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1.
Br J Pharmacol ; 178(22): 4518-4532, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34287836

RESUMO

BACKGROUND AND PURPOSE: Treatment of cardiac arrhythmia remains challenging due to severe side effects of common anti-arrhythmic drugs. We previously demonstrated that mitochondrial Ca2+ uptake in cardiomyocytes represents a promising new candidate structure for safer drug therapy. However, druggable agonists of mitochondrial Ca2+ uptake suitable for preclinical and clinical studies are still missing. EXPERIMENTAL APPROACH: Herewe screened 727 compounds with a history of use in human clinical trials in a three-step screening approach. As a primary screening platform we used a permeabilized HeLa cell-based mitochondrial Ca2+ uptake assay. Hits were validated in cultured HL-1 cardiomyocytes and finally tested for anti-arrhythmic efficacy in three translational models: a Ca2+ overload zebrafish model and cardiomyocytes of both a mouse model for catecholaminergic polymorphic ventricular tachycardia (CPVT) and induced pluripotent stem cell derived cardiomyocytes from a CPVT patient. KEY RESULTS: We identifiedtwo candidate compounds, the clinically approved drugs ezetimibe and disulfiram, which stimulate SR-mitochondria Ca2+ transfer at nanomolar concentrations. This is significantly lower compared to the previously described mitochondrial Ca2+ uptake enhancers (MiCUps) efsevin, a gating modifier of the voltage-dependent anion channel 2, and kaempferol, an agonist of the mitochondrial Ca2+ uniporter. Both substances restored rhythmic cardiac contractions in a zebrafish cardiac arrhythmia model and significantly suppressed arrhythmogenesis in freshly isolated ventricular cardiomyocytes from a CPVT mouse model as well as induced pluripotent stem cell derived cardiomyocytes from a CPVT patient. CONCLUSION AND IMPLICATIONS: Taken together we identified ezetimibe and disulfiram as novel MiCUps and efficient suppressors of arrhythmogenesis and as such as, promising candidates for future preclinical and clinical studies.


Assuntos
Preparações Farmacêuticas , Taquicardia Ventricular , Animais , Arritmias Cardíacas/induzido quimicamente , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Dissulfiram/metabolismo , Dissulfiram/farmacologia , Ezetimiba/metabolismo , Células HeLa , Humanos , Camundongos , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Preparações Farmacêuticas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Taquicardia Ventricular/metabolismo , Peixe-Zebra/metabolismo
2.
Cardiovasc Res ; 113(5): 531-541, 2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28158429

RESUMO

AIMS: Calmodulin (CaM) is a small protein, encoded by three genes (CALM1-3), exerting multiple Ca2+-dependent modulatory roles. A mutation (F142L) affecting only one of the six CALM alleles is associated with long QT syndrome (LQTS) characterized by recurrent cardiac arrests. This phenotypic severity is unexpected from the predicted allelic balance. In this work, the effects of heterozygous CALM1-F142L have been investigated in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from a LQTS patient carrying the F142L mutation, i.e. in the context of native allelic ratio and potential gene modifiers. METHODS AND RESULTS: Skin fibroblasts of the mutation carrier and two unrelated healthy subjects (controls) were reprogrammed to hiPSC and differentiated into hiPSC-CMs. Scanty IK1 expression, an hiPSC-CMs feature potentially biasing repolarization, was corrected by addition of simulated IK1 (Dynamic-Clamp). Abnormalities in repolarization rate-dependency (in single cells and cell aggregates), membrane currents and intracellular Ca2+ dynamics were evaluated as putative arrhythmogenic factors. CALM1-F142L prolonged repolarization, altered its rate-dependency and its response to isoproterenol. This was associated with severe impairment of Ca2+-dependent inactivation (CDI) of ICaL, resulting in augmented inward current during the plateau phase. As a result, the repolarization of mutant cells failed to adapt to high pacing rates, a finding well reproduced by using a recent hiPSC-CM action potential model. The mutation failed to affect IKs and INaL and changed If only marginally. Intracellular Ca2+ dynamics and Ca2+ store stability were not significantly modified. Mutation-induced repolarization abnormalities were reversed by verapamil. CONCLUSION: The main functional derangement in CALM1-F142L was prolonged repolarization with altered rate-dependency and sensitivity to ß-adrenergic stimulation. Impaired CDI of ICaL underlined the electrical abnormality, which was sensitive to ICaL blockade. High mutation penetrance was confirmed in the presence of the native genotype, implying strong dominance of effects.


Assuntos
Sinalização do Cálcio , Calmodulina/genética , Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome do QT Longo/genética , Mutação , Agonistas Adrenérgicos beta/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Estimulação Cardíaca Artificial , Estudos de Casos e Controles , Células Cultivadas , Reprogramação Celular , Técnicas de Reprogramação Celular , Fibroblastos/efeitos dos fármacos , Marcadores Genéticos , Predisposição Genética para Doença , Frequência Cardíaca , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Cinética , Síndrome do QT Longo/metabolismo , Síndrome do QT Longo/fisiopatologia , Potenciais da Membrana , Fenótipo , Pele/citologia , Transfecção
4.
JACC Basic Transl Sci ; 2(6): 737-747, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29354781

RESUMO

Cardiovascular disease-related deaths frequently arise from arrhythmias, but treatment options are limited due to perilous side effects of commonly used antiarrhythmic drugs. Cardiac rhythmicity strongly depends on cardiomyocyte Ca2+ handling and prevalent cardiac diseases are causally associated with perturbations in intracellular Ca2+ handling. Therefore, intracellular Ca2+ transporters are lead candidate structures for novel and safer antiarrhythmic therapies. Mitochondria and mitochondrial Ca2+ transport proteins are important regulators of cardiac Ca2+ handling. Here we evaluated the potential of pharmacological activation of mitochondrial Ca2+ uptake for the treatment of cardiac arrhythmia. To this aim,we tested substances that enhance mitochondrial Ca2+ uptake for their ability to suppress arrhythmia in a murine model for ryanodine receptor 2 (RyR2)-mediated catecholaminergic polymorphic ventricular tachycardia (CPVT) in vitro and in vivo and in induced pluripotent stem cell-derived cardiomyocytes from a CPVT patient. In freshly isolated cardiomyocytes of RyR2R4496C/WT mice efsevin, a synthetic agonist of the voltage-dependent anion channel 2 (VDAC2) in the outer mitochondrial membrane, prevented the formation of diastolic Ca2+ waves and spontaneous action potentials. The antiarrhythmic effect of efsevin was abolished by blockade of the mitochondrial Ca2+ uniporter (MCU), but could be reproduced using the natural MCU activator kaempferol. Both mitochondrial Ca2+ uptake enhancers (MiCUps), efsevin and kaempferol, significantly reduced episodes of stress-induced ventricular tachycardia in RyR2R4496C/WT mice in vivo and abolished diastolic, arrhythmogenic Ca2+ events in human iPSC-derived cardiomyocytes.

5.
J Clin Invest ; 122(7): 2519-30, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22706305

RESUMO

The contraction pattern of the heart relies on the activation and conduction of the electrical impulse. Perturbations of cardiac conduction have been associated with congenital and acquired arrhythmias as well as cardiac arrest. The pattern of conduction depends on the regulation of heterogeneous gene expression by key transcription factors and transcriptional enhancers. Here, we assessed the genome-wide occupation of conduction system-regulating transcription factors TBX3, NKX2-5, and GATA4 and of enhancer-associated coactivator p300 in the mouse heart, uncovering cardiac enhancers throughout the genome. Many of the enhancers colocalized with ion channel genes repressed by TBX3, including the clustered sodium channel genes Scn5a, essential for cardiac function, and Scn10a. We identified 2 enhancers in the Scn5a/Scn10a locus, which were regulated by TBX3 and its family member and activator, TBX5, and are functionally conserved in humans. We also provided evidence that a SNP in the SCN10A enhancer associated with alterations in cardiac conduction patterns in humans disrupts TBX3/TBX5 binding and reduces the cardiac activity of the enhancer in vivo. Thus, the identification of key regulatory elements for cardiac conduction helps to explain how genetic variants in noncoding regulatory DNA sequences influence the regulation of cardiac conduction and the predisposition for cardiac arrhythmias.


Assuntos
Elementos Facilitadores Genéticos , Polimorfismo de Nucleotídeo Único , Canais de Sódio/genética , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Imunoprecipitação da Cromatina , Sequência Consenso , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos , Variação Genética , Sistema de Condução Cardíaco/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Miocárdio/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5 , Canal de Sódio Disparado por Voltagem NAV1.8 , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Análise de Sequência de DNA , Proteínas com Domínio T/metabolismo , Peixe-Zebra
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