The role of spatial organization of Carelease sites in the generation of arrhythmogenic diastolic Carelease in myocytes from failing hearts.

Abstract

In heart failure (HF), dysregulated cardiac ryanodine receptors (RyR2) contribute to the generation of diastolic Cawaves (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 Caimaging were applied to study spatially defined Cahandling in ventricular myocytes isolated from normal (control) and failing canine hearts. Based on their activation time upon electrical stimulation, Carelease sites were categorized as coupled, located in close proximity to the sarcolemmal Cachannels, and uncoupled, the Cachannel-free non-junctional Carelease units. In control myocytes, stimulation of β-adrenergic receptors with isoproterenol (Iso) resulted in a preferential increase in Caspark rate at uncoupled sites. This site-specific effect of Iso was eliminated by the phosphatase inhibitor okadaic acid, which caused similar facilitation of Casparks at coupled and uncoupled sites. Iso-challenged HF myocytes exhibited increased predisposition to DCWs compared to control myocytes. In addition, the overall frequency of Casparks 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 Camapping 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 Carelease sites contributes to Ca-dependent arrhythmogenesis in HF.