hearts with Ca2+/calmodulin kinase II constitutive phosphorylation of ryanodine receptors

Aims Abnormal Ca2+ release from the sarcoplasmic reticulum (SR), associated with Ca2+-calmodulin kinase II (CaMKII)-dependent phosphorylation of RyR2 at Ser2814, has consistently been linked to arrhythmogenesis and ischaemia/reperfusion (I/R)-induced cell death. In contrast, the role played by SR Ca2+ uptake under these stress conditions remains controversial. We tested the hypothesis that an increase in SR Ca2+ uptake is able to attenuate reperfusion arrhythmias and cardiac injury elicited by increased RyR2-Ser2814 phosphorylation. Methods and results We used WT mice, which have been previously shown to exhibit a transient increase in RyR2-Ser2814 phosphorylation at the onset of reperfusion; mice with constitutive pseudo-phosphorylation of RyR2 at Ser2814 (S2814D) to exacerbate CaMKII-dependent reperfusion arrhythmias and cardiac damage, and phospholamban (PLN)-deficient-S2814D knock-in (SDKO) mice resulting from crossbreeding S2814D with phospholamban knockout deficient (PLNKO) mice. At baseline, S2814D and SDKO mice had structurally normal hearts. Moreover none of the strains were arrhythmic before ischaemia. Upon cardiac I/R, WT, and S2814D hearts exhibited abundant arrhythmias that were prevented by PLN ablation. In contrast, PLN ablation increased infarct size compared with WT and S2814D hearts. Mechanistically, the enhanced SR Ca2+ sequestration evoked by PLN ablation in SDKO hearts prevented arrhythmogenic events upon reperfusion by fragmenting SR Ca2+ waves into non-propagated and non-arrhythmogenic events (mini-waves). Conversely, the increase in SR Ca2+ sequestration did not reduce but rather exacerbated I/R-induced SR Ca2+ leak, as well as mitochondrial alterations, which were greatly avoided by inhibition of RyR2. These results indicate that the increase in SR Ca2+ uptake is ineffective in preventing the enhanced SR Ca2+ leak of PLN ablated myocytes from either entering into nearby mitochondria and/or activating additional CaMKII pathways, contributing to cardiac damage. Conclusion Our results demonstrate that increasing SR Ca2+ uptake by PLN ablation can prevent the arrhythmic events triggered by CaMKII-dependent phosphorylation of RyR2-induced SR Ca2+ leak. These findings underscore the benefits of increasing SERCA2a activity in the face of SR Ca2+ triggered arrhythmias. However, enhanced SERCA2a cannot prevent but rather exacerbates I/R cardiac injury.

Nebivolol suppresses cardiac ryanodine receptor-mediated spontaneous Ca2+ release and catecholaminergic polymorphic ventricular tachycardia

β-Blockers are a standard treatment for heart failure and cardiac arrhythmias. There are ∼30 commonly used β-blockers, representing a diverse class of drugs with different receptor affinities and pleiotropic properties. We reported that among 14 β-blockers tested previously, only carvedilol effectively suppressed cardiac ryanodine receptor (RyR2)-mediated spontaneous Ca2+ waves during store Ca2+ overload, also known as store overload-induced Ca2+ release (SOICR). Given the critical role of SOICR in arrhythmogenesis, it is of importance to determine whether there are other β-blockers that suppress SOICR. Here, we assessed the effect of other commonly used β-blockers on RyR2-mediated SOICR in HEK293 cells, using single-cell Ca2+ imaging. Of the 13 β-blockers tested, only nebivolol, a β-1-selective β-blocker with nitric oxide synthase (NOS)-stimulating action, effectively suppressed SOICR. The NOS inhibitor (N-nitro-l-arginine methyl ester) had no effect on nebivolol's SOICR inhibition, and the NOS activator (histamine or prostaglandin E2) alone did not inhibit SOICR. Hence, nebivolol's SOICR inhibition was independent of NOS stimulation. Like carvedilol, nebivolol reduced the opening of single RyR2 channels and suppressed spontaneous Ca2+ waves in intact hearts and catecholaminergic polymorphic ventricular tachycardia (CPVT) in the mice harboring a RyR2 mutation (R4496C). Interestingly, a non-β-blocking nebivolol enantiomer, (l)-nebivolol, also suppressed SOICR and CPVT without lowering heart rate. These data indicate that nebivolol, like carvedilol, possesses a RyR2-targeted action that suppresses SOICR and SOICR-evoked VTs. Thus, nebivolol represents a promising agent for Ca2+-triggered arrhythmias.

Abstract 459: Oxidized CaMKII Causes Atrial Fibrillation Susceptibility in a Diabetic Mouse Model

Background: Atrial fibrillation (AF) and diabetes mellitus (DM) are major, unsolved public health problems. DM is a known risk factor for AF, and both are associated with increased reactive oxygen species (ROS), suggesting a ROS responsive disease signal could be a mechanistic link between them. The multifunctional Ca2+ and calmodulin-dependent protein kinase-II (CaMKII) is activated by oxidation of paired methionines. Oxidized CaMKII (ox-CaMKII) is increased in atria from DM patients and causes ryanodine receptor (RyR2) hyperphosphorylation that promotes pathological intracellular Ca2+ release and Ca2+ triggered arrhythmias. We hypothesize that DM increases myocardial ox-CaMKII, RyR2 hyperphosphorylation and AF. Methods and Results: C57BL/6J mice with streptozocin-induced type 1 DM had increased AF susceptibility following atrial burst pacing compared with citrate buffer-treated wild-type (WT) controls [70% (14/20) vs. 25% (5/20), p = 0.01]. Ox-CaMKII was increased in atrial tissue from diabetic mice compared to controls, consistent with a role for ox-CaMKII in this model. Diabetic ox-CaMKII resistant knock-in (MM-VV) mice (37.5% (9/24) [p < 0.05]) and diabetic mice with myocardium-restricted transgenic overexpression of methionine sulfoxide reductase A (25% (5/20) [p < 0.05]), which reverses ox-CaMKII, were protected from DM increased AF susceptibility compared to diabetic WT controls. Atrial myocytes from diabetic WT mice demonstrated increased RyR2 mediated Ca2+ spark frequency, triggered action potentials and delayed intracellular [Ca2+] decay compared to controls. Diabetic knock-in mice resistant to CaMKII-mediated RyR2 phosphorylation (S2814A) had decreased AF susceptibility (25% (5/20) [p < 0.05]), compared with diabetic WT mice. All groups of diabetic mice had similar increases in plasma glucose. Conclusions: Hyperglycemia increases AF susceptibility and increased ox-CaMKII is associated with increased AF in this diabetic mouse model. Genetic manipulation of an ox-CaMKII pathway can protect against AF susceptibility in DM. These findings suggest that ox-CaMKII is a critical proarrhythmic signal in DM and a potential therapeutic target for AF management in DM patients.

Abstract 17019: Two Distinct mechanisms by which Na+/Ca2+ dysregulation contributes to Arrhythmogenic Diastolic Ca2+ Release

Na + and Ca 2+ imbalance is associated with triggered arrhythmias resulting from diastolic Ca 2+ release (DCR) from sarcoplasmic reticulum (SR). Recent evidence suggests Na + channel blockade to be a promising therapy for pathologies, including catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the specific mechanism(s) as to how Na + /Ca 2+ dysregulation contribute to arrhythmias is unknown. Confocal microscopy of ventricular myocytes isolated from CPVT mice lacking the cardiac calsequestrin was used to assess Ca 2+ handling response to isoproterenol (Iso) and various pharmacological interventions, while electrocardiograms were acquired during catecholamine challenge to assess the roles of various pools of Na + channels in CPVT. We identify two pools of Na + channels: one composed of cardiac-type Na + channels localized to cell periphery, and a ‘ local pool ’ comprised of neuronal Na + channels colocalizing with RyR2 in the T-tubules. Augmenting function of both Na + channel pools with ATX-II in the presence Iso resulted in SR Ca 2+ overload and activation of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), which precipitated DCR. These, in turn, translated into frequent arrhythmias in CPVT mice. Selectively augmenting function of ‘local pool’ neuronal Na + channels with β-Pompilidotoxin (β-PMTX) precipitated DCR on the cellular level causing frequent arrhythmias during catecholamine challenge in vivo . However, increasing local Na + fluxes reduced SR Ca 2+ load suggesting that local elevation in cytosolic Ca 2+ rather than global SR Ca 2+ overload underlies DCR and arrhythmias under such conditions. These data suggest two distinct mechanisms for Na + /Ca 2+ dysregulation-mediated arrhythmias. The first relies on SR Ca 2+ overload and CaMKII activation and the other on local contribution of Na + -Ca 2+ exchange to DCR. Consideration of these divergent mechanisms may enhance individualized approach to arrhythmia management.