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Potenciales de Acción , Células Madre Pluripotentes Inducidas , Síndrome de QT Prolongado , Miocitos Cardíacos , Humanos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Síndrome de QT Prolongado/inducido químicamente , Síndrome de QT Prolongado/fisiopatología , Potenciales de Acción/efectos de los fármacos , Fenotipo , Frecuencia Cardíaca/efectos de los fármacos , Variantes FarmacogenómicasAsunto(s)
Fibrilación Atrial , Mutación con Ganancia de Función , Proteína del Homeodomínio PITX2 , Proteínas de Homeodominio , Factores de Transcripción , Fibrilación Atrial/genética , Fibrilación Atrial/fisiopatología , Fibrilación Atrial/diagnóstico , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Homeodominio/genética , Mitocondrias Cardíacas/genética , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Animales , Potencial de la Membrana Mitocondrial/genética , Predisposición Genética a la Enfermedad , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , FenotipoRESUMEN
BACKGROUND: Despite major advances in the clinical management of long QT syndrome, some patients are not fully protected by beta-blocker therapy. Mexiletine is a well-known sodium channel blocker, with proven efficacy in patients with sodium channel-mediated long QT syndrome type 3. Our aim was to evaluate the efficacy of mexiletine in long QT syndrome type 2 (LQT2) using cardiomyocytes derived from patient-specific human induced pluripotent stem cells, a transgenic LQT2 rabbit model, and patients with LQT2. METHODS: Heart rate-corrected field potential duration, a surrogate for QTc, was measured in human induced pluripotent stem cells from 2 patients with LQT2 (KCNH2-p.A561V, KCNH2-p.R366X) before and after mexiletine using a multiwell multi-electrode array system. Action potential duration at 90% repolarization (APD90) was evaluated in cardiomyocytes isolated from transgenic LQT2 rabbits (KCNH2-p.G628S) at baseline and after mexiletine application. Mexiletine was given to 96 patients with LQT2. Patients were defined as responders in the presence of a QTc shortening ≥40 ms. Antiarrhythmic efficacy of mexiletine was evaluated by a Poisson regression model. RESULTS: After acute treatment with mexiletine, human induced pluripotent stem cells from both patients with LQT2 showed a significant shortening of heart rate-corrected field potential duration compared with dimethyl sulfoxide control. In cardiomyocytes isolated from LQT2 rabbits, acute mexiletine significantly shortened APD90 by 113 ms, indicating a strong mexiletine-mediated shortening across different LQT2 model systems. Mexiletine was given to 96 patients with LQT2 either chronically (n=60) or after the acute oral drug test (n=36): 65% of the patients taking mexiletine only chronically and 75% of the patients who performed the acute oral test were responders. There was a significant correlation between basal QTc and ∆QTc during the test (r= -0.8; P<0.001). The oral drug test correctly predicted long-term effect in 93% of the patients. Mexiletine reduced the mean yearly event rate from 0.10 (95% CI, 0.07-0.14) to 0.04 (95% CI, 0.02-0.08), with an incidence rate ratio of 0.40 (95% CI, 0.16-0.84), reflecting a 60% reduction in the event rate (P=0.01). CONCLUSIONS: Mexiletine significantly shortens cardiac repolarization in LQT2 human induced pluripotent stem cells, in the LQT2 rabbit model, and in the majority of patients with LQT2. Furthermore, mexiletine showed antiarrhythmic efficacy. Mexiletine should therefore be considered a valid therapeutic option to be added to conventional therapies in higher-risk patients with LQT2.
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Animales Modificados Genéticamente , Células Madre Pluripotentes Inducidas , Síndrome de QT Prolongado , Mexiletine , Miocitos Cardíacos , Mexiletine/farmacología , Mexiletine/uso terapéutico , Animales , Humanos , Conejos , Miocitos Cardíacos/efectos de los fármacos , Síndrome de QT Prolongado/tratamiento farmacológico , Síndrome de QT Prolongado/fisiopatología , Síndrome de QT Prolongado/genética , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Masculino , Femenino , Adulto , Potenciales de Acción/efectos de los fármacos , Antiarrítmicos/farmacología , Antiarrítmicos/uso terapéutico , Adolescente , Persona de Mediana Edad , Adulto Joven , Canal de Potasio ERG1/genética , Canal de Potasio ERG1/antagonistas & inhibidores , Canal de Potasio ERG1/metabolismo , Frecuencia Cardíaca/efectos de los fármacos , Modelos Animales de Enfermedad , Niño , Resultado del TratamientoRESUMEN
Atrial fibrillation (AF) is the most common cardiac arrhythmia worldwide; however, the underlying causes of AF initiation are still poorly understood, particularly because currently available models do not allow in distinguishing the initial causes from maladaptive remodeling that induces and perpetuates AF. Lately, the genetic background has been proven to be important in the AF onset. iPSC-derived cardiomyocytes, being patient- and mutation-specific, may help solve this diatribe by showing the initial cell-autonomous changes underlying the development of the disease. Transcription factor paired-like homeodomain 2 (PITX2) has been identified as a key regulator of atrial development/differentiation, and the PITX2 genomic locus has the highest association with paroxysmal AF. PITX2 influences mitochondrial activity, and alterations in either its expression or function have been widely associated with AF. In this work, we investigate the activity of mitochondria in iPSC-derived atrial cardiomyocytes (aCMs) obtained from a young patient (24 years old) with paroxysmal AF, carrying a gain-of-function mutation in PITX2 (rs138163892) and from its isogenic control (CTRL) in which the heterozygous point mutation has been reverted to WT. PITX2 aCMs show a higher mitochondrial content, increased mitochondrial activity, and superoxide production under basal conditions when compared to CTRL aCMs. However, increasing mitochondrial workload by FCCP or ß-adrenergic stimulation allows us to unmask mitochondrial defects in PITX2 aCMs, which are incapable of responding efficiently to the higher energy demand, determining ATP deficiency.
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AIMS: Nfix is a transcription factor belonging to the Nuclear Factor I (NFI) family comprising four members (Nfia, b, c, x). Nfix plays important roles in the development and function of several organs. In muscle development, Nfix controls the switch from embryonic to fetal myogenesis by promoting fast twitching fibres. In the adult muscle, following injury, lack of Nfix impairs regeneration, inducing higher content of slow-twitching fibres. Nfix is expressed also in the heart, but its function has been never investigated before. We studied Nfix role in this organ. METHODS: Using Nfix-null and wild type (WT) mice we analyzed: (1) the expression pattern of Nfix during development by qPCR and (2) the functional alterations caused by its absence, by in vivo telemetry and in vitro patch clamp analysis. RESULTS AND CONCLUSIONS: Nfix expression start in the heart from E12.5. Adult hearts of Nfix-null mice show a hearts morphology and sarcomeric proteins expression similar to WT. However, Nfix-null animals show tachycardia that derives form an intrinsic higher beating rate of the sinus node (SAN). Molecular and functional analysis revealed that sinoatrial cells of Nfix-null mice express a significantly larger L-type calcium current (Cacna1d + Cacna1c). Interestingly, downregulation of Nfix by sh-RNA in primary cultures of neonatal rat ventricular cardiomyocytes induced a similar increase in their spontaneous beating rate and in ICaL current. In conclusion, our data provide the first demonstration of a role of Nfix that, increasing the L-type calcium current, modulates heart rate.
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AIMS: Current long QT syndrome (LQTS) therapy, largely based on beta-blockade, does not prevent arrhythmias in all patients; therefore, novel therapies are warranted. Pharmacological inhibition of the serum/glucocorticoid-regulated kinase 1 (SGK1-Inh) has been shown to shorten action potential duration (APD) in LQTS type 3. We aimed to investigate whether SGK1-Inh could similarly shorten APD in LQTS types 1 and 2. METHODS AND RESULTS: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and hiPSC-cardiac cell sheets (CCS) were obtained from LQT1 and LQT2 patients; CMs were isolated from transgenic LQT1, LQT2, and wild-type (WT) rabbits. Serum/glucocorticoid-regulated kinase 1 inhibition effects (300â nM-10â µM) on field potential durations (FPD) were investigated in hiPSC-CMs with multielectrode arrays; optical mapping was performed in LQT2 CCS. Whole-cell and perforated patch clamp recordings were performed in isolated LQT1, LQT2, and WT rabbit CMs to investigate SGK1-Inh (3â µM) effects on APD. In all LQT2 models across different species (hiPSC-CMs, hiPSC-CCS, and rabbit CMs) and independent of the disease-causing variant (KCNH2-p.A561V/p.A614V/p.G628S/IVS9-28A/G), SGK1-Inh dose-dependently shortened FPD/APD at 0.3-10â µM (by 20-32%/25-30%/44-45%). Importantly, in LQT2 rabbit CMs, 3â µM SGK1-Inh normalized APD to its WT value. A significant FPD shortening was observed in KCNQ1-p.R594Q hiPSC-CMs at 1/3/10â µM (by 19/26/35%) and in KCNQ1-p.A341V hiPSC-CMs at 10â µM (by 29%). No SGK1-Inh-induced FPD/APD shortening effect was observed in LQT1 KCNQ1-p.A341V hiPSC-CMs or KCNQ1-p.Y315S rabbit CMs at 0.3-3â µM. CONCLUSION: A robust SGK1-Inh-induced APD shortening was observed across different LQT2 models, species, and genetic variants but less consistently in LQT1 models. This suggests a genotype- and variant-specific beneficial effect of this novel therapeutic approach in LQTS.
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Células Madre Pluripotentes Inducidas , Síndrome de QT Prolongado , Animales , Humanos , Conejos , Glucocorticoides , Canal de Potasio KCNQ1/genética , Síndrome de QT Prolongado/tratamiento farmacológico , Síndrome de QT Prolongado/genética , Arritmias Cardíacas/genética , Miocitos Cardíacos/fisiología , Potenciales de Acción/fisiologíaRESUMEN
Discovered some 40 years ago, the If current has since been known as the "pacemaker" current due to its role in the initiation and modulation of the heartbeat and of neuronal excitability. But this is not all, the funny current keeps entertaining the researchers; indeed, several data discovering novel and uncanonical roles of f/HCN channel are quickly accumulating. In the present review, we provide an overview of the expression and cellular functions of HCN/f channels in a variety of systems/organs, and particularly in sour taste transduction, hormones secretion, activation of astrocytes and microglia, inhibition of osteoclastogenesis, renal ammonium excretion, and peristalsis in the gastrointestinal and urine systems. We also analyzed the role of HCN channels in sustaining cellular respiration in mitochondria and their participation to mitophagy under specific conditions. The relevance of HCN currents in undifferentiated cells, and specifically in the control of stem cell cycle and in bioelectrical signals driving left/right asymmetry during zygote development, is also considered. Finally, we present novel data concerning the expression of HCN mRNA in human leukocytes. We can thus conclude that the emerging evidence presented in this review clearly points to an increasing interest and importance of the "funny" current that goes beyond its role in cardiac sinoatrial and neuronal excitability regulation.
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Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Canales de Potasio , Corazón , Frecuencia Cardíaca , Humanos , NeuronasRESUMEN
AIMS: PCSK9 is secreted into the circulation, mainly by the liver, and interacts with low-density lipoprotein receptor (LDLR) homologous and non-homologous receptors, including CD36, thus favouring their intracellular degradation. As PCSK9 deficiency increases the expression of lipids and lipoprotein receptors, thus contributing to cellular lipid accumulation, we investigated whether this could affect heart metabolism and function. METHODS AND RESULTS: Wild-type (WT), Pcsk9 KO, Liver conditional Pcsk9 KO and Pcsk9/Ldlr double KO male mice were fed for 20 weeks with a standard fat diet and then exercise resistance, muscle strength, and heart characteristics were evaluated. Pcsk9 KO presented reduced running resistance coupled to echocardiographic abnormalities suggestive of heart failure with preserved ejection fraction (HFpEF). Heart mitochondrial activity, following maximal coupled and uncoupled respiration, was reduced in Pcsk9 KO mice compared to WT mice and was coupled to major changes in cardiac metabolism together with increased expression of LDLR and CD36 and with lipid accumulation. A similar phenotype was observed in Pcsk9/Ldlr DKO, thus excluding a contribution for LDLR to cardiac impairment observed in Pcsk9 KO mice. Heart function profiling of the liver selective Pcsk9 KO model further excluded the involvement of circulating PCSK9 in the development of HFpEF, pointing to a possible role locally produced PCSK9. Concordantly, carriers of the R46L loss-of-function variant for PCSK9 presented increased left ventricular mass but similar ejection fraction compared to matched control subjects. CONCLUSION: PCSK9 deficiency impacts cardiac lipid metabolism in an LDLR independent manner and contributes to the development of HFpEF.
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Insuficiencia Cardíaca , Proproteína Convertasa 9 , Animales , Insuficiencia Cardíaca/genética , Masculino , Ratones , Ratones Noqueados , Proproteína Convertasa 9/genética , Receptores de LDL/genética , Volumen SistólicoRESUMEN
Properties of the funny current (If) have been studied in several animal and cellular models, but so far little is known concerning its properties in human pacemaker cells. This work provides a detailed characterization of If in human-induced pluripotent stem cell (iPSC)-derived pacemaker cardiomyocytes (pCMs), at different time points. Patch-clamp analysis showed that If density did not change during differentiation; however, after day 30, it activates at more negative potential and with slower time constants. These changes are accompanied by a slowing in beating rate. If displayed the voltage-dependent block by caesium and reversed (Erev) at - 22 mV, compatibly with the 3:1 K+/Na+ permeability ratio. Lowering [Na+]o (30 mM) shifted the Erev to - 39 mV without affecting conductance. Increasing [K+]o (30 mM) shifted the Erev to - 15 mV with a fourfold increase in conductance. pCMs express mainly HCN4 and HCN1 together with the accessory subunits CAV3, KCR1, MiRP1, and SAP97 that contribute to the context-dependence of If. Autonomic agonists modulated the diastolic depolarization, and thus rate, of pCMs. The adrenergic agonist isoproterenol induced rate acceleration and a positive shift of If voltage-dependence (EC50 73.4 nM). The muscarinic agonists had opposite effects (Carbachol EC50, 11,6 nM). Carbachol effect was however small but it could be increased by pre-stimulation with isoproterenol, indicating low cAMP levels in pCMs. In conclusion, we demonstrated that pCMs display an If with the physiological properties expected by pacemaker cells and may thus represent a suitable model for studying human If-related sinus arrhythmias.
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Potenciales de Acción/fisiología , Relojes Biológicos/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Miocitos Cardíacos/fisiología , Potenciales de Acción/efectos de los fármacos , Relojes Biológicos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Electrofisiología/métodos , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Atrios Cardíacos/fisiopatología , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Isoproterenol/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp/métodos , Nodo Sinoatrial/efectos de los fármacos , Nodo Sinoatrial/metabolismo , Nodo Sinoatrial/fisiologíaRESUMEN
In the early phases of the COVID-19 pandemic, drug repurposing was widely used to identify compounds that could improve the prognosis of symptomatic patients infected by SARS-CoV-2. Hydroxychloroquine (HCQ) was one of the first drugs used to treat COVID-19 due to its supposed capacity of inhibiting SARS-CoV-2 infection and replication in vitro. While its efficacy is debated, HCQ has been associated with QT interval prolongation and potentially Torsades de Pointes, especially in patients predisposed to developing drug-induced Long QT Syndrome (LQTS) as silent carriers of variants associated with congenital LQTS. If confirmed, these effects represent a limitation to the at-home use of HCQ for COVID-19 infection as adequate ECG monitoring is challenging. We investigated the proarrhythmic profile of HCQ with Multi-Electrode Arrays after exposure of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from two healthy donors, one asymptomatic and two symptomatic LQTS patients. We demonstrated that: I) HCQ induced a concentration-dependent Field Potential Duration (FPD) prolongation and halted the beating at high concentration due to the combined effect of HCQ on multiple ion currents. II) hiPSC-CMs from healthy or asymptomatic carriers tolerated higher concentrations of HCQ and showed lower susceptibility to HCQ-induced electrical abnormalities regardless of baseline FPD. These findings agree with the clinical safety records of HCQ and demonstrated that hiPSC-CMs potentially discriminates symptomatic vs. asymptomatic mutation carriers through pharmacological interventions. Disease-specific cohorts of hiPSC-CMs may be a valid preliminary addition to assess drug safety in vulnerable populations, offering rapid preclinical results with valuable translational relevance for precision medicine.
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GNB5 loss-of-function pathogenic variants cause IDDCA, a rare autosomal recessive human genetic disease characterized by infantile onset of intellectual disability, sinus bradycardia, hypotonia, visual abnormalities, and epilepsy. We generated human induced pluripotent stem cells (hiPSCs) from skin fibroblasts of a patient with the homozygous c.136delG frameshift variant, and a GNB5 knock-out (KO) line by CRISPR/Cas9 editing. hiPSCs express common pluripotency markers and differentiate into the three germ layers. These lines represent a powerful cellular model to study the molecular basis of GNB5-related disorders as well as offer an in vitro model for drug screening.