RESUMEN
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.
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Arritmias Cardíacas , Calmodulina , Pez Cebra , Animales , Calmodulina/metabolismo , Calmodulina/genética , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Mutación , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Humanos , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Mutación Missense , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Calcio/metabolismoRESUMEN
Chondroitin sulfate proteoglycans (CSPGs) inhibit sympathetic reinnervation in rodent hearts post-myocardial infarction (MI), causing regional hypoinnervation that is associated with supersensitivity of ß-adrenergic receptors and increased arrhythmia susceptibility. To investigate the role of CSPGs and hypoinnervation in the heart of larger mammals, we used a rabbit model of reperfused MI and tested electrophysiological responses to sympathetic nerve stimulation (SNS). Innervated hearts from MI and sham rabbits were optically mapped using voltage and Ca2+-sensitive dyes. SNS was performed with electrical stimulation of the spinal cord, and ß-adrenergic responsiveness was tested using isoproterenol. Sympathetic nerve density and CSPG expression were evaluated using immunohistochemistry. CSPGs were robustly expressed in the infarct region of all MI hearts, and the presence of CSPGs was associated with reduced sympathetic nerve density in the infarct versus remote region. Action potential duration (APD) dispersion and tendency for induction of ventricular tachycardia/fibrillation (VT/VF) were increased with SNS in MI but not sham hearts. SNS decreased APD at 80% repolarization (APD80) in MI but not sham hearts, whereas isoproterenol decreased APD80 in both groups. Isoproterenol also shortened Ca2+ transient duration at 80% repolarization in both groups but to a greater extent in MI hearts. Our data suggest that sympathetic remodeling post-MI is similar between rodents and rabbits, with CSPGs associated with sympathetic hypoinnervation. Despite a reduction in sympathetic nerve density, the infarct region of MI hearts remained responsive to both physiological SNS and isoproterenol, potentially through preserved or elevated ß-adrenergic responsiveness, which may underlie increased APD dispersion and tendency for VT/VF.NEW & NOTEWORTHY Here, we show that CSPGs are present in the infarcts of rabbit hearts with reperfused MI, where they are associated with reduced sympathetic nerve density. Despite hypoinnervation, sympathetic responsiveness is maintained or enhanced in MI rabbit hearts, which also demonstrate increased APD dispersion and tendency for arrhythmias following sympathetic modulation. Together, this study indicates that the mechanisms of sympathetic remodeling post-MI are similar between rodents and rabbits, with hypoinnervation likely associated with enhanced ß-adrenergic sensitivity.
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Potenciales de Acción , Modelos Animales de Enfermedad , Infarto del Miocardio , Sistema Nervioso Simpático , Animales , Conejos , Infarto del Miocardio/fisiopatología , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Sistema Nervioso Simpático/fisiopatología , Sistema Nervioso Simpático/metabolismo , Masculino , Isoproterenol/farmacología , Agonistas Adrenérgicos beta/farmacología , Corazón/inervación , Corazón/fisiopatología , Miocardio/metabolismo , Miocardio/patología , Taquicardia Ventricular/fisiopatología , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/etiologíaRESUMEN
Heart Failure with preserved ejection fraction (HFpEF) has a high rate of sudden cardiac death (SCD) and empirical treatment is ineffective. We developed a novel preclinical model of metabolic HFpEF that presents with stress-induced ventricular tachycardia (VT). Mechanistically, we discovered arrhythmogenic changes in intracellular Ca2+ handling distinct from the changes pathognomonic for heart failure with reduced ejection fraction. We further show that dantrolene, a stabilizer of the ryanodine receptor Ca2+ channel, attenuates HFpEF-associated arrhythmogenic Ca2+ handling in vitro and suppresses stress-induced VT in vivo. We propose ryanodine receptor stabilization as a mechanistic approach to mitigation of malignant VT in metabolic HFpEF.
Asunto(s)
Arritmias Cardíacas , Calcio , Dantroleno , Modelos Animales de Enfermedad , Insuficiencia Cardíaca , Canal Liberador de Calcio Receptor de Rianodina , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Calcio/metabolismo , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/fisiopatología , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/tratamiento farmacológico , Dantroleno/farmacología , Volumen Sistólico/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Humanos , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/tratamiento farmacológico , Ratones , Masculino , Señalización del Calcio/efectos de los fármacosRESUMEN
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.
Asunto(s)
Arritmias Cardíacas , Calcio , Dexmedetomidina , Canal Liberador de Calcio Receptor de Rianodina , Animales , Dexmedetomidina/farmacología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Calcio/metabolismo , Ratones , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/tratamiento farmacológico , Arritmias Cardíacas/genética , Potenciales de la Membrana/efectos de los fármacos , Isoproterenol/farmacología , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/genética , Taquicardia Ventricular/tratamiento farmacológico , Antiarrítmicos/farmacología , Masculino , Potenciales de Acción/efectos de los fármacos , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Sacubitril/valsartan (Sac/Val) is superior to angiotensin-converting enzyme inhibitors in reducing the risk of heart failure hospitalization and cardiovascular death, but its mechanistic data on myocardial scar after myocardial infarction (MI) are lacking. The objective of this work was to assess the effects of Sac/Val on inflammation, fibrosis, electrophysiological properties, and ventricular tachycardia inducibility in post-MI scar remodeling in swine. METHODS: After MI, 22 pigs were randomized to receive ß-blocker (BB; control, n=8) or BB+Sac/Val (Sac/Val, n=9). The systemic immune response was monitored. Cardiac magnetic resonance data were acquired at 2-day and 29-day post MI to assess ventricular remodeling. Programmed electrical stimulation and high-density mapping were performed at 30-day post MI to assess ventricular tachycardia inducibility. Myocardial samples were collected for histological analysis. RESULTS: Compared with BB, BB+Sac/Val reduced acute circulating leukocytes (P=0.009) and interleukin-12 levels (P=0.024) at 2-day post MI, decreased C-C chemokine receptor type 2 expression in monocytes (P=0.047) at 15-day post MI, and reduced scar mass (P=0.046) and border zone mass (P=0.043). It also lowered the number and mass of border zone corridors (P=0.009 and P=0.026, respectively), scar collagen I content (P=0.049), and collagen I/III ratio (P=0.040). Sac/Val reduced ventricular tachycardia inducibility (P=0.034) and the number of deceleration zones (P=0.016). CONCLUSIONS: After MI, compared with BB, BB+Sac/Val was associated with reduced acute systemic inflammatory markers, reduced total scar and border zone mass on late gadolinium-enhanced magnetic resonance imaging, and lower ventricular tachycardia inducibility.
Asunto(s)
Aminobutiratos , Compuestos de Bifenilo , Cicatriz , Modelos Animales de Enfermedad , Combinación de Medicamentos , Infarto del Miocardio , Miocardio , Taquicardia Ventricular , Valsartán , Remodelación Ventricular , Animales , Valsartán/farmacología , Aminobutiratos/farmacología , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/fisiopatología , Infarto del Miocardio/complicaciones , Infarto del Miocardio/patología , Cicatriz/fisiopatología , Cicatriz/etiología , Cicatriz/patología , Taquicardia Ventricular/fisiopatología , Taquicardia Ventricular/etiología , Taquicardia Ventricular/tratamiento farmacológico , Taquicardia Ventricular/prevención & control , Taquicardia Ventricular/metabolismo , Remodelación Ventricular/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Miocardio/patología , Miocardio/metabolismo , Antiinflamatorios/farmacología , Tetrazoles/farmacología , Fibrosis , Porcinos , Antiarrítmicos/farmacología , Femenino , Masculino , Factores de Tiempo , Imagen por Resonancia Cinemagnética , Frecuencia Cardíaca/efectos de los fármacosRESUMEN
BACKGROUND: Neural remodeling in the left stellate ganglion (LSG), as mediated by neuroimmune reactions, promotes cardiac sympathetic nerve activity (SNA) and thus increases the incidence of ventricular arrhythmias (VAs). Interleukin-6 (IL-6) is an important factor of the neuroimmune interaction. OBJECTIVE: The present study explored the effects of IL-6 on LSG hyperactivity and the incidence of VAs. METHODS: Eighteen beagles were randomly allocated to a control group (saline with myocardial infarction [MI], n = 6), adeno-associated virus (AAV) group (AAV with MI, n = 6), and IL-6 group (overexpression of IL-6 via AAV vector with MI, n = 6). Ambulatory electrocardiography was performed before and 30 days after AAV microinjection into the LSG. LSG function and ventricular electrophysiology were assessed at 31 days after surgery, and a canine MI model was established. Samples of the LSG were collected for immunofluorescence staining and molecular biological evaluation. Blood samples and 24-hour Holter data were obtained from 24 patients with acute MI on the day after they underwent percutaneous coronary intervention to assess the correlation between IL-6 levels and SNA. RESULTS: IL-6 overexpression increased cardiac SNA and worsened postinfarction VAs. Furthermore, sustained IL-6 overexpression enhanced LSG function, promoted expression of nerve growth factor, c-fos, and fos B in the LSG, and activated the signal transducer and activator of transcription 3/regulator of G protein signalling 4 signaling pathway. Clinical sample analysis revealed a correlation between serum IL-6 levels and heart rate variability frequency domain index as well as T-wave alternans. CONCLUSION: IL-6 levels are correlated with cardiac SNA. Chronic overexpression of IL-6 mediates LSG neural remodeling through the signal transducer and activator of transcription 3/regulator of G protein signalling 4 signaling pathway, elevating the risk of VA after MI.
Asunto(s)
Modelos Animales de Enfermedad , Interleucina-6 , Ganglio Estrellado , Animales , Perros , Interleucina-6/metabolismo , Ganglio Estrellado/metabolismo , Arritmias Cardíacas/etiología , Masculino , Electrocardiografía Ambulatoria/métodos , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatología , Sistema Nervioso Simpático/fisiopatología , Sistema Nervioso Simpático/metabolismo , Neuroinmunomodulación/fisiología , Humanos , Taquicardia Ventricular/etiología , Taquicardia Ventricular/fisiopatología , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/terapiaRESUMEN
Calsequestrin (CASQ) is a key intra-sarcoplasmic reticulum Ca2+-handling protein that plays a pivotal role in the contraction of cardiac and skeletal muscles. Its Ca2+-dependent polymerization dynamics shape the translation of electric excitation signals to the Ca2+-induced contraction of the actin-myosin architecture. Mutations in CASQ are linked to life-threatening pathological conditions, including tubular aggregate myopathy, malignant hyperthermia, and Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). The variability in the penetrance of these phenotypes and the lack of a clear understanding of the disease mechanisms associated with CASQ mutations pose a major challenge to the development of effective therapeutic strategies. In vitro studies have mainly focused on the polymerization and Ca2+-buffering properties of CASQ but have provided little insight into the complex interplay of structural and functional changes that underlie disease. In this review, the biochemical and structural natures of CASQ are explored in-depth, while emphasizing their direct and indirect consequences for muscle Ca2+ physiology. We propose a novel functional classification of CASQ pathological missense mutations based on the structural stability of the monomer, dimer, or linear polymer conformation. We also highlight emerging similarities between polymeric CASQ and polyelectrolyte systems, emphasizing the potential for the use of this paradigm to guide further research.
Asunto(s)
Calsecuestrina , Taquicardia Ventricular , Humanos , Calsecuestrina/genética , Calsecuestrina/metabolismo , Corazón , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Retículo Sarcoplasmático/metabolismo , Mutación Missense , Calcio/metabolismoRESUMEN
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.
Asunto(s)
Canal Liberador de Calcio Receptor de Rianodina , Taquicardia Ventricular , Humanos , Arritmias Cardíacas/genética , Calcio/metabolismo , Células HEK293 , Mutación , Fenotipo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismoRESUMEN
This article reviews progress in the field of cardiac genome editing, in particular, its potential utility in treating cardiac arrhythmias. First, we discuss genome editing methods by which DNA can be disrupted, inserted, deleted, or corrected in cardiomyocytes. Second, we provide an overview of in vivo genome editing in preclinical models of heritable and acquired arrhythmias. Third, we discuss recent advancements in cardiac gene transfer, including delivery methods, gene expression optimization, and potential adverse effects associated with therapeutic somatic genome editing. While genome editing for cardiac arrhythmias is still in its infancy, this approach holds great promise, especially for inherited arrhythmia syndromes with a defined genetic defect.
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Edición Génica , Taquicardia Ventricular , Humanos , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/terapia , Arritmias Cardíacas/genética , Arritmias Cardíacas/terapia , Arritmias Cardíacas/metabolismo , Miocitos Cardíacos/metabolismoRESUMEN
We illustrate use of induced pluripotent stem cells (iPSCs) as platforms for investigating cardiomyocyte phenotypes in a human family pedigree exemplified by novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants occurring alone and in combination. The proband, a four-month-old boy, presented with polymorphic ventricular tachycardia. Genetic tests revealed double novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants inherited from his father (F) and mother (M), respectively. His father showed ventricular premature beats; his mother was asymptomatic. Molecular biological characterizations demonstrated greater TNNT2 messenger RNA (mRNA) expression in the iPSCs-induced cardiomyocytes (iPS-CMs) than in the iPSCs. Cardiac troponin Ts became progressively organized but cytoplasmic RYR2 and SCN10A aggregations occurred in the iPS-CMs. Proband-specific iPS-CMs showed decreased RYR2 and SCN10A mRNA expression. The RYR2-A1855D variant resulted in premature spontaneous sarcoplasmic reticular Ca2+ transients, Ca2+ oscillations and increased action potential durations. SCN10A-Q1362H did not confer any specific phenotype. However, the combined heterozygous RYR2-A1855D and SCN10A-Q1362H variants in the proband iPS-CMs resulted in accentuated Ca2+ homeostasis disorders, action potential prolongation and susceptibility to early afterdepolarizations at high stimulus frequencies. These findings attribute the clinical phenotype in the proband to effects of the heterozygous RYR2 variant exacerbated by heterozygous SCN10A modification. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.
Asunto(s)
Células Madre Pluripotentes Inducidas , Taquicardia Ventricular , Humanos , Lactante , Masculino , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Calcio/metabolismo , Homeostasis , Mutación , Canal de Sodio Activado por Voltaje NAV1.8/genética , Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/farmacología , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismoRESUMEN
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.
Asunto(s)
Canal Liberador de Calcio Receptor de Rianodina , Taquicardia Ventricular , Ratones , Animales , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Antiarrítmicos/farmacología , Antiarrítmicos/uso terapéutico , Arritmias Cardíacas/tratamiento farmacológico , Taquicardia Ventricular/tratamiento farmacológico , Taquicardia Ventricular/metabolismo , Miocitos Cardíacos/metabolismoRESUMEN
BACKGROUND: CaM (calmodulin) is a ubiquitously expressed, multifunctional Ca2+ sensor protein that regulates numerous proteins. Recently, CaM missense variants have been identified in patients with malignant inherited arrhythmias, such as long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the exact mechanism of CaM-related CPVT in human cardiomyocytes remains unclear. In this study, we sought to investigate the arrhythmogenic mechanism of CPVT caused by a novel variant using human induced pluripotent stem cell (iPSC) models and biochemical assays. METHODS: We generated iPSCs from a patient with CPVT bearing CALM2 p.E46K. As comparisons, we used 2 control lines including an isogenic line, and another iPSC line from a patient with long QT syndrome bearing CALM2 p.N98S (also reported in CPVT). Electrophysiological properties were investigated using iPSC-cardiomyocytes. We further examined the RyR2 (ryanodine receptor 2) and Ca2+ affinities of CaM using recombinant proteins. RESULTS: We identified a novel de novo heterozygous variant, CALM2 p.E46K, in 2 unrelated patients with CPVT accompanied by neurodevelopmental disorders. The E46K-cardiomyocytes exhibited more frequent abnormal electrical excitations and Ca2+ waves than the other lines in association with increased Ca2+ leakage from the sarcoplasmic reticulum via RyR2. Furthermore, the [3H]ryanodine binding assay revealed that E46K-CaM facilitated RyR2 function especially by activating at low [Ca2+] levels. The real-time CaM-RyR2 binding analysis demonstrated that E46K-CaM had a 10-fold increased RyR2 binding affinity compared with wild-type CaM which may account for the dominant effect of the mutant CaM. Additionally, the E46K-CaM did not affect CaM-Ca2+ binding or L-type calcium channel function. Finally, antiarrhythmic agents, nadolol and flecainide, suppressed abnormal Ca2+ waves in E46K-cardiomyocytes. CONCLUSIONS: We, for the first time, established a CaM-related CPVT iPSC-CM model which recapitulated severe arrhythmogenic features resulting from E46K-CaM dominantly binding and facilitating RyR2. In addition, the findings in iPSC-based drug testing will contribute to precision medicine.
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Células Madre Pluripotentes Inducidas , Síndrome de QT Prolongado , Taquicardia Ventricular , Humanos , Calmodulina/genética , Calmodulina/metabolismo , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Taquicardia Ventricular/metabolismo , Arritmias Cardíacas , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/metabolismo , Calcio/metabolismo , MutaciónRESUMEN
Calcium ions (Ca2+) are the basis of a unique and potent array of cellular responses. Calmodulin (CaM) is a small but vital protein that is able to rapidly transmit information about changes in Ca2+ concentrations to its regulatory targets. CaM plays a critical role in cellular Ca2+ signaling, and interacts with a myriad of target proteins. Ca2+-dependent modulation by CaM is a major component of a diverse array of processes, ranging from gene expression in neurons to the shaping of the cardiac action potential in heart cells. Furthermore, the protein sequence of CaM is highly evolutionarily conserved, and identical CaM proteins are encoded by three independent genes (CALM1-3) in humans. Mutations within any of these three genes may lead to severe cardiac deficits including severe long QT syndrome (LQTS) and/or catecholaminergic polymorphic ventricular tachycardia (CPVT). Research into disease-associated CaM variants has identified several proteins modulated by CaM that are likely to underlie the pathogenesis of these calmodulinopathies, including the cardiac L-type Ca2+ channel (LTCC) CaV1.2, and the sarcoplasmic reticulum Ca2+ release channel, ryanodine receptor 2 (RyR2). Here, we review the research that has been done to identify calmodulinopathic CaM mutations and evaluate the mechanisms underlying their role in disease.
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Síndrome de QT Prolongado , Taquicardia Ventricular , Humanos , Calmodulina/genética , Calmodulina/metabolismo , Mutación , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/metabolismo , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Calcio/metabolismoRESUMEN
Flecainide, a cardiac class 1C blocker of the surface membrane sodium channel (NaV1.5), has also been reported to reduce cardiac ryanodine receptor (RyR2)-mediated sarcoplasmic reticulum (SR) Ca2+ release. It has been introduced as a clinical antiarrhythmic agent for catecholaminergic polymorphic ventricular tachycardia (CPVT), a condition most commonly associated with gain-of-function RyR2 mutations. Current debate concerns both cellular mechanisms of its antiarrhythmic action and molecular mechanisms of its RyR2 actions. At the cellular level, it targets NaV1.5, RyR2, Na+/Ca2+ exchange (NCX), and additional proteins involved in excitation-contraction (EC) coupling and potentially contribute to the CPVT phenotype. This Viewpoint primarily addresses the various direct molecular actions of flecainide on isolated RyR2 channels in artificial lipid bilayers. Such studies demonstrate different, multifarious, flecainide binding sites on RyR2, with voltage-dependent binding in the channel pore or voltage-independent binding at distant peripheral sites. In contrast to its single NaV1.5 pore binding site, flecainide may bind to at least four separate inhibitory sites on RyR2 and one activation site. None of these binding sites have been specifically located in the linear RyR2 sequence or high-resolution structure. Furthermore, it is not clear which of the inhibitory sites contribute to flecainide's reduction of spontaneous Ca2+ release in cellular studies. A confounding observation is that flecainide binding to voltage-dependent inhibition sites reduces cation fluxes in a direction opposite to physiological Ca2+ flow from SR lumen to cytosol. This may suggest that, rather than directly blocking Ca2+ efflux, flecainide can reduce Ca2+ efflux by blocking counter currents through the pore which otherwise limit SR membrane potential change during systolic Ca2+ efflux. In summary, the antiarrhythmic effects of flecainide in CPVT seem to involve multiple components of EC coupling and multiple actions on RyR2. Their clarification may identify novel specific drug targets and facilitate flecainide's clinical utilization in CPVT.
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Flecainida , Taquicardia Ventricular , Antiarrítmicos/farmacología , Calcio/metabolismo , Flecainida/metabolismo , Flecainida/farmacología , Humanos , Miocitos Cardíacos/metabolismo , Rianodina/metabolismo , Rianodina/farmacología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Sodio/metabolismo , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismoRESUMEN
Sudden cardiac death (SCD) caused by ventricular arrhythmias is the leading cause of mortality of cardiovascular disease. Mutation in TECRL, an endoplasmic reticulum protein, was first reported in catecholaminergic polymorphic ventricular tachycardia during which a patient succumbed to SCD. Using loss- and gain-of-function approaches, we investigated the role of TECRL in murine and human cardiomyocytes. Tecrl (knockout, KO) mouse shows significantly aggravated cardiac dysfunction, evidenced by the decrease of ejection fraction and fractional shortening. Mechanistically, TECRL deficiency impairs mitochondrial respiration, which is characterized by reduced adenosine triphosphate production, increased fatty acid synthase (FAS) and reactive oxygen species production, along with decreased MFN2, p-AKT (Ser473), and NRF2 expressions. Overexpression of TECRL induces mitochondrial respiration, in PI3K/AKT dependent manner. TECRL regulates mitochondrial function mainly through PI3K/AKT signaling and the mitochondrial fusion protein MFN2. Apoptosis inducing factor (AIF) and cytochrome C (Cyc) is released from the mitochondria into the cytoplasm after siTECRL infection, as demonstrated by immunofluorescent staining and western blotting. Herein, we propose a previously unrecognized TECRL mechanism in regulating CPVT and may provide possible support for therapeutic target in CPVT.
Asunto(s)
Mitocondrias , Miocitos Cardíacos , Oxidorreductasas , Taquicardia Ventricular , Animales , Humanos , Ratones , Mitocondrias/enzimología , Mitocondrias/metabolismo , Mitocondrias/patología , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Oxidorreductasas/deficiencia , Oxidorreductasas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Taquicardia Ventricular/enzimología , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patologíaRESUMEN
Inherited arrhythmias are the leading causes for cardiac arrest and sudden cardiac death (SCD). Other than ion channel mutations, inherited arrhythmias including catecholaminergic polymorphic ventricular tachycardia (CPVT), long QT syndrome (LQTS), idiopathic ventricular fibrillation (IVF) and arrhythmogenic right ventricular cardiomyopathy (ARVC/D) may also be instigated by genetic mutations of sarcoplasmic reticulum (SR) proteins, including ryanodine receptor type-2 (RyR2), calsequestrin 2, SR Ca2+-ATPase type-2a (SERCA2a) and phospholamban. In cardiomyocytes, Ca2+ is an essential ion in addition to Na+ and K+ ions with vital roles in arrhythmogenesis. SR plays a critical role in the maintenance of Ca2+ homeostasis which can be disrupted by mutations in SR Ca2+ regulatory proteins or abnormal SR-intracellular organelle interaction. Early afterdepolarizations, delayed afterdepolarizations and reentry are three primary mechanisms contributing to arrhythmias elicited by SR Ca2+ dysregulation in cardiomyocytes. In this review, we will aim to summarize normal SR Ca2+ regulation in cardiomyocytes, mechanisms of how Ca2+ triggers arrhythmias and involvements of SR gene mutations in inherited arrhythmias as well as the possible arrhythmogenic effects of these mutations.
Asunto(s)
Retículo Sarcoplasmático , Taquicardia Ventricular , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Calcio/metabolismo , Humanos , Mutación , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismoRESUMEN
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.
Asunto(s)
Canal Liberador de Calcio Receptor de Rianodina , Taquicardia Ventricular , Displasia Ventricular Derecha Arritmogénica , Calcio/metabolismo , Señalización del Calcio , Humanos , Mutación , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Taquicardia Ventricular/etiología , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/terapiaRESUMEN
Type 2 ryanodine receptor (RYR2) is a cardiac Ca2+ release channel in the ER. Mutations in RYR2 are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT is associated with enhanced spontaneous Ca2+ release, which tends to occur when [Ca2+]ER reaches a threshold. Mutations lower the threshold [Ca2+]ER by increasing luminal Ca2+ sensitivity or enhancing cytosolic [Ca2+] ([Ca2+]cyt)-dependent activity. Here, to establish the mechanism relating the change in [Ca2+]cyt-dependent activity of RYR2 and the threshold [Ca2+]ER, we carried out cell-based experiments and in silico simulations. We expressed WT and CPVT-linked mutant RYR2s in HEK293 cells and measured [Ca2+]cyt and [Ca2+]ER using fluorescent Ca2+ indicators. CPVT RYR2 cells showed higher oscillation frequency and lower threshold [Ca2+]ER than WT cells. The [Ca2+]cyt-dependent activity at resting [Ca2+]cyt, Arest, was greater in CPVT mutants than in WT, and we found an inverse correlation between threshold [Ca2+]ER and Arest. In addition, lowering RYR2 expression increased the threshold [Ca2+]ER and a product of Arest, and the relative expression level for each mutant correlated with threshold [Ca2+]ER, suggesting that the threshold [Ca2+]ER depends on the net Ca2+ release rate via RYR2. Modeling reproduced Ca2+ oscillations with [Ca2+]cyt and [Ca2+]ER changes in WT and CPVT cells. Interestingly, the [Ca2+]cyt-dependent activity of specific mutations correlated with the age of disease onset in patients carrying them. Our data suggest that the reduction in threshold [Ca2+]ER for spontaneous Ca2+ release by CPVT mutation is explained by enhanced [Ca2+]cyt-dependent activity without requiring modulation of the [Ca2+]ER sensitivity of RYR2.
Asunto(s)
Canal Liberador de Calcio Receptor de Rianodina , Taquicardia Ventricular , Calcio/metabolismo , Células HEK293 , Humanos , Mutación , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismoRESUMEN
Arrhythmias originating in scarred ventricular myocardium are a major cause of death, but the underlying mechanism allowing these rhythms to exist remains unknown. This gap in knowledge critically limits identification of at-risk patients and treatment once arrhythmias become manifest. Here we show that potassium voltage-gated channel subfamily E regulatory subunits 3 and 4 (KCNE3, KCNE4) are uniquely upregulated at arrhythmia sites within scarred myocardium. Ventricular arrhythmias occur in areas with a distinctive cardiomyocyte repolarization pattern, where myocyte tracts with short repolarization times connect to myocytes tracts with long repolarization times. We found this unique pattern of repolarization heterogeneity only in ventricular arrhythmia circuits. In contrast, conduction abnormalities were ubiquitous within scar. These repolarization heterogeneities are consistent with known functional effects of KCNE3 and KCNE4 on the slow delayed-rectifier potassium current. We observed repolarization heterogeneity using conventional cardiac electrophysiologic techniques that could potentially translate to identification of at-risk patients. The neutralization of the repolarization heterogeneities could represent a potential strategy for the elimination of ventricular arrhythmia circuits.
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Cicatriz/fisiopatología , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatología , Animales , Arritmias Cardíacas/fisiopatología , Electrocardiografía , Técnicas Electrofisiológicas Cardíacas , Femenino , Cobayas , Ventrículos Cardíacos/fisiopatología , Humanos , Canal de Potasio KCNQ1 , Masculino , Miocardio/patología , Canales de Potasio con Entrada de Voltaje/metabolismoRESUMEN
Mutations in the calcium-sensing protein calmodulin (CaM) have been linked to two cardiac arrhythmia diseases, Long QT Syndrome 14 (LQT14) and Catecholaminergic Polymorphic Ventricular Tachycardia Type 4 (CPVT4), with varying degrees of severity. Functional characterization of the CaM mutants most strongly associated with LQT14 show a clear disruption of the calcium-dependent inactivation (CDI) of the L-Type calcium channel (LCC). CPVT4 mutants on the other hand are associated with changes in their affinity to the ryanodine receptor. In clinical studies, some variants have been associated with both CPVT4 and LQT15. This study uses simulations in a model for excitation-contraction coupling in the rat ventricular myocytes to understand how LQT14 variant might give the functional phenotype similar to CPVT4. Changing the CaM-dependent transition rate by a factor of 0.75 corresponding to the D96V variant and by a factor of 0.90 corresponding to the F142L or N98S variants, in a physiologically based stochastic model of the LCC prolonger, the action potential duration changed by a small amount in a cardiac myocyte but did not disrupt CICR at 1, 2, and 4 Hz. Under beta-adrenergic simulation abnormal excitation-contraction coupling was observed above 2 Hz pacing for the mutant CaM. The same conditions applied under beta-adrenergic stimulation led to the rapid onset of arrhythmia in the mutant CaM simulations. Simulations with the LQT14 mutations under the conditions of rapid pacing with beta-adrenergic stimulation drives the cardiac myocyte toward an arrhythmic state known as Ca2+ overload. These simulations provide a mechanistic link to a disease state for LQT14-associated mutations in CaM to yield a CPVT4 phenotype. The results show that small changes to the CaM-regulated inactivation of LCC promote arrhythmia and underscore the significance of CDI in proper heart function.