RESUMEN
Epitranscriptomic modifications of mRNA are important regulators of gene expression. While internal 2'-O-methylation (Nm) has been discovered on mRNA, questions remain about its origin and function in cells and organisms. Here, we show that internal Nm modification can be guided by small nucleolar RNAs (snoRNAs), and that these Nm sites can regulate mRNA and protein expression. Specifically, two box C/D snoRNAs (SNORDs) and the 2'-O-methyltransferase fibrillarin lead to Nm modification in the protein-coding region of peroxidasin (Pxdn). The presence of Nm modification increases Pxdn mRNA expression but inhibits its translation, regulating PXDN protein expression and enzyme activity both in vitro and in vivo. Our findings support a model in which snoRNA-guided Nm modifications of mRNA can regulate physiologic gene expression by altering mRNA levels and tuning protein translation.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Peroxidasa/genética , ARN Mensajero/genética , ARN Nucleolar Pequeño/genética , Proteínas de la Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Humanos , Metilación , Metiltransferasas/metabolismo , Peroxidasa/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN Nucleolar Pequeño/metabolismo , PeroxidasinaRESUMEN
BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic syndrome characterized by sudden death. There are several genetic forms of CPVT associated with mutations in genes encoding the cardiac ryanodine receptor (RyR2) and its auxiliary proteins including calsequestrin (CASQ2) and calmodulin (CaM). It has been suggested that impairment of the ability of RyR2 to stay closed (ie, refractory) during diastole may be a common mechanism for these diseases. Here, we explore the possibility of engineering CaM variants that normalize abbreviated RyR2 refractoriness for subsequent viral-mediated delivery to alleviate arrhythmias in non-CaM-related CPVT. METHODS AND RESULTS: To that end, we have designed a CaM protein (GSH-M37Q; dubbed as therapeutic CaM or T-CaM) that exhibited a slowed N-terminal Ca dissociation rate and prolonged RyR2 refractoriness in permeabilized myocytes derived from CPVT mice carrying the CASQ2 mutation R33Q. This T-CaM was introduced to the heart of R33Q mice through recombinant adeno-associated viral vector serotype 9. Eight weeks postinfection, we performed confocal microscopy to assess Ca handling and recorded surface ECGs to assess susceptibility to arrhythmias in vivo. During catecholamine stimulation with isoproterenol, T-CaM reduced isoproterenol-promoted diastolic Ca waves in isolated CPVT cardiomyocytes. Importantly, T-CaM exposure abolished ventricular tachycardia in CPVT mice challenged with catecholamines. CONCLUSIONS: Our results suggest that gene transfer of T-CaM by adeno-associated viral vector serotype 9 improves myocyte Ca handling and alleviates arrhythmias in a calsequestrin-associated CPVT model, thus supporting the potential of a CaM-based antiarrhythmic approach as a therapeutic avenue for genetically distinct forms of CPVT.
Asunto(s)
Calmodulina/genética , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Frecuencia Cardíaca , Taquicardia Ventricular/terapia , Animales , Señalización del Calcio , Calmodulina/biosíntesis , Calsecuestrina/deficiencia , Calsecuestrina/genética , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Fenotipo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatologíaRESUMEN
BACKGROUND: Long-term aerobic exercise alters autonomic balance, which may not be favorable in heart rate (HR)-dependent arrhythmic diseases including catecholaminergic polymorphic ventricular tachycardia (CPVT) because of preexisting bradycardia and increased sensitivity to parasympathetic stimulation. OBJECTIVE: The purpose of this study was to determine whether long-term exercise-induced autonomic adaptations modify CPVT susceptibility. METHODS: We determined exercise-induced parasympathetic effects on HR, arrhythmia incidence, and intracellular sarcoplasmic reticulum (SR) Ca2+ leak in atrial (ACM) and ventricular (VCM) cardiomyocytes, in exercised (EX) calsequestrin knockout (CASQ2-/-) mice, a model of CPVT. RESULTS: Although 8-week treadmill running improved exercise capacity in EX CPVT mice, the incidence and duration of ventricular tachycardia also increased. HR variability analyses revealed an increased high-frequency component of the power spectrum and root mean square of successive differences in R-R intervals indicating accentuated vagal antagonism during ß-adrenergic stimulation resulting in negligible HR acceleration. In EX CASQ2-/- VCM, peak amplitude of Ca2+ transient (CaT) increased, whereas SR Ca2+ content decreased. Aberrant Ca2+ sparks occurred at baseline, which was exacerbated with isoproterenol. Notably, although 10 µM of the cholinergic agonist carbachol prevented isoproterenol-induced Ca2+ waves in ACM, CaT amplitude, SR Ca2+ load, and isoproterenol-induced Ca2+ waves paradoxically increased in VCM. In parallel, ventricular ryanodine receptor (RyR2) protein expression increased, whereas protein kinase A- and calmodulin-dependent protein kinase II-mediated phosphorylation of RyR2 was not significantly altered, which could imply an increased number of "leaky" channels. CONCLUSION: Our novel results suggest that long-term exercise in CASQ2-/- mice increases susceptibility to ventricular arrhythmias by accentuating vagal antagonism during ß-adrenergic challenge, which prevents HR acceleration and exacerbates abnormal RyR2 Ca2+ leak in EX CASQ2-/- VCM.
Asunto(s)
Calsecuestrina/metabolismo , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Taquicardia Ventricular/metabolismo , Nervio Vago/fisiopatología , Animales , Modelos Animales de Enfermedad , Estudios de Seguimiento , Masculino , Ratones , Ratones Noqueados , Miocitos Cardíacos/patología , Condicionamiento Físico Animal , Taquicardia Ventricular/patología , Taquicardia Ventricular/fisiopatología , Factores de TiempoRESUMEN
In heart failure (HF), dysregulated cardiac ryanodine receptors (RyR2) contribute to the generation of diastolic Ca2+ waves (DCWs), thereby predisposing adrenergically stressed failing hearts to life-threatening arrhythmias. However, the specific cellular, subcellular, and molecular defects that account for cardiac arrhythmia in HF remain to be elucidated. Patch-clamp techniques and confocal Ca2+ imaging were applied to study spatially defined Ca2+ handling in ventricular myocytes isolated from normal (control) and failing canine hearts. Based on their activation time upon electrical stimulation, Ca2+ release sites were categorized as coupled, located in close proximity to the sarcolemmal Ca2+ channels, and uncoupled, the Ca2+ channel-free non-junctional Ca2+ release units. In control myocytes, stimulation of ß-adrenergic receptors with isoproterenol (Iso) resulted in a preferential increase in Ca2+ spark rate at uncoupled sites. This site-specific effect of Iso was eliminated by the phosphatase inhibitor okadaic acid, which caused similar facilitation of Ca2+ sparks at coupled and uncoupled sites. Iso-challenged HF myocytes exhibited increased predisposition to DCWs compared to control myocytes. In addition, the overall frequency of Ca2+ sparks was increased in HF cells due to preferential stimulation of coupled sites. Furthermore, coupled sites exhibited accelerated recovery from functional refractoriness in HF myocytes compared to control myocytes. Spatially resolved subcellular Ca2+ mapping revealed that DCWs predominantly originated from coupled sites. Inhibition of CaMKII suppressed DCWs and prevented preferential stimulation of coupled sites in Iso-challenged HF myocytes. These results suggest that CaMKII- (and phosphatase)-dependent dysregulation of junctional Ca2+ release sites contributes to Ca2+-dependent arrhythmogenesis in HF.
Asunto(s)
Arritmias Cardíacas/metabolismo , Señalización del Calcio , Calcio/metabolismo , Insuficiencia Cardíaca/metabolismo , Frecuencia Cardíaca , Microdominios de Membrana/metabolismo , Miocitos Cardíacos/metabolismo , Función Ventricular Izquierda , Agonistas Adrenérgicos beta/farmacología , Animales , Arritmias Cardíacas/fisiopatología , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio/efectos de los fármacos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Estimulación Cardíaca Artificial , Diástole , Modelos Animales de Enfermedad , Perros , Femenino , Insuficiencia Cardíaca/fisiopatología , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Potenciales de la Membrana , Miocitos Cardíacos/efectos de los fármacos , Periodo Refractario Electrofisiológico , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Sarcolema/metabolismo , Sus scrofa , Factores de Tiempo , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
BACKGROUND: Cardiac arrhythmias are a leading cause of death in the US. Vast majority of these arrhythmias including catecholaminergic polymorphic ventricular tachycardia (CPVT) are associated with increased levels of circulating catecholamines and involve abnormal impulse formation secondary to aberrant Ca2+ and Na+ handling. However, the mechanistic link between ß-AR stimulation and the subcellular/molecular arrhythmogenic trigger(s) remains elusive. METHODS AND RESULTS: We performed functional and structural studies to assess Ca2+ and Na+ signaling in ventricular myocyte as well as surface electrocardiograms in mouse models of cardiac calsequestrin (CASQ2)-associated CPVT. We demonstrate that a subpopulation of Na+ channels (neuronal Na+ channels; nNav) that colocalize with RyR2 and Na+/Ca2+ exchanger (NCX) are a part of the ß-AR-mediated arrhythmogenic process. Specifically, augmented Na+ entry via nNav in the settings of genetic defects within the RyR2 complex and enhanced sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA)-mediated SR Ca2+ refill is both an essential and a necessary factor for the arrhythmogenesis. Furthermore, we show that augmentation of Na+ entry involves ß-AR-mediated activation of CAMKII subsequently leading to nNav augmentation. Importantly, selective pharmacological inhibition as well as silencing of Nav1.6 inhibit myocyte arrhythmic potential and prevent arrhythmias in vivo. CONCLUSION: These data suggest that the arrhythmogenic alteration in Na+/Ca2+ handling evidenced ruing ß-AR stimulation results, at least in part, from enhanced Na+ influx through nNav. Therefore, selective inhibition of these channels and Nav1.6 in particular can serve as a potential antiarrhythmic therapy.
RESUMEN
Although the effects and the underlying mechanism of sympathetic stimulation on cardiac Ca handling are relatively well established both in health and disease, the modes of action and mechanisms of parasympathetic modulation are poorly defined. Here, we demonstrate that parasympathetic stimulation initiates a novel mode of excitation-contraction coupling that enhances the efficiency of cardiac sarcoplasmic reticulum Ca store utilization. This efficient mode of excitation-contraction coupling involves reciprocal changes in the phosphorylation of ryanodine receptor 2 at Ser-2808 and Ser-2814. Specifically, Ser-2808 phosphorylation was mediated by muscarinic receptor subtype 2 and activation of PKG (protein kinase G), whereas dephosphorylation of Ser-2814 involved activation of muscarinic receptor subtype 3 and decreased reactive oxygen species-dependent activation of CaMKII (Ca/calmodulin-dependent protein kinase II). The overall effect of these changes in phosphorylation of ryanodine receptor 2 is an increase in systolic Ca release at the low sarcoplasmic reticulum Ca content and a paradoxical reduction in aberrant Ca leak. Accordingly, cholinergic stimulation of cardiomyocytes isolated from failing hearts improved Ca cycling efficiency by restoring altered ryanodine receptor 2 phosphorylation balance.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calcio/metabolismo , Miocitos Cardíacos/metabolismo , Receptores Muscarínicos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Células Cultivadas , Colinérgicos/farmacología , Modelos Animales de Enfermedad , Perros , Acoplamiento Excitación-Contracción/fisiología , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Ratones , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Sensibilidad y EspecificidadRESUMEN
Treatment for heart disease, the leading cause of death in the world, has progressed little for several decades. Here we develop a protein engineering approach to directly tune in vivo cardiac contractility by tailoring the ability of the heart to respond to the Ca(2+) signal. Promisingly, our smartly formulated Ca(2+)-sensitizing TnC (L48Q) enhances heart function without any adverse effects that are commonly observed with positive inotropes. In a myocardial infarction (MI) model of heart failure, expression of TnC L48Q before the MI preserves cardiac function and performance. Moreover, expression of TnC L48Q after the MI therapeutically enhances cardiac function and performance, without compromising survival. We demonstrate engineering TnC can specifically and precisely modulate cardiac contractility that when combined with gene therapy can be employed as a therapeutic strategy for heart disease.
Asunto(s)
Calcio/metabolismo , Ventrículos Cardíacos/metabolismo , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Ingeniería de Proteínas , Troponina C/genética , Función Ventricular , Animales , Señalización del Calcio , Electrocardiografía , Prueba de Esfuerzo , Tolerancia al Ejercicio , Terapia Genética , Vectores Genéticos , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Contracción Miocárdica , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/fisiopatología , Imagen Óptica , Conejos , Troponina C/metabolismo , UltrasonografíaRESUMEN
AIMS: Cardiac calsequestrin (CASQ2) and histidine-rich Ca-binding protein (HRC) are sarcoplasmic reticulum (SR) Ca-binding proteins that regulate SR Ca release in mammalian heart. Deletion of either CASQ2 or HRC results in relatively mild phenotypes characterized by preserved cardiac structure and function, although CASQ2 knockout (KO), or Cnull, shows increased arrhythmia burden under conditions of catecholaminergic stress. We hypothesized that given the apparent overlap of functions of CASQ2 and HRC, simultaneous ablation of both would deteriorate the cardiac phenotype compared with the single knockouts. METHODS AND RESULTS: In contrast to this expectation, double knockout (DKO) mice lacking both CASQ2 and HRC exhibited normal cardiac ejection fraction and ultrastructure. Moreover, the predisposition to catecholamine-dependent arrhythmia that characterizes the Cnull phenotype was alleviated in the DKO mice. At the myocyte level, DKO mice displayed Ca transients of normal amplitude; additionally, the frequency of spontaneous Ca waves and sparks in the presence of isoproterenol were decreased markedly compared with Cnull. Furthermore, restitution of SR Ca release was slowed in DKO myocytes compared with Cnull cells. CONCLUSION: Our results suggest that rather than being functionally redundant, CASQ2 and HRC modulate cardiac ryanodine receptor-mediated (RyR2) Ca release in an opposing manner. In particular, while CASQ2 stabilizes RyR2 rendering it refractory in the diastolic phase, HRC enhances RyR2 activity facilitating RyR2 recovery from refractoriness.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Calsecuestrina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Taquicardia Ventricular/metabolismo , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Calsecuestrina/genética , Modelos Animales de Enfermedad , Ecocardiografía , Isoproterenol , Ratones Noqueados , Contracción Miocárdica , Miocitos Cardíacos/metabolismoRESUMEN
Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme composed of a catalytic, scaffolding, and regulatory subunit. In the heart, PP2A activity is requisite for cardiac excitation-contraction coupling and central in adrenergic signaling. We found that mice deficient in the PP2A regulatory subunit B56α (1 of 13 regulatory subunits) had altered PP2A signaling in the heart that was associated with changes in cardiac physiology, suggesting that the B56α regulatory subunit had an autoinhibitory role that suppressed excess PP2A activity. The increase in PP2A activity in the mice with reduced B56α expression resulted in slower heart rates and increased heart rate variability, conduction defects, and increased sensitivity of heart rate to parasympathetic agonists. Increased PP2A activity in B56α(+/-) myocytes resulted in reduced Ca(2+) waves and sparks, which was associated with decreased phosphorylation (and thus decreased activation) of the ryanodine receptor RyR2, an ion channel on intracellular membranes that is involved in Ca(2+) regulation in cardiomyocytes. In line with an autoinhibitory role for B56α, in vivo expression of B56α in the absence of altered abundance of other PP2A subunits decreased basal phosphatase activity. Consequently, in vivo expression of B56α suppressed parasympathetic regulation of heart rate and increased RyR2 phosphorylation in cardiomyocytes. These data show that an integral component of the PP2A holoenzyme has an important inhibitory role in controlling PP2A enzyme activity in the heart.
Asunto(s)
Señalización del Calcio , Proteínas Musculares/metabolismo , Miocardio/enzimología , Miocitos Cardíacos/enzimología , Proteína Fosfatasa 2/metabolismo , Animales , Ratones , Ratones Noqueados , Proteínas Musculares/genética , Proteína Fosfatasa 2/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
Excessive oxidative stress in the heart results in contractile dysfunction. While antioxidant therapies have been a disappointment clinically, exercise has shown beneficial results, in part by reducing oxidative stress. We have previously shown that neuronal nitric oxide synthase (nNOS) is essential for cardioprotective adaptations caused by exercise. We hypothesize that part of the cardioprotective role of nNOS is via the augmentation of the antioxidant defense with exercise by positively shifting the nitroso-redox balance. Our results show that nNOS is indispensable for the augmented anti-oxidant defense with exercise. Furthermore, exercise training of nNOS knockout mice resulted in a negative shift in the nitroso-redox balance resulting in contractile dysfunction. Remarkably, overexpressing nNOS (conditional cardiac-specific nNOS overexpression) was able to mimic exercise by increasing VO2max. This study demonstrates that exercise results in a positive shift in the nitroso-redox balance that is nNOS-dependent. Thus, targeting nNOS signaling may mimic the beneficial effects of exercise by combating oxidative stress and may be a viable treatment strategy for heart disease.
Asunto(s)
Contracción Miocárdica/fisiología , Miocardio/metabolismo , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico/biosíntesis , Condicionamiento Físico Animal , Adaptación Fisiológica , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Regulación de la Expresión Génica , Ratones , Ratones Noqueados , Miocardio/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Óxido Nítrico Sintasa de Tipo I/deficiencia , Oxidación-Reducción , Estrés Oxidativo , Cultivo Primario de Células , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
AIMS: Sudden death resulting from cardiac arrhythmias is the most common consequence of cardiac disease. Certain arrhythmias caused by abnormal impulse formation including catecholaminergic polymorphic ventricular tachycardia (CPVT) are associated with delayed afterdepolarizations resulting from diastolic Ca2+ release (DCR) from the sarcoplasmic reticulum (SR). Despite high response of CPVT to agents directly affecting Ca2+ cycling, the incidence of refractory cases is still significant. Surprisingly, these patients often respond to treatment with Na+ channel blockers. However, the relationship between Na+ influx and disturbances in Ca2+ handling immediately preceding arrhythmias in CPVT remains poorly understood and is the object of this study. METHODS AND RESULTS: We performed optical Ca2+ and membrane potential imaging in ventricular myocytes and intact cardiac muscles as well as surface ECGs on a CPVT mouse model with a mutation in cardiac calsequestrin. We demonstrate that a subpopulation of Na+ channels (neuronal Na+ channels; nNav) colocalize with ryanodine receptor Ca2+ release channels (RyR2). Disruption of the crosstalk between nNav and RyR2 by nNav blockade with riluzole reduced and also desynchronized DCR in isolated cardiomyocytes and in intact cardiac tissue. Such desynchronization of DCR on cellular and tissue level translated into decreased arrhythmias in CPVT mice. CONCLUSIONS: Thus, our study offers the first evidence that nNav contribute to arrhythmogenic DCR, thereby providing a conceptual basis for mechanism-based antiarrhythmic therapy.
Asunto(s)
Arritmias Cardíacas/metabolismo , Calcio/metabolismo , Neuronas/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacocinética , Taquicardia Ventricular/metabolismo , Animales , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatología , Calsecuestrina/genética , Diástole/fisiología , Modelos Animales de Enfermedad , Masculino , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Neuronas/fisiología , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Retículo Sarcoplasmático/metabolismo , Taquicardia Ventricular/genética , Taquicardia Ventricular/fisiopatologíaRESUMEN
Phosphorylation of the cardiac ryanodine receptor (RyR2) by protein kinase A (PKA) at Ser-2808 is suggested to mediate the physiological 'fight or flight' response and contribute to heart failure by rendering the sarcoplasmic reticulum (SR) leaky for Ca(2+). In the present study, we examined the potential role of RyR2 phosphorylation at Ser-2808 in the progression of Ca(2+)-dependent cardiomyopathy (CCM) by using mice genetically modified to feature elevated SR Ca(2+) leak while expressing RyR2s that cannot be phosphorylated at this site (S2808A). Surprisingly, rather than alleviating the disease phenotype, constitutive dephosphorylation of Ser-2808 aggravated CCM as manifested by shortened survival, deteriorated in vivo cardiac function, exacerbated SR Ca(2+) leak and mitochondrial injury. Notably, the deteriorations of cardiac function, myocyte Ca(2+) handling, and mitochondria integrity were consistently worse in mice with heterozygous ablation of Ser-2808 than in mice with complete ablation. Wild-type (WT) and CCM myocytes expressing unmutated RyR2s exhibited a high level of baseline phosphorylation at Ser-2808. Exposure of these CCM cells to protein phosphatase 1 caused a transitory increase in Ca(2+) leak attributable to partial dephosphorylation of RyR2 tetramers at Ser-2808 from more fully phosphorylated state. Thus, exacerbated Ca(2+) leak through partially dephosphorylated RyR2s accounts for the prevalence of the disease phenotype in the heterozygous S2808A CCM mice. These results do not support the importance of RyR2 hyperphosphorylation in Ca(2+)-dependent heart disease, and rather suggest roles for the opposite process, the RyR2 dephosphorylation at this residue in physiological and pathophysiological Ca(2+) signalling.
Asunto(s)
Calcio/metabolismo , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Serina/genética , Animales , Femenino , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Fosforilación/fisiologíaRESUMEN
INTRODUCTION: Bisphosphonates, including ibandronate, are used in the prevention and treatment of osteoporosis. METHODS AND RESULTS: We report a case of suspected ibandronate-associated arrhythmia, following a single dose of ibandronate in a 55-year-old female. ECG at presentation revealed frequent ectopy and QT/QTc interval prolongation; at follow-up 9 months later the QT/QTc intervals were normalized. Proarrhythmic potential of ibandronate was assessed with a combination of in vivo and in vitro approaches in canines and canine ventricular myocytes. We observed late onset in vivo repolarization instability after ibandronate treatment. Myocytes superfused with ibandronate exhibited action potential duration (APD) prolongation and variability, increased early afterdepolarizations (EADs) and reduced Ito (P < 0.05), with no change in IKr . Ibandronate-induced APD changes and EADs were prevented by inhibition of intracellular calcium cycling. Ibandronate increased sarcoplasmic reticulum calcium load; during washout there was an increase in calcium spark frequency and spontaneous calcium waves. Computational modeling was used to examine the observed effects of ibandronate. While reductions in Ito alone had modest effects on APD, when combined with altered RyR inactivation kinetics, the model predicted effects on APD and SR Ca(2+) load consistent with observed experimental results. CONCLUSION: Ibandronate may increase the susceptibility to ventricular ectopy and arrhythmias. Collectively these data suggest that reduced Ito combined with abnormal RyR calcium handling may result in a previously unrecognized form of drug-induced proarrhythmia.
Asunto(s)
Conservadores de la Densidad Ósea/efectos adversos , Difosfonatos/efectos adversos , Fibrilación Ventricular/inducido químicamente , Fibrilación Ventricular/diagnóstico , Animales , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Células Cultivadas , Perros , Femenino , Humanos , Ácido Ibandrónico , Masculino , Persona de Mediana Edad , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Fibrilación Ventricular/fisiopatologíaRESUMEN
AIMS: Recent studies suggest that proarrhythmic effects of cardiac glycosides (CGs) on cardiomyocyte Ca(2+) handling involve generation of reactive oxygen species (ROS). However, the specific pathway(s) of ROS production and the subsequent downstream molecular events that mediate CG-dependent arrhythmogenesis remain to be defined. METHODS AND RESULTS: We examined the effects of digitoxin (DGT) on Ca(2+) handling and ROS production in cardiomyocytes using a combination of pharmacological approaches and genetic mouse models. Myocytes isolated from mice deficient in NADPH oxidase type 2 (NOX2KO) and mice transgenically overexpressing mitochondrial superoxide dismutase displayed markedly increased tolerance to the proarrhythmic action of DGT as manifested by the inhibition of DGT-dependent ROS and spontaneous Ca(2+) waves (SCW). Additionally, DGT-induced mitochondrial membrane potential depolarization was abolished in NOX2KO cells. DGT-dependent ROS was suppressed by the inhibition of PI3K, PKC, and the mitochondrial KATP channel, suggesting roles for these proteins, respectively, in activation of NOX2 and in mitochondrial ROS generation. Western blot analysis revealed increased levels of oxidized CaMKII in WT but not in NOX2KO hearts treated with DGT. The DGT-induced increase in SCW frequency was abolished in myocytes isolated from mice in which the Ser 2814 CaMKII phosphorylation site on RyR2 is constitutively inactivated. CONCLUSION: These results suggest that the arrhythmogenic adverse effects of CGs on Ca(2+) handling involve PI3K- and PKC-mediated stimulation of NOX2 and subsequent NOX2-dependent ROS release from the mitochondria; mitochondria-derived ROS then activate CaMKII with consequent phosphorylation of RyR2 at Ser 2814.
Asunto(s)
Antiarrítmicos/toxicidad , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Digitoxina/toxicidad , Miocitos Cardíacos/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Arritmias Cardíacas/inducido químicamente , Calcio/metabolismo , Masculino , Potencial de la Membrana Mitocondrial , Ratones , Ratones Noqueados , NADPH Oxidasas/metabolismo , Fosforilación , Superóxido Dismutasa/metabolismoRESUMEN
It has been proposed that dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) can reduce the risk of ventricular arrhythmias in post-MI patients. Abnormal Ca(2+) handling has been implicated in the genesis of post-MI ventricular arrhythmias. Therefore, we tested the hypothesis that dietary n-3 PUFAs alter the vulnerability of ventricular myocytes to cellular arrhythmia by stabilizing intracellular Ca(2+) cycling. To test this hypothesis, we used a canine model of post-MI ventricular fibrillation (VF) and assigned the animals to either placebo (1 g/day corn oil) or n-3 PUFAs (1-4 g/day) groups. Using Ca(2+) imaging techniques, we examined the intracellular Ca(2+) handling in myocytes isolated from post-MI hearts resistant (VF-) and susceptible (VF+) to VF. Frequency of occurrence of diastolic Ca(2+) waves (DCWs) in VF+ myocytes from placebo group was significantly higher than in placebo-treated VF- myocytes. n-3 PUFA treatment did not decrease frequency of DCWs in VF+ myocytes. In contrast, VF- myocytes from the n-3 PUFA group had a significantly higher frequency of DCWs than myocytes from the placebo group. In addition, n-3 PUFA treatment increased beat-to-beat alterations in the amplitude of Ca(2+) transients (Ca(2+) alternans) in VF- myocytes. These n-3 PUFAs effects in VF- myocytes were associated with an increased Ca(2+) spark frequency and reduced sarcoplasmic reticulum Ca(2+) content, indicative of increased activity of ryanodine receptors. Thus, dietary n-3 PUFAs do not alleviate intracellular Ca(2+) cycling remodeling in myocytes isolated from post-MI VF+ hearts. Furthermore, dietary n-3 PUFAs increase vulnerability of ventricular myocytes to cellular arrhythmia in post-MI VF- hearts by destabilizing intracellular Ca(2+) handling.
Asunto(s)
Arritmias Cardíacas/prevención & control , Calcio/metabolismo , Muerte Súbita Cardíaca/patología , Ácidos Grasos Omega-3/administración & dosificación , Ácidos Grasos Omega-3/metabolismo , Infarto del Miocardio/metabolismo , Animales , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatología , Dieta/métodos , Perros , Corazón/fisiopatología , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Fibrilación Ventricular/metabolismo , Fibrilación Ventricular/fisiopatologíaRESUMEN
Dysregulated intracellular Ca(2+) signaling is implicated in a variety of cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia. Spontaneous diastolic Ca(2+) release (DCR) can induce arrhythmogenic plasma membrane depolarizations, although the mechanism responsible for DCR synchronization among adjacent myocytes required for ectopic activity remains unclear. We investigated the synchronization mechanism(s) of DCR underlying untimely action potentials and diastolic contractions (DCs) in a catecholaminergic polymorphic ventricular tachycardia mouse model with a mutation in cardiac calsequestrin. We used a combination of different approaches including single ryanodine receptor channel recording, optical imaging (Ca(2+) and membrane potential), and contractile force measurements in ventricular myocytes and intact cardiac muscles. We demonstrate that DCR occurs in a temporally and spatially uniform manner in both myocytes and intact myocardial tissue isolated from cardiac calsequestrin mutation mice. Such synchronized DCR events give rise to triggered electrical activity that results in synchronous DCs in the myocardium. Importantly, we establish that synchronization of DCR is a result of a combination of abbreviated ryanodine receptor channel refractoriness and the preceding synchronous stimulated Ca(2+) release/reuptake dynamics. Our study reveals how aberrant DCR events can become synchronized in the intact myocardium, leading to triggered activity and the resultant DCs in the settings of a cardiac rhythm disorder.
Asunto(s)
Señalización del Calcio/fisiología , Calsecuestrina/genética , Corazón/fisiología , Canal Liberador de Calcio Receptor de Rianodina/genética , Taquicardia Ventricular/fisiopatología , Animales , Calcio/metabolismo , Calsecuestrina/fisiología , Diástole/fisiología , Modelos Animales de Enfermedad , Ventrículos Cardíacos/citología , Masculino , Ratones , Ratones Mutantes , Mutación , Miocitos Cardíacos/fisiología , Músculos Papilares/citología , Músculos Papilares/fisiología , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Retículo Sarcoplasmático/fisiología , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismoRESUMEN
The risk of sudden cardiac death is increased following myocardial infarction. Exercise training reduces arrhythmia susceptibility, but the mechanism is unknown. We used a canine model of sudden cardiac death (healed infarction, with ventricular tachyarrhythmias induced by an exercise plus ischemia test, VF+); we previously reported that endurance exercise training was antiarrhythmic in this model (Billman GE. Am J Physiol Heart Circ Physiol 297: H1171-H1193, 2009). A total of 41 VF+ animals were studied, after random assignment to 10 wk of endurance exercise training (EET; n = 21) or a matched sedentary period (n = 20). Following (>1 wk) the final attempted arrhythmia induction, isolated myocytes were used to test the hypotheses that the endurance exercise-induced antiarrhythmic effects resulted from normalization of cellular electrophysiology and/or normalization of calcium handling. EET prevented VF and shortened in vivo repolarization (P < 0.05). EET normalized action potential duration and variability compared with the sedentary group. EET resulted in a further decrement in transient outward current compared with the sedentary VF+ group (P < 0.05). Sedentary VF+ dogs had a significant reduction in repolarizing K(+) current, which was restored by exercise training (P < 0.05). Compared with controls, myocytes from the sedentary VF+ group displayed calcium alternans, increased calcium spark frequency, and increased phosphorylation of S2814 on ryanodine receptor 2. These abnormalities in intracellular calcium handling were attenuated by exercise training (P < 0.05). Exercise training prevented ischemically induced VF, in association with a combination of beneficial effects on cellular electrophysiology and calcium handling.
Asunto(s)
Señalización del Calcio , Muerte Súbita Cardíaca/prevención & control , Terapia por Ejercicio , Miocitos Cardíacos/metabolismo , Resistencia Física , Fibrilación Ventricular/prevención & control , Potenciales de Acción , Animales , Muerte Súbita Cardíaca/etiología , Modelos Animales de Enfermedad , Perros , Electrocardiografía , Frecuencia Cardíaca , Infarto del Miocardio/complicaciones , Técnicas de Placa-Clamp , Fosforilación , Potasio/metabolismo , Canales de Potasio/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Factores de Tiempo , Fibrilación Ventricular/diagnóstico , Fibrilación Ventricular/etiología , Fibrilación Ventricular/metabolismo , Fibrilación Ventricular/fisiopatologíaRESUMEN
RATIONALE: Diastolic spontaneous Ca(2+) waves (DCWs) are recognized as important contributors to triggered arrhythmias. DCWs are thought to arise when [Ca(2+)] in sarcoplasmic reticulum ([Ca(2+)](SR)) reaches a certain threshold level, which might be reduced in cardiac disease as a consequence of sensitization of ryanodine receptors (RyR2s) to luminal Ca(2+). OBJECTIVE: We investigated the mechanisms of DCW generation in myocytes from normal and diseased hearts, using a canine model of post-myocardial infarction ventricular fibrillation (VF). METHODS AND RESULTS: The frequency of DCWs, recorded during periodic pacing in the presence of a ß-adrenergic receptor agonist isoproterenol, was significantly higher in VF myocytes than in normal controls. Rather than occurring immediately on reaching a final [Ca(2+)](SR), DCWs arose with a distinct time delay after attaining steady [Ca(2+)](SR) in both experimental groups. Although the rate of [Ca(2+)](SR) recovery after the SR Ca(2+) release was similar between the groups, in VF myocytes the latency to DCWs was shorter, and the [Ca(2+)](SR) at DCW initiation was lower. The restitution of depolarization-induced Ca(2+) transients, assessed by a 2-pulse protocol, was significantly faster in VF myocytes than in controls. The VF-related alterations in myocyte Ca(2+) cycling were mimicked by the RyR2 agonist, caffeine. The reducing agent, mercaptopropionylglycine, or the CaMKII inhibitor, KN93, decreased DCW frequency and normalized restitution of Ca(2+) release in VF myocytes. CONCLUSIONS: The attainment of a certain threshold [Ca(2+)](SR) is not sufficient for the generation of DCWs. Postrelease Ca(2+) signaling refractoriness critically influences the occurrence of DCWs. Shortened Ca(2+) signaling refractoriness due to RyR2 phosphorylation and oxidation is responsible for the increased rate of DCWs observed in VF myocytes and could provide a substrate for synchronization of arrhythmogenic events at the tissue level in hearts prone to VF.
Asunto(s)
Señalización del Calcio , Muerte Súbita Cardíaca/etiología , Infarto del Miocardio/complicaciones , Miocitos Cardíacos/metabolismo , Fibrilación Ventricular/etiología , Agonistas Adrenérgicos beta , Animales , Bencilaminas/farmacología , Cafeína/farmacología , Agonistas de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Estimulación Cardíaca Artificial , Modelos Animales de Enfermedad , Perros , Acoplamiento Excitación-Contracción , Femenino , Isoproterenol , Masculino , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/efectos de los fármacos , Oxidación-Reducción , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Tiempo de Reacción , Sustancias Reductoras/farmacología , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Sulfonamidas/farmacología , Factores de Tiempo , Tiopronina/farmacología , Fibrilación Ventricular/metabolismo , Fibrilación Ventricular/fisiopatologíaRESUMEN
Nitric oxide (NO) and superoxide (O(2) (-)) are important cardiac signaling molecules that regulate myocyte contraction. For appropriate regulation, NO and O(2) (.-) must exist at defined levels. Unfortunately, the NO and O(2) (.-) levels are altered in many cardiomyopathies (heart failure, ischemia, hypertrophy, etc.) leading to contractile dysfunction and adverse remodeling. Hence, rescuing the nitroso-redox levels is a potential therapeutic strategy. Nitrone spin traps have been shown to scavenge O(2) (.-) while releasing NO as a reaction byproduct; and we synthesized a novel, cell permeable nitrone, 2-2-3,4-dihydro-2H-pyrrole 1-oxide (EMEPO). We hypothesized that EMEPO would improve contractile function in myocytes with altered nitroso-redox levels. Ventricular myocytes were isolated from wildtype (C57Bl/6) and NOS1 knockout (NOS1(-/-)) mice, a known model of NO/O(2) (.-) imbalance, and incubated with EMEPO. EMEPO significantly reduced O(2) (.-) (lucigenin-enhanced chemiluminescence) and elevated NO (DAF-FM diacetate) levels in NOS1(-/-) myocytes. Furthermore, EMEPO increased NOS1(-/-) myocyte basal contraction (Ca(2+) transients, Fluo-4AM; shortening, video-edge detection), the force-frequency response and the contractile response to ß-adrenergic stimulation. EMEPO had no effect in wildtype myocytes. EMEPO also increased ryanodine receptor activity (sarcoplasmic reticulum Ca(2+) leak/load relationship) and phospholamban Serine16 phosphorylation (Western blot). We also repeated our functional experiments in a canine post-myocardial infarction model and observed similar results to those seen in NOS1(-/-) myocytes. In conclusion, EMEPO improved contractile function in myocytes experiencing an imbalance of their nitroso-redox levels. The concurrent restoration of NO and O(2) (.-) levels may have therapeutic potential in the treatment of various cardiomyopathies.
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Calcio/metabolismo , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Óxido Nítrico Sintasa de Tipo I/fisiología , Óxido Nítrico/metabolismo , Óxidos de Nitrógeno/farmacología , Retículo Sarcoplasmático/metabolismo , Animales , Esterificación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Oxidación-Reducción , Retículo Sarcoplasmático/efectos de los fármacos , Marcadores de Spin , Superóxidos/metabolismoRESUMEN
In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca(2+) waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca(2+)/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca(2+) waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca(2+) waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca(2+) cycling, and increased propensity to arrhythmogenesis.