RESUMEN
Genome-wide association studies have reported a correlation between a SNP of the RING finger E3 ubiquitin protein ligase rififylin (RFFL) and QT interval variability in humans (Newton-Cheh et al., 2009). Previously, we have shown that RFFL downregulates expression and function of the human-like ether-a-go-go-related gene potassium channel and corresponding rapidly activating delayed rectifier potassium current (IKr) in adult rabbit ventricular cardiomyocytes. Here, we report that RFFL also affects the transient outward current (Ito), but in a peculiar way. RFFL overexpression in adult rabbit ventricular cardiomyocytes significantly decreases the contribution of its fast component (Ito,f) from 35% to 21% and increases the contribution of its slow component (Ito,s) from 65% to 79%. Since Ito,f in rabbits is mainly conducted by Kv4.3, we investigated the effect of RFFL on Kv4.3 expressed in HEK293A cells. We found that RFFL overexpression reduced Kv4.3 expression and corresponding Ito,f in a RING domain-dependent manner in the presence or absence of its accessory subunit Kv channel-interacting protein 2. On the other hand, RFFL overexpression in Kv1.4-expressing HEK cells leads to an increase in both Kv1.4 expression level and Ito,s, similarly in a RING domain-dependent manner. Our physiologically detailed rabbit ventricular myocyte computational model shows that these yin and yang effects of RFFL overexpression on Ito,f, and Ito,s affect phase 1 of the action potential waveform and slightly decrease its duration in addition to suppressing IKr. Thus, RFFL modifies cardiac repolarization reserve via ubiquitination of multiple proteins that differently affect various potassium channels and cardiac action potential duration.
Asunto(s)
Miocitos Cardíacos , Canales de Potasio Shal , Ubiquitina-Proteína Ligasas , Animales , Humanos , Conejos , Potenciales de Acción/fisiología , Estudio de Asociación del Genoma Completo , Miocitos Cardíacos/metabolismo , Potasio/metabolismo , Canales de Potasio Shal/genética , Canales de Potasio Shal/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Células HEK293RESUMEN
The QT interval is a recording of cardiac electrical activity. Previous genome-wide association studies identified genetic variants that modify the QT interval upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor-α factor), a protein encoding a regulator of endosomal trafficking. However, it was not clear how LITAF might impact cardiac excitation. We investigated the effect of LITAF on the voltage-gated sodium channel Nav1.5, which is critical for cardiac depolarization. We show that overexpressed LITAF resulted in a significant increase in the density of Nav1.5-generated voltage-gated sodium current INa and Nav1.5 surface protein levels in rabbit cardiomyocytes and in HEK cells stably expressing Nav1.5. Proximity ligation assays showed co-localization of endogenous LITAF and Nav1.5 in cardiomyocytes, whereas co-immunoprecipitations confirmed they are in the same complex when overexpressed in HEK cells. In vitro data suggest that LITAF interacts with the ubiquitin ligase NEDD4-2, a regulator of Nav1.5. LITAF overexpression down-regulated NEDD4-2 in cardiomyocytes and HEK cells. In HEK cells, LITAF increased ubiquitination and proteasomal degradation of co-expressed NEDD4-2 and significantly blunted the negative effect of NEDD4-2 on INa We conclude that LITAF controls cardiac excitability by promoting degradation of NEDD4-2, which is essential for removal of surface Nav1.5. LITAF-knockout zebrafish showed increased variation in and a nonsignificant 15% prolongation of action potential duration. Computer simulations using a rabbit-cardiomyocyte model demonstrated that changes in Ca2+ and Na+ homeostasis are responsible for the surprisingly modest action potential duration shortening. These computational data thus corroborate findings from several genome-wide association studies that associated LITAF with QT interval variation.
Asunto(s)
Endosomas/metabolismo , Miocitos Cardíacos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina/metabolismo , Potenciales de Acción , Animales , Estudio de Asociación del Genoma Completo , Humanos , Miocitos Cardíacos/citología , Canal de Sodio Activado por Voltaje NAV1.5/genética , Ubiquitina-Proteína Ligasas Nedd4/genética , Proteínas Nucleares/genética , Unión Proteica , Transporte de Proteínas , Conejos , Factores de Transcripción/genética , Ubiquitinación , Pez CebraRESUMEN
BACKGROUND: Long QT syndrome has been associated with sudden cardiac death likely caused by early afterdepolarizations (EADs) and polymorphic ventricular tachycardias (PVTs). Suppressing the late sodium current (INaL) may counterbalance the reduced repolarization reserve in long QT syndrome and prevent EADs and PVTs. METHODS: We tested the effects of the selective INaL blocker GS967 on PVT induction in a transgenic rabbit model of long QT syndrome type 2 using intact heart optical mapping, cellular electrophysiology and confocal Ca2+ imaging, and computer modeling. RESULTS: GS967 reduced ventricular fibrillation induction under a rapid pacing protocol (n=7/14 hearts in control versus 1/14 hearts at 100 nmol/L) without altering action potential duration or restitution and dispersion. GS967 suppressed PVT incidences by reducing Ca2+-mediated EADs and focal activity during isoproterenol perfusion (at 30 nmol/L, n=7/12 and 100 nmol/L n=8/12 hearts without EADs and PVTs). Confocal Ca2+ imaging of long QT syndrome type 2 myocytes revealed that GS967 shortened Ca2+ transient duration via accelerating Na+/Ca2+ exchanger (INCX)-mediated Ca2+ efflux from cytosol, thereby reducing EADs. Computer modeling revealed that INaL potentiates EADs in the long QT syndrome type 2 setting through (1) providing additional depolarizing currents during action potential plateau phase, (2) increasing intracellular Na+ (Nai) that decreases the depolarizing INCX thereby suppressing the action potential plateau and delaying the activation of slowly activating delayed rectifier K+ channels (IKs), suggesting important roles of INaL in regulating Nai. CONCLUSIONS: Selective INaL blockade by GS967 prevents EADs and abolishes PVT in long QT syndrome type 2 rabbits by counterbalancing the reduced repolarization reserve and normalizing Nai. Graphic Abstract: A graphic abstract is available for this article.
Asunto(s)
Antiarrítmicos/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Síndrome de QT Prolongado/tratamiento farmacológico , Miocitos Cardíacos/efectos de los fármacos , Piridinas/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/efectos de los fármacos , Taquicardia Ventricular/prevención & control , Triazoles/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Animales Modificados Genéticamente , Señalización del Calcio/efectos de los fármacos , Simulación por Computador , Canales de Potasio de Tipo Rectificador Tardío/metabolismo , Modelos Animales de Enfermedad , Femenino , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/metabolismo , Síndrome de QT Prolongado/fisiopatología , Masculino , Modelos Cardiovasculares , Miocitos Cardíacos/metabolismo , Conejos , Canales de Sodio/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatología , Factores de Tiempo , Fibrilación Ventricular/genética , Fibrilación Ventricular/metabolismo , Fibrilación Ventricular/fisiopatología , Fibrilación Ventricular/prevención & controlRESUMEN
BACKGROUND: The turnover of cardiac ion channels underlying action potential duration is regulated by ubiquitination. Genome-wide association studies of QT interval identified several single-nucleotide polymorphisms located in or near genes involved in protein ubiquitination. A genetic variant upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor) gene prompted us to determine its role in modulating cardiac excitation. METHODS: Optical mapping was performed in zebrafish hearts to determine Ca2+ transients. Live-cell confocal calcium imaging was performed on adult rabbit cardiomyocytes to determine intracellular Ca2+handling. L-type calcium channel (LTCC) current (ICa,L) was measured using whole-cell recording. To study the effect of LITAF on Cav1.2 (L-type voltage-gated calcium channel 1.2) channel expression, surface biotinylation, and Westerns were performed. LITAF interactions were studied using coimmunoprecipitation and in situ proximity ligation assay. RESULTS: LITAF knockdown in zebrafish resulted in a robust increase in calcium transients. Overexpressed LITAF in 3-week-old rabbit cardiomyocytes resulted in a decrease in ICa,L and Cavα1c abundance, whereas LITAF knockdown increased ICa,L and Cavα1c protein. LITAF-overexpressing decreases calcium transients in adult rabbit cardiomyocytes, which was associated with lower Cavα1c levels. In tsA201 cells, overexpressed LITAF downregulated total and surface pools of Cavα1c via increased Cavα1c ubiquitination and its subsequent lysosomal degradation. We observed colocalization between LITAF and LTCC in tsA201 and cardiomyocytes. In tsA201, NEDD (neural precursor cell expressed developmentally downregulated protein) 4-1, but not its catalytically inactive form NEDD4-1-C867A, increased Cavα1c ubiquitination. Cavα1c ubiquitination was further increased by coexpressed LITAF and NEDD4-1 but not NEDD4-1-C867A. NEDD4-1 knockdown abolished the negative effect of LITAF on ICa,L and Cavα1c levels in 3-week-old rabbit cardiomyocytes. Computer simulations demonstrated that a decrease of ICa,L current associated with LITAF overexpression simultaneously shortened action potential duration and decreased calcium transients in rabbit cardiomyocytes. CONCLUSIONS: LITAF acts as an adaptor protein promoting NEDD4-1-mediated ubiquitination and subsequent degradation of LTCC, thereby controlling LTCC membrane levels and function and thus cardiac excitation.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de la Membrana/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/genética , Proteínas de Unión al ADN/genética , Corazón/embriología , Humanos , Proteínas de la Membrana/genética , Miocitos Cardíacos/enzimología , Ubiquitina-Proteína Ligasas Nedd4/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conejos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitinación , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/genéticaRESUMEN
Long QT syndrome type 2 (LQT2) is a congenital disease characterized by loss of function mutations in hERG potassium channels (IKr). LQT2 is associated with fatal ventricular arrhythmias promoted by triggered activity in the form of early afterdepolarizations (EADs). We previously demonstrated that intracellular Ca2+ handling is remodeled in LQT2 myocytes. Remodeling leads to aberrant late RyR-mediated Ca2+ releases that drive forward-mode Na+-Ca2+ exchanger (NCX) current and slow repolarization to promote reopening of L-type calcium channels and EADs. Forward-mode NCX was found to be enhanced despite the fact that these late releases do not significantly alter the whole-cell cytosolic calcium concentration during a vulnerable period of phase 2 of the action potential corresponding to the onset of EADs. Here, we use a multiscale ventricular myocyte model to explain this finding. We show that because the local NCX current is a saturating nonlinear function of the local submembrane calcium concentration, a larger number of smaller-amplitude discrete Ca2+ release events can produce a large increase in whole-cell forward-mode NCX current without increasing significantly the whole-cell cytosolic calcium concentration. Furthermore, we develop novel insights, to our knowledge, into how alterations of stochastic RyR activity at the single-channel level cause late aberrant Ca2+ release events. Experimental measurements in transgenic LTQ2 rabbits confirm the critical arrhythmogenic role of NCX and identify this current as a potential target for antiarrhythmic therapies in LQT2.
Asunto(s)
Calcio/metabolismo , Espacio Intracelular/metabolismo , Síndrome de QT Prolongado/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Intercambiador de Sodio-Calcio/metabolismo , Animales , Citosol/metabolismo , Ventrículos Cardíacos/patología , Activación del Canal Iónico , Modelos Biológicos , Fenotipo , Probabilidad , Conejos , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
BACKGROUND: Sudden death in long-QT syndrome type 1 (LQT1), an inherited disease caused by loss-of-function mutations in KCNQ1, is triggered by early afterdepolarizations (EADs) that initiate polymorphic ventricular tachycardia (pVT). We investigated ionic mechanisms that underlie pVT in LQT1 using a transgenic rabbit model of LQT1. METHODS: Optical mapping, cellular patch clamping, and computer modeling were used to elucidate the mechanisms of EADs in transgenic LQT1 rabbits. RESULTS: The results showed that shorter action potential duration in the right ventricle (RV) was associated with focal activity during pVT initiation. RV cardiomyocytes demonstrated higher incidence of EADs under 50 nmol/L isoproterenol. Voltage-clamp studies revealed that the transient outward potassium current (Ito) magnitude was 28% greater in RV associated with KChiP2 but with no differences in terms of calcium-cycling kinetics and other sarcolemmal currents. Perfusing with the Ito blocker 4-aminopyridine changed the initial focal sites of pVT from the RV to the left ventricle, corroborating the role of Ito in pVT initiation. Computer modeling showed that EADs occur preferentially in the RV because of the larger conductance of the slow-inactivating component of Ito, which repolarizes the membrane potential sufficiently rapidly to allow reactivation of ICa,L before IKr has had sufficient time to activate. CONCLUSIONS: Ito heterogeneity creates both triggers and an arrhythmogenic substrate in LQT1. In the absence of IKs, Ito interactions with ICa,L and IKr promote EADs in the RV while prolonging action potential duration in the left ventricle. This heterogeneity of action potential enhances dispersion of refractoriness and facilitates conduction blocks that initiate pVTs.