Background In catecholaminergic polymorphic ventricular tachycardia (CPVT) cardiac Purkinje cells (PCs) appear more susceptible to Ca2+ dysfunction than ventricular myocytes (VMs). The underlying mechanisms remain unknown. Using a CPVT mouse (RyR2R4496C+/Cx40eGFP), we tested whether PC intracellular Ca2+ ([Ca2+]i) dysregulation results from a constitutive [Na+]i surplus relative to VMs. Methods and Results Simultaneous optical mapping of voltage and [Ca2+]i in CPVT hearts showed that spontaneous Ca2+ release (SCR) preceded pacing-induced triggered activity at subendocardial PCs. On simultaneous current-clamp and Ca2+ imaging early (EADs) and delayed (DADs) afterdepolarizations trailed SCR and were more frequent in CPVT PCs than CPVT VMs. As a result of increased activity of mutant RyR2 channels, sarcoplasmic reticulum (SR) Ca2+ load, measured by caffeine-induced Ca2+ transients, was lower in CPVT VM and PCs than respective controls (Ctrls) and SR fractional release was greater in both CPVT PCs and VMs than respective Ctrls. [Na+]i was higher in both Ctrl and CPVT PCs than VMs, whereas the density of the Na+-Ca2+ exchanger (NCX) current was not different between PCs and VMs. Computer simulations using a PC model predicted that the elevated [Na+]i of PCs promoted DADs, which were always preceded by SCR events from hyperactive RyR2 channels. Increasing [Na+]i monotonically increased DAD frequency. Confocal imaging experiments showed that post-pacing Ca2+ spark frequency was highest in intact CPVT PCs, but such differences were reversed upon saponin-induced membrane permeabilization, indicating that differences in [Na+]i played a central role. Conclusions In CPVT mice, the constitutive [Na+]i excess of PCs promotes triggered activity and arrhythmogenesis at lower levels of stress than VMs.

Cardiac Purkinje cells (PCs) are morphologically and electrophysiologically different from ventricular myocytes and, importantly, exhibit distinct calcium (Ca2+) homeostasis. Recent studies suggest that PCs are more susceptible to action potential (AP) abnormalities than ventricular myocytes; however, the exact mechanisms are poorly understood. In this study, we utilized a detailed biophysical mathematical model of a murine PC to systematically examine the role of cytosolic Ca2+ diffusion in shaping the AP in PCs. A biphasic spatiotemporal Ca2+ diffusion process, as recorded experimentally, was implemented in the model. In this study, we investigated the role of cytosolic Ca2+ dynamics on AP and ionic current properties by varying the effective Ca2+ diffusion rate. It was observed that AP morphology, specifically the plateau, was affected due to changes in the intracellular Ca2+ dynamics. Elevated Ca2+ concentration in the sarcolemmal region activated inward sodium–Ca2+ exchanger (NCX) current, resulting in a prolongation of the AP plateau at faster diffusion rates. Artificially clamping the NCX current to control values completely reversed the alterations in the AP plateau, thus confirming the role of NCX in modifying the AP morphology. Our results demonstrate that cytosolic Ca2+ diffusion waves play a significant role in shaping APs of PCs and could provide mechanistic insights in the increased arrhythmogeneity of PCs.

Cardiac Purkinje cells (PCs) are more susceptible to action potential abnormalities as compared to ventricular myocytes (VMs), which could be associated with their distinct intracellular calcium handling. We developed a detailed biophysical model of a mouse cardiac PC, which importantly reproduces the experimentally observed biphasic cytosolic calcium waves. The model includes a stochastic gating formulation for the opening and closing of ryanodine receptor (RyR) channels, simulated with a Monte Carlo method, to accurately reproduce cytosolic calcium wave propagation and the effects of spontaneous calcium release events. Simulations predict that during an action potential, smaller cytosolic calcium wavelets propagated from the sarcolemma towards the center of the cell and initiated larger magnitude cell‐wide calcium waves via a calcium‐induced‐calcium release mechanism. In the presence of RyR mutations, frequent spontaneous calcium leaks from sarcoplasmic reticulum (SR) initiated calcium waves, which upon reaching the cell periphery produced delayed afterdepolarizations (DADs) via sodium‐calcium exchanger (NCX) and T‐type calcium (ICaT) channel activation. In the presence of isoproterenol‐mediated effects, DADs induced triggered activity by reactivation of fast sodium channels. Based on our model, we found that the activation of either L‐type calcium channels (ICaL), ICaT, sodium‐potassium exchanger (INaK) or NCX is sufficient for occurrence of triggered activity; however, a partial blockade of ICaT or INaK is essential for its successful termination. Our modeling study highlights valuable insights into the mechanisms of DAD‐induced triggered activity mediated via cytosolic calcium waves in cardiac PCs and may elucidate the increased arrhythmogeneity in PCs.

The Cardiac Purkinje cells (PCs) exhibit distinct calcium (Ca2+) homeostasis than that in ventricular myocytes (VMs). Due to lack of t-tubules in PCs, the Ca2+ ions entering the cell have to diffuse through the cytoplasm to reach the sarcoplasmic reticulum (SR) before triggering Ca2+-induced-Ca2+-release (CICR). In recent experimental studies PCs have been shown to be more susceptible to action potential (AP) abnormalities than the VMs, however the exact mechanisms are poorly understood. In this study, we utilize morphologically realistic detailed biophysical mathematical model of a murine PC to systematically examine the role intracellular Ca2+ diffusion in the APs of PCs. A biphasic spatiotemporal Ca2+ diffusion process, as observed experimentally, was implemented in the model which includes radial Ca2+ wavelets and cell wide longitudinal Ca2+ diffusion wave (CWW). The AP morphology, specifically plateau, is affected due to changes in intracellular Ca2+ dynamics. When Ca2+ concentration in sarcolemmal region is elevated, it activated inward sodium Ca2+ exchanger (NCX) current resulting into prolongation of the plateau at faster diffusion rates. Our results demonstrate that the cytosolic Ca2+ diffusion waves play a significant role in shaping APs of PCs and could provide mechanistic insights into the increased arrhythmogeneity of PCs.