RESUMEN
BACKGROUND: Postoperative arrhythmias such as junctional ectopic tachycardia and atrioventricular block are serious postoperative complications for children with congenital heart disease. We hypothesize that ischemia-reperfusion (I/R) related changes exacerbate these postoperative arrhythmias in the neonate heart and administration of postoperative inotropes is contributory. OBJECTIVE: The purpose of this study was to study the effects of I/R and postischemic dopamine application on automaticity and rhythmicity in immature and mature pacemaker cells and whole heart preparations. METHODS: Single pacemaker cells and whole heart models of postoperative arrhythmias were generated in a rabbit model encompassing 3 primary risk factors: age, I/R exposure, and dopamine application. Single cells were studied using current clamp and line scan confocal microscopy, whereas whole hearts were studied using optical mapping. RESULTS: Four responses were observed in neonatal atrioventricular nodal cells (AVNCs): slowing of AVNC automaticity (from 62±10 to 36 ± 12 action potentials per minute, P<.05); induction of arrhythmicity or increased beat-to-beat variability (0.08 ± 0.04 to 3.83 ± 1.79, P<.05); altered automaticity (subthreshold electrical fluctuations); and disruption of calcium transients. In contrast, these responses were not observed in mature AVNCs or neonatal sinoatrial cells. In whole heart experiments, neonatal hearts experienced persistent postischemia arrhythmias of varying severity, whereas mature hearts exhibited no arrhythmias or relatively transient ones. CONCLUSION: Neonatal pacemaker cells and whole hearts demonstrate a susceptibility to I/R insults resulting in alterations in automaticity, which may predispose neonates to postoperative arrhythmias such as junctional ectopic tachycardia and atrioventricular block.
Asunto(s)
Bloqueo Atrioventricular , Nodo Atrioventricular , Cardiopatías Congénitas/cirugía , Daño por Reperfusión Miocárdica , Complicaciones Posoperatorias , Taquicardia Ectópica de Unión , Animales , Animales Recién Nacidos , Bloqueo Atrioventricular/etiología , Bloqueo Atrioventricular/fisiopatología , Bloqueo Atrioventricular/prevención & control , Nodo Atrioventricular/patología , Nodo Atrioventricular/fisiopatología , Modelos Animales de Enfermedad , Cardiopatías Congénitas/fisiopatología , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/etiología , Daño por Reperfusión Miocárdica/fisiopatología , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/fisiopatología , Complicaciones Posoperatorias/prevención & control , Conejos , Taquicardia Ectópica de Unión/etiología , Taquicardia Ectópica de Unión/fisiopatología , Taquicardia Ectópica de Unión/prevención & controlRESUMEN
Although the L-type Ca(2+) current (ICa,L) plays an important role in cardiac contractility and pacemaking, its role in embryonic stem-cell derived cardiomyocytes (ESC-CMs) has not yet been explored in detail. We used patch-clamp techniques to characterize ICa,L, action potential properties, and nifedipine (an ICa,L blocker) sensitivity on spontaneously contracting embryoid bodies (EBs) or isolated ESC-CMs. Cellular preparations exhibited differential sensitivity to nifedipine, with substantial variation in the dose required to abolish automaticity. Isolated ESC-CMs expressing nodal-like action potentials were highly sensitive to nifedipine; 1 nM significantly decreased firing rate, diastolic depolarization rate (DDR), and upstroke velocity, and 10 nM completely abolished spontaneous activity. In contrast, ESC-CMs expressing atrial-like action potentials were relatively nifedipine-resistant, requiring 10 µM to arrest automaticity; 1 µM significantly decreased upstroke velocity while the firing rate and DDR were unaffected. Nodal-like cells exhibited a more negative voltage for half-maximal ICa activation (-30 ± 1 mV vs. -20 ± 3 mV; p<0.05) and slower inactivation (71 ± 10 ms vs. 43 ± 3 ms; p<0.05) than atrial-like cells. Our data indicate that ICa,L differentially regulates automaticity and chronotropy in nodal-like ESC-CMs, and primarily links excitation to contraction in atrial-like ESC-CMs by contributing to the upstroke phase of the action potential.
RESUMEN
BACKGROUND: The properties of the atrioventricular (AV) node in the neonate heart and its role in unique pediatric cardiac arrhythmias such as junctional ectopic tachycardia (JET) are poorly understood. This is due in large part to the dearth of information on the structure and physiology of the AV node in the immature myocardium. METHODS AND RESULTS: Sinoatrial nodal cells (SANCs), AV nodal tissues, and myocytes (AVNCs) were obtained from neonatal (10-day-old) rabbits, and the histological, immunohistological, and electrophysiological properties were characterized in detail. Masson's trichrome histological staining clearly delineated AV nodal structures including the inferior nodal extension, compact node, and the bundle of His region. AV tissue sections and AVNCs were immunolabeled against neurofilament 160 (NF160), connexin 43 (Cx43), hyperpolarization-activated, cyclic nucleotide modulated channel (HCN4), sodium/calcium exchanger, ryanodine receptor, sarcoplasmic/endoplasmic reticulum Ca(2+) pump (SERCA), and phospholamban (PLB). In AVNCs with triple-positive NF160, SERCA, and PLB labeling, SERCA and PLB were found with high degrees of colocalization. The majority (59%) of NF160-positive AVNCs were found to coexpress HCN4. NF160 and HCN4 expression was found to be even higher in SANCs, where 88% of SANCs exhibited coexpression. Spontaneous action potentials recorded from isolated neonatal AVNCs were uniformly of the atrionodal type, showing none of the action potential heterogeneities found in the mature heart. Current recordings found the hyperpolarization-activated funny current (I(f)) in 55% (11 of 21 cells) of AVNCs, consistent with the immunocytochemistry results. CONCLUSIONS: This represents the first detailed electrophysiological and immunohistological report of the neonatal AV node and lays the groundwork for a better understanding of heart rate regulation and unique arrhythmias in the neonate heart.
Asunto(s)
Nodo Atrioventricular/citología , Separación Celular , Potenciales de Acción , Animales , Animales Recién Nacidos , Nodo Atrioventricular/metabolismo , Relojes Biológicos , Proteínas de Unión al Calcio/metabolismo , Conexina 43/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Conductividad Eléctrica , Impedancia Eléctrica , Inmunohistoquímica , Proteínas de Neurofilamentos/metabolismo , Técnicas de Placa-Clamp , Conejos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Factores de TiempoRESUMEN
In adult cardiac cells the established mechanism of excitation-contraction coupling is by calcium-induced calcium release (CICR) mediated by L-type Ca(2+) channels. However, in neonate cardiomyocytes, a CICR modality involving reverse mode Na(+)-Ca(2+) exchanger (NCX) activity predominates. This has been hypothesized to be due, in part, to the high expression levels of NCX in the neonate heart which drop several fold during ontogeny. Very little is known about the nature of NCX distribution within the cardiomyocyte and how this might change with development given the significant differences in gene expression. We investigated the spatial arrangements of NCX in developing rabbit ventricular myocytes with traditional as well as novel image processing and analysis techniques. Using image segmentation, colocalization analysis was conducted at the whole cell, compartmental (cell periphery and cell interior) and object levels. Because NCX has been suggested to colocalize with caveolin-3 (cav-3) and perhaps form a signaling unit within caveolae, the spatial relationship of NCX relative to cav-3 was also examined in detail. NCX and cav-3 objects were found to be isolated islands of lit voxels that are present after thresholding. These objects were categorized into non-colocalized (0%), lowly colocalized (<50%) and highly colocalized (>50%) subpopulations in both the interior and peripheral compartments. Our results show that NCX and cav-3 are distributed on the peripheral membrane as discrete objects and are not highly colocalized throughout development. 3D distance analysis revealed that NCX and cav-3 objects are organized with a longitudinal and lateral periodicity of about 1mum and that NCX and cav-3 cluster appear to be mutually exclusive on the cell periphery. We conclude that despite the very significant decrease in NCX expression with maturation, qualitatively there were no differences in NCX surface distribution or in the spatial relationship to caveolin 3.
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Caveolina 3/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Animales , Compartimento Celular , Ventrículos Cardíacos/citología , Transporte de Proteínas , ConejosRESUMEN
Mechanisms of cardiac excitation-contraction coupling in neonates are still not clearly defined. Previous work in neonates shows reverse-mode Na(+)-Ca(2+) exchange to be the primary route of Ca(2+) entry during systole and the neonatal sarcoplasmic reticulum to have similar capability as that of adult in storing and releasing Ca(2+). We investigated Na(+)-Ca(2+) exchanger (NCX) and ryanodine receptor (RyR) distribution in developing ventricular myocytes using immunofluorescence, confocal microscopy, and digital image analysis. In neonates, both NCX and RyR clusters on the surface of the cell displayed a short longitudinal periodicity of approximately 0.7 microm. However, by adulthood, both proteins were also found in the interior. In the adult, clusters of NCX on the surface of the cell retained the approximately 0.7-microm periodicity whereas clusters of RyR adopted a longer longitudinal periodicity of approximately 2.0 microm. This suggests that neonatal myocytes also have a peri-M-line RyR distribution that is absent in adult myocytes. NCX and RyR colocalized voxel density was maximal in neonates and declined significantly with ontogeny. We conclude in newborns, Ca(2+) influx via NCX could potentially activate the dense network of peripheral Ca(2+) stores via peripheral couplings, evoking Ca(2+)-induced Ca(2+) release.
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Biofisica/métodos , Regulación del Desarrollo de la Expresión Génica , Canal Liberador de Calcio Receptor de Rianodina/química , Intercambiador de Sodio-Calcio/química , Animales , Calcio/metabolismo , Análisis por Conglomerados , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Modelos Estadísticos , Células Musculares/metabolismo , Conformación Proteica , Conejos , Retículo Sarcoplasmático/metabolismoRESUMEN
Rapid, nongenomic effects of 17 beta-estradiol (E(2)) in endothelial cells are postulated to arise from membrane-associated estrogen receptors (ERs), which have not been visualized in vascular tissue. To identify membrane ERs, we used multiple site-directed ER alpha or ER beta antibodies to label en face rat cerebral and coronary arterial endothelia. Western blots revealed a novel 55-kDa ER alpha isoform. Three-dimensional images of cells labeled with these antibodies and markers for the nucleus and caveolin-1 were acquired with a wide-field microscope, deconvolved, and numerically analyzed. We found ER alpha in the nucleus and cell periphery, where one-third colocalized with caveolin-1. The receptor location was dependent on the epitope of the antibody. Human ovarian surface epithelium produced similar results; but in rat myometrium, the distribution was epitope independent and nuclear. ER beta distribution was predominantly intranuclear and epitope independent. A small amount of ER alpha colocalized with ER beta within the nucleus. The results were identical in both arterial preparations and insensitive to E(2). We postulate that the different ER alpha conformations at the membrane, in the nucleus, and between different cell types allow E(2) to trigger cell- and location-specific signaling cascades.