RESUMEN
The sodium channel NaV1.8, encoded by the SCN10A gene, has recently emerged as a potential regulator of cardiac electrophysiology. We have previously shown that NaV1.8 contributes to arrhythmogenesis by inducing a persistent Na+ current (late Na+ current, INaL) in human atrial and ventricular cardiomyocytes (CM). We now aim to further investigate the contribution of NaV1.8 to human ventricular arrhythmogenesis at the CM-specific level using pharmacological inhibition as well as a genetic knockout (KO) of SCN10A in induced pluripotent stem cell CM (iPSC-CM). In functional voltage-clamp experiments, we demonstrate that INaL was significantly reduced in ventricular SCN10A-KO iPSC-CM and in control CM after a specific pharmacological inhibition of NaV1.8. In contrast, we did not find any effects on ventricular APD90. The frequency of spontaneous sarcoplasmic reticulum Ca2+ sparks and waves were reduced in SCN10A-KO iPSC-CM and control cells following the pharmacological inhibition of NaV1.8. We further analyzed potential triggers of arrhythmias and found reduced delayed afterdepolarizations (DAD) in SCN10A-KO iPSC-CM and after the specific inhibition of NaV1.8 in control cells. In conclusion, we show that NaV1.8-induced INaL primarily impacts arrhythmogenesis at a subcellular level, with minimal effects on systolic cellular Ca2+ release. The inhibition or knockout of NaV1.8 diminishes proarrhythmic triggers in ventricular CM. In conjunction with our previously published results, this work confirms NaV1.8 as a proarrhythmic target that may be useful in an anti-arrhythmic therapeutic strategy.
Asunto(s)
Arritmias Cardíacas , Ventrículos Cardíacos , Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Canal de Sodio Activado por Voltaje NAV1.8 , Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , Canal de Sodio Activado por Voltaje NAV1.8/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/citología , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/genética , Arritmias Cardíacas/etiología , Potenciales de Acción/efectos de los fármacosRESUMEN
Filamin C (FLNC) is a highly important actin crosslinker and multi-adaptor protein in striated skeletal and cardiac muscle. Mutations have been linked to a range of cardiomyopathy types. Here, we generated induced pluripotent stem cells (iPSC) from a patient with dilated cardiomyopathy (DCM) harboring a new, unique heterozygous FLNC mutation p.R2187P. From this patient-specific iPSC line, a corresponding isogenic control line was created by CRISPR/Cas9 genome editing. Both, the patient-specific and isogenic-control iPSC maintained full pluripotency, genomic integrity, and in vitro differentiation capacity. All iPSC lines differentiate into iPSC-cardiomyocytes, hence providing the possibility to study the pathogenesis of FLNC-mediated DCM further.
Asunto(s)
Sistemas CRISPR-Cas , Cardiomiopatía Dilatada , Filaminas , Células Madre Pluripotentes Inducidas , Humanos , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Sistemas CRISPR-Cas/genética , Células Madre Pluripotentes Inducidas/metabolismo , Filaminas/genética , Filaminas/metabolismo , Mutación , Diferenciación Celular , Línea Celular , MasculinoRESUMEN
RBM20 mutations account for 3 % of genetic cardiomypathies and manifest with high penetrance and arrhythmogenic effects. Numerous mutations in the conserved RS domain have been described as causing dilated cardiomyopathy (DCM), whereas a particular mutation (p.R634L) drives development of a different cardiac phenotype: left-ventricular non-compaction cardiomyopathy. We generated a mutation-induced pluripotent stem cell (iPSC) line in which the RBM20-LVNC mutation p.R634L was introduced into a DCM patient line with rescued RBM20-p.R634W mutation. These DCM-634L-iPSC can be differentiated into functional cardiomyocytes to test whether this RBM20 mutation induces development of the LVNC phenotype within the genetic context of a DCM patient.
Asunto(s)
Cardiomiopatías , Cardiomiopatía Dilatada , Células Madre Pluripotentes Inducidas , Proteínas de Unión al ARN , Humanos , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatía Dilatada/genética , Antecedentes Genéticos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación/genética , Miocitos Cardíacos/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
While excessive production of reactive oxygen species (ROS) is a characteristic hallmark of numerous diseases, clinical approaches that ameliorate oxidative stress have been unsuccessful. Here, utilizing multi-omics, we demonstrate that in cardiomyocytes, mitochondrial isocitrate dehydrogenase (IDH2) constitutes a major antioxidative defense mechanism. Paradoxically reduced expression of IDH2 associated with ventricular eccentric hypertrophy is counterbalanced by an increase in the enzyme activity. We unveil redox-dependent sex dimorphism, and extensive mutual regulation of the antioxidative activities of IDH2 and NRF2 by a feedforward network that involves 2-oxoglutarate and L-2-hydroxyglutarate and mediated in part through unconventional hydroxy-methylation of cytosine residues present in introns. Consequently, conditional targeting of ROS in a murine model of heart failure improves cardiac function in sex- and phenotype-dependent manners. Together, these insights may explain why previous attempts to treat heart failure with antioxidants have been unsuccessful and open new approaches to personalizing and, thereby, improving such treatment.
Asunto(s)
Insuficiencia Cardíaca , Estrés Oxidativo , Ratones , Animales , Especies Reactivas de Oxígeno/metabolismo , Antioxidantes/metabolismo , Oxidación-Reducción , Insuficiencia Cardíaca/genética , Cardiomegalia , Epigénesis Genética , Isocitrato Deshidrogenasa/genéticaRESUMEN
Mutations in the mitochondrial-DNA or mitochondria related nuclear-encoded-DNA lead to various multisystemic disorders collectively termed mitochondrial diseases. One in three cases of mitochondrial disease affects the heart muscle, which is called mitochondrial cardiomyopathy (MCM) and is associated with hypertrophic, dilated, and noncompact cardiomyopathy. The heart is an organ with high energy demand, and mitochondria occupy 30%-40% of its cardiomyocyte-cell volume. Mitochondrial dysfunction leads to energy depletion and has detrimental effects on cardiac performance. However, disease development and progression in the context of mitochondrial and nuclear DNA mutations, remains incompletely understood. The system of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) is an excellent platform to study MCM since the unique genetic identity to their donors enables a robust recapitulation of the predicted phenotypes in a dish on a patient-specific level. Here, we focus on recent insights into MCM studied by patient-specific iPSC-CM and further discuss research gaps and advances in metabolic maturation of iPSC-CM, which is crucial for the study of mitochondrial dysfunction and to develop novel therapeutic strategies.