Divergent Biochemical Properties and Disparate Impact of Arrhythmogenic Calmodulin Mutations on Zebrafish Cardiac Function.

Calmodulin (CaM) is a ubiquitous, small cytosolic calcium (Ca2+)-binding sensor that plays a vital role in many cellular processes by binding and regulating the activity of over 300 protein targets. In cardiac muscle, CaM modulates directly or indirectly the activity of several proteins that play a key role in excitation-contraction coupling (ECC), such as ryanodine receptor type 2 (RyR2), l-type Ca2+ (Cav1.2), sodium (NaV1.5) and potassium (KV7.1) channels. Many recent clinical and genetic studies have reported a series of CaM mutations in patients with life-threatening arrhythmogenic syndromes, such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). We recently showed that four arrhythmogenic CaM mutations (N98I, D132E, D134H, and Q136P) significantly reduce the binding of CaM to RyR2. Herein, we investigate in vivo functional effects of these CaM mutations on the normal zebrafish embryonic heart function by microinjecting complementary RNA corresponding to CaMN98I, CaMD132E, CaMD134H, and CaMQ136P mutants. Expression of CaMD132E and CaMD134H mutants results in significant reduction of the zebrafish heart rate, mimicking a severe form of human bradycardia, whereas expression of CaMQ136P results in an increased heart rate mimicking human ventricular tachycardia. Moreover, analysis of cardiac ventricular rhythm revealed that the CaMD132E and CaMN98I zebrafish groups display an irregular pattern of heart beating and increased amplitude in comparison to the control groups. Furthermore, circular dichroism spectroscopy experiments using recombinant CaM proteins reveals a decreased structural stability of the four mutants compared to the wild-type CaM protein in the presence of Ca2+. Finally, Ca2+-binding studies indicates that all CaM mutations display reduced CaM Ca2+-binding affinities, with CaMD132E exhibiting the most prominent change. Our data suggest that CaM mutations can trigger different arrhythmogenic phenotypes through multiple and complex molecular mechanisms.

Dexmedetomidine inhibited arrhythmia susceptibility to adrenergic stress in RyR2R2474S mice through regulating the coupling of membrane potential and intracellular calcium.

BACKGROUND: Dexmedetomidine (DEX), a highly selective α2-adrenoceptor agonist, can decrease the incidence of arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the underlying mechanisms by which DEX affects cardiac electrophysiological function remain unclear. METHODS: Ryanodine receptor (RyR2) heterozygous R2474S mice were used as a model for CPVT. WT and RyR2R2474S/+ mice were treated with isoproterenol (ISO) and DEX, and electrocardiograms were continuously monitored during both in vivo and ex vivo experiments. Dual-dye optical mapping was used to explore the anti-arrhythmic mechanism of DEX. RESULTS: DEX significantly reduced the occurrence and duration of ISO-induced of VT/VF in RyR2R2474S/+ mice in vivo and ex vivo. DEX remarkably prolonged action potential duration (APD80) and calcium transient duration (CaTD80) in both RyR2R2474S/+ and WT hearts, whereas it reduced APD heterogeneity and CaT alternans in RyR2R2474S/+ hearts. DEX inhibited ectopy and reentry formation, and stabilized voltage-calcium latency. CONCLUSION: DEX exhibited an antiarrhythmic effect through stabilizing membrane voltage and intracellular Ca2+. DEX can be used as a beneficial perioperative anesthetic for patients with CPVT or other tachy-arrhythmias.

RyR2 C-terminal truncating variants identified in patients with arrhythmic phenotypes exert a dominant negative effect through formation of wildtype-truncation heteromers.

Gain-of-function missense variants in the cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), whereas RyR2 loss-of-function missense variants cause Ca2+ release deficiency syndrome (CRDS). Recently, truncating variants in RyR2 have also been associated with ventricular arrhythmias (VAs) and sudden cardiac death. However, there are limited insights into the potential clinical relevance and in vitro functional impact of RyR2 truncating variants. We performed genetic screening of patients presenting with syncope, VAs, or unexplained sudden death and in vitro characterization of the expression and function of RyR2 truncating variants in HEK293 cells. We identified two previously unknown RyR2 truncating variants (Y4591Ter and R4663Ter) and one splice site variant predicted to result in a frameshift and premature termination (N4717 + 15Ter). These 3 new RyR2 truncating variants and a recently reported RyR2 truncating variant, R4790Ter, were generated and functionally characterized in vitro. Immunoprecipitation and immunoblotting analyses showed that all 4 RyR2 truncating variants formed heteromers with the RyR2-wildtype (WT) protein. Each of these C-terminal RyR2 truncations was non-functional and suppressed [3H]ryanodine binding to RyR2-WT and RyR2-WT mediated store overload induced spontaneous Ca2+ release activity in HEK293 cells. The expression of these RyR2 truncating variants in HEK293 cells was markedly reduced compared with that of the full-length RyR2 WT protein. Our data indicate that C-terminal RyR2 truncating variants are non-functional and can exert a dominant negative impact on the function of the RyR2 WT protein through formation of heteromeric WT/truncation complex.

In Vivo Pharmacokinetic and Pharmacodynamic Properties of the Antiarrhythmic Molecule ent-Verticilide.

The unnatural verticilide enantiomer (ent-verticilide) is a selective and potent inhibitor of cardiac ryanodine receptor (RyR2) calcium release channels and exhibits antiarrhythmic activity in a murine model of catecholaminergic polymorphic ventricular tachycardia (CPVT). To determine verticilide's pharmacokinetic and pharmacodynamic properties in vivo, we developed a bioassay to measure nat- and ent-verticilide in murine plasma and correlated plasma concentrations with antiarrhythmic efficacy in a mouse model of CPVT. nat-Verticilide rapidly degraded in plasma in vitro, showing >95% degradation within 5 minutes, whereas ent-verticilide showed <1% degradation over 6 hours. Plasma was collected from mice following intraperitoneal administration of ent-verticilide at two doses (3 mg/kg, 30 mg/kg). Peak C max and area under the plasma-concentration time curve (AUC) scaled proportionally to dose, and the half-life was 6.9 hours for the 3-mg/kg dose and 6.4 hours for the 30-mg/kg dose. Antiarrhythmic efficacy was examined using a catecholamine challenge protocol at time points ranging from 5 to 1440 minutes after intraperitoneal dosing. ent-Verticilide inhibited ventricular arrhythmias as early as 7 minutes after administration in a concentration-dependent manner, with an estimated potency (IC50) of 266 ng/ml (312 nM) and an estimated maximum inhibitory effect of 93.5%. Unlike the US Food and Drug Administration-approved pan-RyR blocker dantrolene, the RyR2-selective blocker ent-verticilide (30 mg/kg) did not reduce skeletal muscle strength in vivo. We conclude that ent-verticilide has favorable pharmacokinetic properties and reduces ventricular arrhythmias with an estimated potency in the nanomolar range, warranting further drug development. SIGNIFICANCE STATEMENT: ent-Verticilide has therapeutic potential to treat cardiac arrhythmias, but little is known about its pharmacological profile in vivo. The primary purpose of this study is to determine the systemic exposure and pharmacokinetics of ent-verticilide in mice and estimate its efficacy and potency in vivo. The current work suggests ent-verticilide has favorable pharmacokinetic properties and reduces ventricular arrhythmias with an estimated potency in the nanomolar range, warranting further drug development.

Impaired Binding to Junctophilin-2 and Nanostructural Alteration in CPVT Mutation.

[Figure: see text].

RYR2 Channel Inhibition Is the Principal Mechanism of Flecainide Action in CPVT.

RATIONALE: The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive RyR2 (cardiac ryanodine receptor) mediated calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro, reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide's efficacy in CPVT. OBJECTIVE: To determine whether RyR2 block independently contributes to flecainide's efficacy for suppressing spontaneous sarcoplasmic reticulum Ca release and for preventing ventricular tachycardia in vivo. METHODS AND RESULTS: We synthesized N-methylated flecainide analogues (QX-flecainide and N-methyl flecainide) and showed that N-methylation reduces flecainide's inhibitory potency on RyR2 channels incorporated into artificial lipid bilayers. N-methylation did not alter flecainide's inhibitory activity on human cardiac sodium channels expressed in HEK293T cells. Antiarrhythmic efficacy was tested utilizing a Casq2 (cardiac calsequestrin) knockout (Casq2-/-) CPVT mouse model. In membrane-permeabilized Casq2-/- cardiomyocytes-lacking intact sarcolemma and devoid of sodium channel contribution-flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2-/- cardiomyocytes pretreated with tetrodotoxin to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous sarcoplasmic reticulum Ca release, while QX-flecainide and N-methyl flecainide did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2-/- mice, whereas N-methyl flecainide had no significant effect on arrhythmia burden, despite comparable sodium channel block. CONCLUSIONS: Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone did not prevent ventricular tachycardia. Hence, RyR2 channel inhibition likely constitutes the principal mechanism of antiarrhythmic action of flecainide in CPVT.

Therapeutic Approaches of Ryanodine Receptor-Associated Heart Diseases.

Cardiac diseases are the leading causes of death, with a growing number of cases worldwide, posing a challenge for both healthcare and research. Therefore, the most relevant aim of cardiac research is to unravel the molecular pathomechanisms and identify new therapeutic targets. Cardiac ryanodine receptor (RyR2), the Ca2+ release channel of the sarcoplasmic reticulum, is believed to be a good therapeutic target in a group of certain heart diseases, collectively called cardiac ryanopathies. Ryanopathies are associated with the impaired function of the RyR, leading to heart diseases such as congestive heart failure (CHF), catecholaminergic polymorphic ventricular tachycardia (CPVT), arrhythmogenic right ventricular dysplasia type 2 (ARVD2), and calcium release deficiency syndrome (CRDS). The aim of the current review is to provide a short insight into the pathological mechanisms of ryanopathies and discuss the pharmacological approaches targeting RyR2.

Molecular remodeling of Cx43, but not structural remodeling, promotes arrhythmias in an arrhythmogenic canine model of nonischemic heart failure.

BACKGROUND: Both gap junctional remodeling and interstitial fibrosis have been linked to impaired electrical conduction velocity (CV) and fatal ventricular arrhythmias in nonischemic heart failure (HF). However, the arrhythmogenic role of the ventricular gap junctional Cx43 in nonischemic HF remains in debate. Here, we assessed this in a newly developed arrhythmogenic canine model of nonischemic HF. METHODS AND RESULTS: Nonischemic HF was induced in canines by combined aortic valve insufficiency and aortic constriction. Left ventricular (LV) myocardium from HF dogs showed similar pathological changes to that of humans. HF dogs had reduced LV function, widened QRS complexes, and spontaneous nonsustained ventricular tachycardia. CV was measured in intact LV epicardium with high-density grid mapping. Total (Cx43-T) and nonphosphorylated Cx43 (Cx43-NP) and histological interstitial fibrosis were assessed from these mapped LV tissues. Longitudinal CV, which was slowed in HF (49 ± 1 vs. 65 ± 2 cm/s in Ctl), was positively correlated with reduced total junctional Cx43 and negatively correlated with markedly increased junctional Cx43-NP (2-fold) in HF. Cx43 dephosphorylation in HF was associated with enhanced colocalization of PP2A at the level of Cx43. Unchanged action potential upstroke and transverse CV were associated with unaltered Cx43 lateralization and interstitial fibrosis in the nonischemic HF canine LV. CONCLUSION: Our unique arrhythmogenic canine model of HF resembles human nonischemic HF (prior to the end stage). Cx43 remodeling occurs prior to the structural remodeling (with lack of fibrosis) in HF and it is crucial in slowed CV and ventricular arrhythmia development. Our findings suggest that altered Cx43 alone is arrhythmogenic and modulation of Cx43 has the anti-arrhythmic therapeutic potential for HF patients.

Integrin ß1D Deficiency-Mediated RyR2 Dysfunction Contributes to Catecholamine-Sensitive Ventricular Tachycardia in Arrhythmogenic Right Ventricular Cardiomyopathy.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary heart disease characterized by fatty infiltration, life-threatening arrhythmias, and increased risk of sudden cardiac death. The guideline for management of ARVC in patients is to improve quality of life by reducing arrhythmic symptoms and to prevent sudden cardiac death. However, the mechanism underlying ARVC-associated cardiac arrhythmias remains poorly understood. METHODS: Using protein mass spectrometry analyses, we identified that integrin ß1 is downregulated in ARVC hearts without changes to Ca2+-handling proteins. As adult cardiomyocytes express only the ß1D isoform, we generated a cardiac specific ß1D knockout mouse model and performed functional imaging and biochemical analyses to determine the consequences of integrin ß1D loss on function in the heart in vivo and in vitro. RESULTS: Integrin ß1D deficiency and RyR2 Ser-2030 hyperphosphorylation were detected by Western blotting in left ventricular tissues from patients with ARVC but not in patients with ischemic or hypertrophic cardiomyopathy. Using lipid bilayer patch clamp single channel recordings, we found that purified integrin ß1D protein could stabilize RyR2 function by decreasing RyR2 open probability, mean open time, and increasing mean close time. Also, ß1D knockout mice exhibited normal cardiac function and morphology but presented with catecholamine-sensitive polymorphic ventricular tachycardia, consistent with increased RyR2 Ser-2030 phosphorylation and aberrant Ca2+ handling in ß1D knockout cardiomyocytes. Mechanistically, we revealed that loss of DSP (desmoplakin) induces integrin ß1D deficiency in ARVC mediated through an ERK1/2 (extracellular signal-regulated kinase 1 and 2)-fibronectin-ubiquitin/lysosome pathway. CONCLUSIONS: Our data suggest that integrin ß1D deficiency represents a novel mechanism underlying the increased risk of ventricular arrhythmias in patients with ARVC.

Fibroblast growth factor 23 (FGF23) induces ventricular arrhythmias and prolongs QTc interval in mice in an FGF receptor 4-dependent manner.

Fibroblast growth factor 23 (FGF23) is a phosphate regulating protein hormone released by osteocytes. FGF23 becomes markedly elevated in chronic kidney disease (CKD), for which the leading cause of death is cardiovascular disease, particularly sudden cardiac death. Previously, we found that FGF23 increases intracellular Ca2+ in cardiomyocytes and alters contractility in mouse ventricles ex vivo via FGF receptor 4 (FGFR4). In the present study, we demonstrate that FGF23 induces cardiac arrhythmias and prolongs QTc interval in mice, and we tested whether these effects are mediated through FGFR4. In isolated Langendorff perfused hearts, FGF23 perfusion increased mechanical arrhythmias in the form of premature ventricular beats (PVBs), and induced runs of ventricular tachycardia in 6 of 11 animals, which were attenuated with pretreatment of an anti-FGFR4 blocking antibody. Ex vivo ECG analysis of isolated intact hearts showed increased ventricular arrhythmias and QTc prolongation after FGF23 infusion compared with vehicle. In vivo, injection of FGF23 into the jugular vein led to the emergence of premature ventricular contractions (PVCs) in 5 out of 11 experiments. FGF23 also produced a significant lengthening effect upon QTc interval in vivo. In vivo FGFR4 blockade ameliorated the arrhythmogenic and QTc prolonging effects of FGF23. Finally, FGF23 increased cardiomyocyte Ca2+ levels in intact left ventricular muscle which was inhibited by FGR4 blockade. We conclude that FGF23/FGFR4 signaling in the heart may contribute to ventricular arrhythmogenesis and repolarization disturbances commonly observed in patients with CKD via Ca2+ overload and may be an important therapeutic target to reduce cardiac mortality in CKD.NEW & NOTEWORTHY Here we provide direct evidence that fibroblast growth factor 23 (FGF23), a phosphaturic hormone elevated in chronic kidney disease, is proarrhythmic. FGF23 acutely triggered ventricular arrhythmias and prolonged corrected QT interval (QTc) in isolated mouse hearts and in vivo. FGF23 also increased Ca2+ levels in ventricular muscle tissue. Blockade of the FGF receptor 4 signaling pathway using a monoclonal antibody ameliorated ventricular arrhythmias, QTc prolongation, and elevated ventricular Ca2+ induced by FGF23, and may represent a potential therapeutic target in chronic kidney disease.

In vitro and in vivo cardiac toxicity of flavored electronic nicotine delivery systems.

The usage of flavored electronic nicotine delivery systems (ENDS) is popular, specifically in the teen and young adult age-groups. The possible cardiac toxicity of the flavoring aspect of ENDS is largely unknown. Vaping, a form of electronic nicotine delivery, uses "e-liquid" to generate "e-vapor," an aerosolized mixture of nicotine and/or flavors. We report our investigation into the cardiotoxic effects of flavored e-liquids. E-vapors containing flavoring aldehydes such as vanillin and cinnamaldehyde, as indicated by mass spectrometry, were more toxic in HL-1 cardiomyocytes than fruit-flavored e-vapor. Exposure of human induced pluripotent stem cell-derived cardiomyocytes to cinnamaldehyde or vanillin-flavored e-vapor affected the beating frequency and prolonged the field potential duration of these cells more than fruit-flavored e-vapor. In addition, vanillin aldehyde-flavored e-vapor reduced the human ether-à-go-go-related gene (hERG)-encoded potassium current in transfected human embryonic kidney cells. In mice, inhalation exposure to vanillin aldehyde-flavored e-vapor for 10 wk caused increased sympathetic predominance in heart rate variability measurements. In vivo inducible ventricular tachycardia was significantly longer, and in optical mapping, the magnitude of ventricular action potential duration alternans was significantly larger in the vanillin aldehyde-flavored e-vapor-exposed mice than in controls. We conclude that the widely popular flavored ENDS are not harm free, and they have a potential for cardiac harm. More studies are needed to further assess their cardiac safety profile and long-term health effects.NEW & NOTEWORTHY The use of electronic nicotine delivery systems (ENDS) is not harm free. It is not known whether ENDS negatively affect cardiac electrophysiological function. Our study in cell lines and in mice shows that ENDS can compromise cardiac electrophysiology, leading to action potential instability and inducible ventricular arrhythmias. Further investigations are necessary to assess the long-term cardiac safety profile of ENDS products in humans and to better understand how individual components of ENDS affect cardiac toxicity.

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. The aim of this study was to test whether the suppression of RyR2 activity caused by inhibition of CaMKII increases propensity for Ca2+ alternans. 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 ß-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. In conclusion, 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.NEW & NOTEWORTHY Genetically increased RyR2 activity promotes arrhythmogenic Ca2+ release. Inhibition of CaMKII suppresses RyR2 activity and arrhythmogenic Ca2+ release. Suppression of RyR2 activity prolongs refractoriness of Ca2+ release. Prolonged refractoriness of Ca2+ release leads to arrhythmogenic Ca2+ alternans. CaMKII inhibition promotes Ca2+ alternans by prolonging Ca2+ release refractoriness.

Macrophage depletion in stellate ganglia alleviates cardiac sympathetic overactivation and ventricular arrhythmogenesis by attenuating neuroinflammation in heart failure.

Cardiac sympathetic overactivation is involved in arrhythmogenesis in patients with chronic heart failure (CHF). Inflammatory infiltration in the stellate ganglion (SG) is a critical factor for cardiac sympathoexcitation in patients with ventricular arrhythmias. This study aims to investigate if macrophage depletion in SGs decreases cardiac sympathetic overactivation and ventricular arrhythmogenesis in CHF. Surgical ligation of the coronary artery was used for induction of CHF. Clodronate liposomes were microinjected into bilateral SGs of CHF rats for macrophage depletion. Using cytokine array, immunofluorescence staining, and Western blot analysis, we found that macrophage expansion and expression of TNFα and IL-1ß in SGs were markedly increased in CHF rats. Flow cytometry data confirmed that the percentage of macrophages in SGs was higher in CHF rats than that in sham rats. Clodronate liposomes significantly reduced CHF-elevated proinflammatory cytokine levels and macrophage expansion in SGs. Clodronate liposomes also reduced CHF-increased N-type Ca2+ currents and excitability of cardiac sympathetic postganglionic neurons and inhibited CHF-enhanced cardiac sympathetic nerve activity. ECG data from 24-h, continuous telemetry recording in conscious rats demonstrated that clodronate liposomes not only restored CHF-induced heterogeneity of ventricular electrical activities, but also decreased the incidence and duration of ventricular tachycardia/fibrillation in CHF. Macrophage depletion with clodronate liposomes attenuated CHF-induced cardiac sympathetic overactivation and ventricular arrhythmias through reduction of macrophage expansion and neuroinflammation in SGs.

Ca2+-dependent modulation of voltage-gated myocyte sodium channels.

Voltage-dependent Na+ channel activation underlies action potential generation fundamental to cellular excitability. In skeletal and cardiac muscle this triggers contraction via ryanodine-receptor (RyR)-mediated sarcoplasmic reticular (SR) Ca2+ release. We here review potential feedback actions of intracellular [Ca2+] ([Ca2+]i) on Na+ channel activity, surveying their structural, genetic and cellular and functional implications, translating these to their possible clinical importance. In addition to phosphorylation sites, both Nav1.4 and Nav1.5 possess potentially regulatory binding sites for Ca2+ and/or the Ca2+-sensor calmodulin in their inactivating III-IV linker and C-terminal domains (CTD), where mutations are associated with a range of skeletal and cardiac muscle diseases. We summarize in vitro cell-attached patch clamp studies reporting correspondingly diverse, direct and indirect, Ca2+ effects upon maximal Nav1.4 and Nav1.5 currents (Imax) and their half-maximal voltages (V1/2) characterizing channel gating, in cellular expression systems and isolated myocytes. Interventions increasing cytoplasmic [Ca2+]i down-regulated Imax leaving V1/2 constant in native loose patch clamped, wild-type murine skeletal and cardiac myocytes. They correspondingly reduced action potential upstroke rates and conduction velocities, causing pro-arrhythmic effects in intact perfused hearts. Genetically modified murine RyR2-P2328S hearts modelling catecholaminergic polymorphic ventricular tachycardia (CPVT), recapitulated clinical ventricular and atrial pro-arrhythmic phenotypes following catecholaminergic challenge. These accompanied reductions in action potential conduction velocities. The latter were reversed by flecainide at RyR-blocking concentrations specifically in RyR2-P2328S as opposed to wild-type hearts, suggesting a basis for its recent therapeutic application in CPVT. We finally explore the relevance of these mechanisms in further genetic paradigms for commoner metabolic and structural cardiac disease.

Excess ischemic tachyarrhythmias trigger protection against myocardial infarction in hypertensive rats.

Increased level of C-reactive protein (CRP) is a risk factor for cardiovascular diseases, including myocardial infarction and hypertension. Here, we analyzed the effects of CRP overexpression on cardiac susceptibility to ischemia/reperfusion (I/R) injury in adult spontaneously hypertensive rats (SHR) expressing human CRP transgene (SHR-CRP). Using an in vivo model of coronary artery occlusion, we found that transgenic expression of CRP predisposed SHR-CRP to repeated and prolonged ventricular tachyarrhythmias. Excessive ischemic arrhythmias in SHR-CRP led to a significant reduction in infarct size (IS) compared with SHR. The proarrhythmic phenotype in SHR-CRP was associated with altered heart and plasma eicosanoids, myocardial composition of fatty acids (FAs) in phospholipids, and autonomic nervous system imbalance before ischemia. To explain unexpected IS-limiting effect in SHR-CRP, we performed metabolomic analysis of plasma before and after ischemia. We also determined cardiac ischemic tolerance in hearts subjected to remote ischemic perconditioning (RIPer) and in hearts ex vivo. Acute ischemia in SHR-CRP markedly increased plasma levels of multiple potent cardioprotective molecules that could reduce IS at reperfusion. RIPer provided IS-limiting effect in SHR that was comparable with myocardial infarction observed in naïve SHR-CRP. In hearts ex vivo, IS did not differ between the strains, suggesting that extra-cardiac factors play a crucial role in protection. Our study shows that transgenic expression of human CRP predisposes SHR-CRP to excess ischemic ventricular tachyarrhythmias associated with a drop of pump function that triggers myocardial salvage against lethal I/R injury likely mediated by protective substances released to blood from hypoxic organs and tissue at reperfusion.

Resveratrol Confers Protection Against Ischemia/Reperfusion Injury by Increase of Angiotensin (1-7) Expression in a Rat Model of Myocardial Hypertrophy.

ABSTRACT: Left ventricular hypertrophy (LVH) makes the heart vulnerable to ischemia/reperfusion (IR) injury. Angiotensin (Ang) (1-7) is recognized as a cardioprotective peptide. We investigated the effect of polyphenol resveratrol on myocardial IR injury after hypertrophy and examined cardiac content of Ang (1-7) and transcription of its receptor (MasR). Rats were divided into sham-operated, LVH, IR, LVH + IR, and resveratrol + LVH + IR groups. Myocardial hypertrophy and IR models were created by abdominal aortic banding and left coronary artery occlusion, respectively. To evaluate the electrocardiogram parameters and incidence of arrhythmias, electrocardiogram was recorded by subcutaneous leads (lead II). Blood pressure was measured through the left carotid artery. Infarct size was determined by the triphenyl tetrazolium chloride staining. The Ang (1-7) level was evaluated by immunohistochemistry. The Mas receptor mRNA level was assessed by the real-time real time reverse transcription polymerase chain reaction technique. QT-interval duration, infarct size, and incidence of ischemia-induced arrhythmia were significantly higher in the LVH + IR group. However, in the resveratrol-treated group, these parameters were decreased significantly. The cardiac level of Ang (1-7) was decreased in untreated hypertrophied hearts (LVH and LVH + IR groups). Pretreatment with resveratrol normalized the cardiac level of Ang (1-7). The mRNA level of Mas receptor was increased in all of hypertrophied hearts in the presence or absence of resveratrol. Resveratrol can decrease IR injury in rats with LVH. The anti-ischemic effect of resveratrol may be related to the enhancement of Ang (1-7)/MasR axis.

Interplay between Triadin and Calsequestrin in the Pathogenesis of CPVT in the Mouse.

Recessive forms of catecholaminergic polymorphic ventricular tachycardia (CPVT) are induced by mutations in genes encoding triadin or calsequestrin, two proteins that belong to the Ca2+ release complex, responsible for intracellular Ca2+ release triggering cardiac contractions. To better understand the mechanisms of triadin-induced CPVT and to assay multiple therapeutic interventions, we used a triadin knockout mouse model presenting a CPVT-like phenotype associated with a decrease in calsequestrin protein level. We assessed different approaches to rescue protein expression and to correct intracellular Ca2+ release and cardiac function: pharmacological treatment with kifunensine or a viral gene transfer-based approach, using adeno-associated virus serotype 2/9 (AAV2/9) encoding the triadin or calsequestrin. We observed that the levels of triadin and calsequestrin are intimately linked, and that reduction of both proteins contributes to the CPVT phenotype. Different combinations of triadin and calsequestrin expression level were obtained using these therapeutic approaches. A full expression of each is not necessary to correct the phenotype; a fine-tuning of the relative re-expression of both triadin and calsequestrin is required to correct the CPVT phenotype and rescue the cardiac function. AAV-mediated gene delivery of calsequestrin or triadin and treatment with kifunensine are potential treatments for recessive forms of CPVT due to triadin mutations.

Effect of icosapent ethyl on susceptibility to ventricular arrhythmias in postinfarcted rat hearts: Role of GPR120-mediated connexin43 phosphorylation.

The ω-3 fatty acids exert as an antioxidant via the G protein-coupled receptor 120 (GPR120). Icosapent ethyl, a purified eicosapentaenoic acid, showed a marked reduction in sudden cardiac death. Connexin43 is sensitive to redox status. We assessed whether icosapent ethyl attenuates fatal arrhythmias after myocardial infarction, a status of high oxidative stress, through increased connexin43 expression and whether the GPR120 signalling is involved in the protection. Male Wistar rats after ligating coronary artery were assigned to either vehicle or icosapent ethyl for 4 weeks. The postinfarction period was associated with increased oxidative-nitrosative stress. In concert, myocardial connexin43 levels revealed a significant decrease in vehicle-treated infarcted rats compared with sham. These changes of oxidative-nitrosative stress and connexin43 levels were blunted after icosapent ethyl administration. Provocative arrhythmias in the infarcted rats treated with icosapent ethyl were significantly improved than vehicle. Icosapent ethyl significantly increased GPR120 compared to vehicle after infarction. The effects of icosapent ethyl on superoxide and connexin43 were similar to GPR120 agonist GW9508. Besides, the effects of icosapent ethyl on oxidative-nitrosative stress and connexin43 phosphorylation were abolished by administering AH-7614, an inhibitor of GPR120. SIN-1 abolished the Cx43 phosphorylation of icosapent ethyl without affecting GPR120 levels. Taken together, chronic use of icosapent ethyl after infarction is associated with up-regulation of connexin43 phosphorylation through a GPR120-dependent antioxidant pathway and thus plays a beneficial effect on arrhythmogenic response to programmed electrical stimulation.

Concurrent increases in post-pacing action potential duration and contractility predict occurrence of ventricular arrhythmia.

Excitation-contraction coupling from the integration of action potential duration (APD) and muscle contractility plays an important role in arrhythmogenesis. We aimed to determine whether distinctive excitation-contraction coupling contributes to the genesis of ventricular tachycardias (VTs). Action potential (AP) and mechanical activity were simultaneously recorded under electrical pacing (cycle lengths from 1000 to 100 ms) in the tissue model created from isolated rabbit right ventricular outflow tracts treated with NS 5806 (10 µM, transient outward potassium current enhancer), pinacidil (2 µM, ATP-sensitive potassium channel opener), and pilsicainide (5 µM, sodium channel blocker). There were 15 (9.9%) inducible VT episodes (group 1) and 136 (90.1%) non-inducible VT episodes (group 2) in our tissue model. Group 1 had greater post-pacing increases of the first occurrence of AP at 90% repolarization (ΔAPD90, p < 0.001) and contractility (ΔContractility, p = 0.003) compared with group 2. Triggered VT episodes were common (72.7%) in cases with a ΔAPD90 > 15% and a ΔContractility > 270%, but were undetectable in those with a ΔAPD90 < 15% and a ΔContractility < 270%. In those with pacing-induced VTs, KB-R7943 (10 µM, a Na+-Ca2+ exchanger inhibitor, NCX inhibitor) significantly reduced the occurrence of VTs from 100.0 to 20.0% (15/15 to 3/15 episodes, p < 0.001). Concurrent increases in both post-pacing APD and contractility resulted in the occurrence of ventricular arrhythmias. NCX inhibition may be a potential therapeutic strategy for ventricular arrhythmias.

Conditional ablation and conditional rescue models for Casq2 elucidate the role of development and of cell-type specific expression of Casq2 in the CPVT2 phenotype.

Cardiac calsequestrin (Casq2) associates with the ryanodine receptor 2 channel in the junctional sarcoplasmic reticulum to regulate Ca2+ release into the cytoplasm. Patients carrying mutations in CASQ2 display low resting heart rates under basal conditions and stress-induced polymorphic ventricular tachycardia (CPVT). In this study, we generate and characterize novel conditional deletion and conditional rescue mouse models to test the influence of developmental programs on the heart rate and CPVT phenotypes. We also compare the requirements for Casq2 function in the cardiac conduction system (CCS) and in working cardiomyocytes. Our study shows that the CPVT phenotype is dependent upon concurrent loss of Casq2 function in both the CCS and in working cardiomyocytes. Accordingly, restoration of Casq2 in only the CCS prevents CPVT. In addition, occurrence of CPVT is independent of the developmental history of Casq2-deficiency. In contrast, resting heart rate depends upon Casq2 gene activity only in the CCS and upon developmental history. Finally, our data support a model where low basal heart rate is a significant risk factor for CPVT.