Bupleurum exerts antiarrhythmic effects by inhibiting L-type calcium channels in mouse ventricular myocytes.

BACKGROUND: Bupleurum (Bup), is a traditional effective medicine to treat colds and fevers in clinics. Multiple studies have demonstrated that Bup exhibites various biological activities, including cardioprotective effects, anti-inflammatory, anticancer, antipyretic, antimicrobial, and antiviral effects, etc. Currently, the effects of Bup on cardiac electrophysiology have not been reported yet. METHODS: Electrocardiogram recordings were used to investigate the effects of Bup on aconitine-induced arrhythmias. Patch-clamp techniques were used to explore the effects of Bup on APs and ion currents. RESULTS: Bup reduced the incidence of ventricular fibrillation (VF) and delayed the onset time of ventricular tachycardia (VT) in mice. Additionally, Bup (40 mg/mL) suppressed DADs induced by high-Ca2+ and shortened action potential duration at 50 % completion of repolarization (APD50) and action potential duration at 90 % completion of repolarization (APD90) to 60.89 % ± 8.40 % and 68.94 % ± 3.24 % of the control, respectively. Moreover, Bup inhibited L-type calcium currents (ICa.L) in a dose-dependent manner, with an IC50 value of 25.36 mg/mL. Furthermore, Bup affected the gated kinetics of L-type calcium channels by slowing down steady-state activation, accelerating the steady-state inactivation, and delaying the inactivation-recovery process. However, Bup had no effects on the Transient sodium current (INa.T), ATX II-increased late sodium current (INa.L), transient outward current (Ito), delayed rectifier potassium current (IK), or inward rectifier potassium current (IK1). CONCLUSION: Bup is an antiarrhythmic agent that may exert its antiarrhythmic effects by inhibiting L-type calcium channels.

Injection of IK1 through dynamic clamp can make all the difference in patch-clamp studies on hiPSC-derived cardiomyocytes.

Human-induced stem cell-derived cardiomyocytes (hiPSC-CMs) are a valuable tool for studying development, pharmacology, and (inherited) arrhythmias. Unfortunately, hiPSC-CMs are depolarized and spontaneously active, even the working cardiomyocyte subtypes such as atrial- and ventricular-like hiPSC-CMs, in contrast to the situation in the atria and ventricles of adult human hearts. Great efforts have been made, using many different strategies, to generate more mature, quiescent hiPSC-CMs with more close-to-physiological resting membrane potentials, but despite promising results, it is still difficult to obtain hiPSC-CMs with such properties. The dynamic clamp technique allows to inject a current with characteristics of the inward rectifier potassium current (IK1), computed in real time according to the actual membrane potential, into patch-clamped hiPSC-CMs during action potential measurements. This results in quiescent hiPSC-CMs with a close-to-physiological resting membrane potential. As a result, action potential measurements can be performed with normal ion channel availability, which is particularly important for the physiological functioning of the cardiac SCN5A-encoded fast sodium current (INa). We performed in vitro and in silico experiments to assess the beneficial effects of the dynamic clamp technique in dissecting the functional consequences of the SCN5A-1795insD+/- mutation. In two separate sets of patch-clamp experiments on control hiPSC-CMs and on hiPSC-CMs with mutations in ACADVL and GNB5, we assessed the value of dynamic clamp in detecting delayed afterdepolarizations and in investigating factors that modulate the resting membrane potential. We conclude that the dynamic clamp technique has highly beneficial effects in all of the aforementioned settings and should be widely used in patch-clamp studies on hiPSC-CMs while waiting for the ultimate fully mature hiPSC-CMs.

Effects of apocynin on ISO-induced delayed afterdepolarizations in rat atrial myocytes and the underlying mechanisms.

We aimed to investigate the effect of apocynin (APO) on delayed afterdepolarizations (DADs) in rat atrial myocytes and the underlying mechanisms. Rat atrial myocytes were isolated by a Langendorff perfusion apparatus. DADs were induced by isoproterenol (ISO). Action potentials (APs) and ion currents were recorded by the whole-cell clamp technique. The fluorescent indicator fluo-4 was used to visualize intracellular Ca2+ transients, and western blotting was used to measure the expression of related proteins. The incidence of DADs in rat atrial myocytes increased significantly after ISO treatment, leading to an increased incidence of triggered activity (TA). The incidence of ISO-induced DADs and TA were reduced by 100.0 µM APO from 48.89% to 25.56% and 17.78% to 5.56%, respectively. In the range of 3.0 µM-300.0 µM, the effect of APO was concentration dependent, with a half maximal inhibitory concentration (IC50) of 120.1 µM and a Hill coefficient of 1.063. APO reversed the increase in transient inward current (Iti) and Na+/Ca2+-exchange current (INCX) densities induced by ISO in atrial myocytes. The frequency of spontaneous Ca2+ transients in atrial myocytes was reduced by 100.0 µM APO. Compared with ISO, APO downregulated the expression of NOX2 and increased the phosphorylation of PLNSer16 and the sarcoplasmic reticulum Ca2+-ATPase-2a (SERCA2a) level; however, it had little effect on ryanodine-receptor channel type-2 (RyR2). These findings showed that APO may block Iti and INCX and reduce intracellular Ca2+ levels in rat atrial myocytes, thus reducing the incidence of ISO-induced DADs and TA.

Molecular, Subcellular, and Arrhythmogenic Mechanisms in Genetic RyR2 Disease.

The ryanodine receptor (RyR2) has a critical role in controlling Ca2+ release from the sarcoplasmic reticulum (SR) throughout the cardiac cycle. RyR2 protein has multiple functional domains with specific roles, and four of these RyR2 protomers are required to form the quaternary structure that comprises the functional channel. Numerous mutations in the gene encoding RyR2 protein have been identified and many are linked to a wide spectrum of arrhythmic heart disease. Gain of function mutations (GoF) result in a hyperactive channel that causes excessive spontaneous SR Ca2+ release. This is the predominant cause of the inherited syndrome catecholaminergic polymorphic ventricular tachycardia (CPVT). Recently, rare hypoactive loss of function (LoF) mutations have been identified that produce atypical effects on cardiac Ca2+ handling that has been termed calcium release deficiency syndrome (CRDS). Aberrant Ca2+ release resulting from both GoF and LoF mutations can result in arrhythmias through the Na+/Ca2+ exchange mechanism. This mini-review discusses recent findings regarding the role of RyR2 domains and endogenous regulators that influence RyR2 gating normally and with GoF/LoF mutations. The arrhythmogenic consequences of GoF/LoF mutations will then be discussed at the macromolecular and cellular level.

Sarcoplasmic Reticulum Calcium Release Is Required for Arrhythmogenesis in the Mouse.

Dysfunctional sarcoplasmic reticulum Ca2+ handling is commonly observed in heart failure, and thought to contribute to arrhythmogenesis through several mechanisms. Some time ago we developed a cardiomyocyte-specific inducible SERCA2 knockout mouse, which is remarkable in the degree to which major adaptations to sarcolemmal Ca2+ entry and efflux overcome the deficit in SR reuptake to permit relatively normal contractile function. Conventionally, those adaptations would also be expected to dramatically increase arrhythmia susceptibility. However, that susceptibility has never been tested, and it is possible that the very rapid repolarization of the murine action potential (AP) allows for large changes in sarcolemmal Ca2+ transport without substantially disrupting electrophysiologic stability. We investigated this hypothesis through telemetric ECG recording in the SERCA2-KO mouse, and patch-clamp electrophysiology, Ca2+ imaging, and mathematical modeling of isolated SERCA2-KO myocytes. While the SERCA2-KO animals exhibit major (and unique) electrophysiologic adaptations at both the organ and cell levels, they remain resistant to arrhythmia. A marked increase in peak L-type calcium (I CaL) current and slowed I CaL decay elicited pronounced prolongation of initial repolarization, but faster late repolarization normalizes overall AP duration. Early afterdepolarizations were seldom observed in KO animals, and those that were observed exhibited a mechanism intermediate between murine and large mammal dynamical properties. As expected, spontaneous SR Ca2+ sparks and waves were virtually absent. Together these findings suggest that intact SR Ca2+ handling is an absolute requirement for triggered arrhythmia in the mouse, and that in its absence, dramatic changes to the major inward currents can be resisted by the substantial K+ current reserve, even at end-stage disease.

Two-hit mechanism of cardiac arrhythmias in diabetic hyperglycaemia: reduced repolarization reserve, neurohormonal stimulation, and heart failure exacerbate susceptibility.

AIMS: Diabetic hyperglycaemia is associated with increased arrhythmia risk. We aimed to investigate whether hyperglycaemia alone can be accountable for arrhythmias or whether it requires the presence of additional pathological factors. METHODS AND RESULTS: Action potentials (APs) and arrhythmogenic spontaneous diastolic activities were measured in isolated murine ventricular, rabbit atrial, and ventricular myocytes acutely exposed to high glucose. Acute hyperglycaemia increased the short-term variability (STV) of action potential duration (APD), enhanced delayed afterdepolarizations, and the inducibility of APD alternans during tachypacing in both murine and rabbit atrial and ventricular myocytes. Hyperglycaemia also prolonged APD in mice and rabbit atrial cells but not in rabbit ventricular myocytes. However, rabbit ventricular APD was more strongly depressed by block of late Na+ current (INaL) during hyperglycaemia, consistent with elevated INaL in hyperglycaemia. All the above proarrhythmic glucose effects were Ca2+-dependent and abolished by CaMKII inhibition. Importantly, when the repolarization reserve was reduced by pharmacological inhibition of K+ channels (either Ito, IKr, IKs, or IK1) or hypokalaemia, acute hyperglycaemia further prolonged APD and further increased STV and alternans in rabbit ventricular myocytes. Likewise, when rabbit ventricular myocytes were pretreated with isoproterenol or angiotensin II, hyperglycaemia significantly prolonged APD, increased STV and promoted alternans. Moreover, acute hyperglycaemia markedly prolonged APD and further enhanced STV in failing rabbit ventricular myocytes. CONCLUSION: We conclude that even though hyperglycaemia alone can enhance cellular proarrhythmic mechanisms, a second hit which reduces the repolarization reserve or stimulates G protein-coupled receptor signalling greatly exacerbates cardiac arrhythmogenesis in diabetic hyperglycaemia.

Isoliensinine Eliminates Afterdepolarizations Through Inhibiting Late Sodium Current and L-Type Calcium Current.

Isoliensinine (IL) extracted from lotus seed has a good therapeutic effect on cardiovascular diseases. However, its effect on ion channels of ventricular myocytes is still unclear. We used whole-cell patch-clamp techniques to detect the effects of IL on transmembrane ion currents and action potential (AP) in isolated rabbit left ventricular myocytes. IL inhibited the transient sodium current (INaT), late sodium current (INaL) enlarged by sea anemone toxin (ATX II) and L-type calcium current (ICaL) in a concentration-dependent manner without affecting inward rectifier potassium current (IK1) and delayed rectifier potassium current (IK). These inhibitory effects are mainly manifested as reduced the AP amplitude (APA) and maximum depolarization velocity (Vmax) and shortened the action potential duration (APD), but had no significant effect on the resting membrane potential (RMP). Moreover, IL significantly eliminated ATX II-induced early afterdepolarizations (EADs) and high extracellular calcium-induced delayed afterdepolarizations (DADs). These results revealed that IL effectively eliminated EADs and DADs through inhibiting INaL and ICaL in ventricular myocytes, which indicates it has potential antiarrhythmic action.

Afterdepolarizations and abnormal calcium handling in atrial myocytes with modulated SERCA uptake: a sensitivity analysis of calcium handling channels.

Delayed afterdepolarizations (DADs) and spontaneous depolarizations (SDs) are typically triggered by spontaneous diastolic Ca2+ release from the sarcoplasmic reticulum (SR) which is caused by an elevated SR Ca2+-ATPase (SERCA) uptake and dysfunctional ryanodine receptors. However, recent studies on the T-box transcription factor gene (TBX5) demonstrated that abnormal depolarizations could occur despite a reduced SERCA uptake. Similar findings have also been reported in experimental or clinical studies of diabetes and heart failure. To investigate the sensitivity of SERCA in the genesis of DADs/SDs as well as its dependence on other Ca2+ handling channels, we performed systematic analyses using the Maleckar et al. model. Results showed that the modulation of SERCA alone cannot trigger abnormal depolarizations, but can instead affect the interdependency of other Ca2+ handling channels in triggering DADs/SDs. Furthermore, we discovered the existence of a threshold value for the intracellular concentration of Ca2+ ([Ca2+]i) for abnormal depolarizations, which is modulated by the maximum SERCA uptake and the concentration of Ca2+ in the uptake and release compartments in the SR ([Ca2+]up and [Ca2+]rel). For the first time, our modelling study reconciles different mechanisms of abnormal depolarizations in the setting of 'lone' AF, reduced TBX5, diabetes and heart failure, and may lead to more targeted treatment for these patients. This article is part of the theme issue 'Uncertainty quantification in cardiac and cardiovascular modelling and simulation'.

Delayed afterdepolarization-induced triggered activity in cardiac purkinje cells mediated through cytosolic calcium diffusion waves.

Cardiac Purkinje cells (PCs) are more susceptible to action potential abnormalities as compared to ventricular myocytes (VMs), which could be associated with their distinct intracellular calcium handling. We developed a detailed biophysical model of a mouse cardiac PC, which importantly reproduces the experimentally observed biphasic cytosolic calcium waves. The model includes a stochastic gating formulation for the opening and closing of ryanodine receptor (RyR) channels, simulated with a Monte Carlo method, to accurately reproduce cytosolic calcium wave propagation and the effects of spontaneous calcium release events. Simulations predict that during an action potential, smaller cytosolic calcium wavelets propagated from the sarcolemma towards the center of the cell and initiated larger magnitude cell-wide calcium waves via a calcium-induced-calcium release mechanism. In the presence of RyR mutations, frequent spontaneous calcium leaks from sarcoplasmic reticulum (SR) initiated calcium waves, which upon reaching the cell periphery produced delayed afterdepolarizations (DADs) via sodium-calcium exchanger (NCX) and T-type calcium (ICaT ) channel activation. In the presence of isoproterenol-mediated effects, DADs induced triggered activity by reactivation of fast sodium channels. Based on our model, we found that the activation of either L-type calcium channels (ICaL ), ICaT , sodium-potassium exchanger (INaK ) or NCX is sufficient for occurrence of triggered activity; however, a partial blockade of ICaT or INaK is essential for its successful termination. Our modeling study highlights valuable insights into the mechanisms of DAD-induced triggered activity mediated via cytosolic calcium waves in cardiac PCs and may elucidate the increased arrhythmogeneity in PCs.

Ranolazine is an Effective and Safe Treatment of Adults with Symptomatic Premature Ventricular Contractions due to Triggered Ectopy.

Early and delayed afterdepolarizations (EAD/DAD) cause triggered ventricular ectopy. Because ranolazine (RAN) suppresses EAD/DAD, we postulated that RAN might be effective in reducing premature ventricular contractions (PVCs). To assess the effect of RAN in patients with symptomatic PVCs due to triggered ectopy and its safety and tolerability. A total of 59 patients with symptomatic PVCs were identified from full-disclosure Holters. Doses of 500 and 1,000 mg offlabel RAN, daily, were given to 34 and 66% patients, respectively, and repeat Holters were performed prospectively during mean followup of 3.1 months. The two Holters were retrospectively compared. Congestive heart failure (CHF) was defined as symptoms including dyspnea, orthopnea, paroxysmal nocturnal dyspnea, and fatigue, with a brain natriuretic peptide > 400. Systolic (heart failure with reduced ejection fraction) versus diastolic (heart failure with preserved ejection fraction, HFpEF) CHF depended upon an echocardiographic left ventricular ejection fraction (LVEF) at least 50% by apical two- and four-chamber Simpson's method (HFpEF). The mean age of the patients was 63 years, 60% were males, mean left ventricular ejection fraction was 60%, with 34% having coronary artery disease, 73% were hypertensive, 24% had type 2 diabetic, and 34% were on beta blockers. Upon repeat Holters at a mean of 3.1 months after initiating RAN, 95% (56/59) of the patients had their PVC count reduced as follows: 24% (14/59) had more than 90% decrease, 34% (20/59) had 71 to 90% decrease, and 17% (10/59) had 50 to 70% decrease. In the entire group, RAN reduced PVCs by 71% (mean: 13,329 to 3,837; p < 0.001). Ventricular bigeminy was reduced by 80% (4,168 to 851; p < 0.001), ventricular coupletswere reduced by 78% (374 to 81; p < 0.001), and ventricular tachycardiawas reduced by 91% (56 to 5; p < 0.001). The PVC reduction was dose dependent. Off-label RAN offers an effective and safe pharmacologic treatment for symptomatic triggered PVCs. A large, prospective randomized study is needed.

Mutation E169K in junctophilin-2 causes atrial fibrillation due to impaired RyR2 stabilization.

OBJECTIVES: This study sought to study the role of junctophilin-2 (JPH2) in atrial fibrillation (AF). BACKGROUND: JPH2 is believed to have an important role in sarcoplasmic reticulum (SR) Ca(2+) handling and modulation of ryanodine receptor Ca(2+) channels (RyR2). Whereas defective RyR2-mediated Ca(2+) release contributes to the pathogenesis of AF, nothing is known about the potential role of JPH2 in atrial arrhythmias. METHODS: Screening 203 unrelated hypertrophic cardiomyopathy patients uncovered a novel JPH2 missense mutation (E169K) in 2 patients with juvenile-onset paroxysmal AF (pAF). Pseudoknock-in (PKI) mouse models were generated to determine the molecular defects underlying the development of AF caused by this JPH2 mutation. RESULTS: PKI mice expressing E169K mutant JPH2 exhibited a higher incidence of inducible AF than wild type (WT)-PKI mice, whereas A399S-PKI mice expressing a hypertrophic cardiomyopathy-linked JPH2 mutation not associated with atrial arrhythmias were not significantly different from WT-PKI. E169K-PKI but not A399A-PKI atrial cardiomyocytes showed an increased incidence of abnormal SR Ca(2+) release events. These changes were attributed to reduced binding of E169K-JPH2 to RyR2. Atrial JPH2 levels in WT-JPH2 transgenic, nontransgenic, and JPH2 knockdown mice correlated negatively with the incidence of pacing-induced AF. Ca(2+) spark frequency in atrial myocytes and the open probability of single RyR2 channels from JPH2 knockdown mice was significantly reduced by a small JPH2-mimicking oligopeptide. Moreover, patients with pAF had reduced atrial JPH2 levels per RyR2 channel compared to sinus rhythm patients and an increased frequency of spontaneous Ca(2+) release events. CONCLUSIONS: Our data suggest a novel mechanism by which reduced JPH2-mediated stabilization of RyR2 due to loss-of-function mutation or reduced JPH2/RyR2 ratios can promote SR Ca(2+) leak and atrial arrhythmias, representing a potential novel therapeutic target for AF.

The effects of omega-3 polyunsaturated fatty acids on cardiac rhythm: a critical reassessment.

Although epidemiological studies provide strong evidence for an inverse relationship between omega-3 polyunsaturated fatty acids (n-3 PUFAs) and cardiac mortality, inconsistent and often conflicting results have been obtained from both animal studies and clinical prevention trials. Despite these heterogeneous results, some general conclusions can be drawn from these studies: 1) n-PUFAs have potent effects on ion channels and calcium regulatory proteins that vary depending on the route of administration. Circulating (acute administration) n-3 PUFAs affect ion channels directly while incorporation (long-term supplementation) of these lipids into cell membranes indirectly alter cardiac electrical activity via alteration of membrane properties. 2) n-3 PUFAs reduce baseline HR and increase HRV via alterations in intrinsic pacemaker rate rather than from changes in cardiac autonomic neural regulation. 3) n-3 PUFAs may be only effective if given before electrophysiological or structural remodeling has begun and have no efficacy against atrial fibrillation. 5) Despite initial encouraging results, more recent clinical prevention and animal studies have not only failed to reduce sudden cardiac death but actually increased mortality in angina patients and increased rather than decreased malignant arrhythmias in animal models of regional ischemia. 6) Given the inconsistent benefits reported in clinical and experimental studies and the potential adverse actions on cardiac rhythm noted during myocardial ischemia, n-3 PUFA must be prescribed with caution and generalized recommendations to increase fish intake or to take n-3 PUFA supplements need to be reconsidered.

Modeling Catecholaminergic Polymorphic Ventricular Tachycardia using Induced Pluripotent Stem Cell-derived Cardiomyocytes.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic cardiac disorder characterized by life-threatening arrhythmias induced by physical or emotional stress, in the absence structural heart abnormalities. The arrhythmias may cause syncope or degenerate into cardiac arrest and sudden death which usually occurs during childhood. Recent studies have shown that CPVT is caused by mutations in the cardiac ryanodine receptor type 2 (RyR2) or calsequestrin 2 (CASQ2) genes. Both proteins are key contributors to the intracellular Ca(2+) handling process and play a pivotal role in Ca(2+) release from the sarcoplasmic reticulum to the cytosol during systole. Although the molecular pathogenesis of CPVT is not entirely clear, it was suggested that the CPVT mutations promote excessive sarcoplasmic reticulum Ca(2+) leak, which initiates delayed afterdepolarizations (DADs) and triggered arrhythmias in cardiac myocytes. The recent breakthrough discovery of induced pluripotent stem cells (iPSC) generated from somatic cells (e.g. fibroblasts, keratinocytes) now enables researches to investigate mutated cardiomyocytes generated from the patient's iPSC. To this end, in the present article we review recent studies on CPVT iPSC-derived cardiomyocytes, thus demonstrating in the mutated cells catecholamine-induced DADs and triggered arrhythmias.