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Background: Collecting electrophysiological and molecular data from the murine conduction system presents technical challenges. Thus, only little advantage has been taken of numerous genetically engineered murine models to study excitation through the cardiac conduction system of the mouse., Objective: To develop an approach for isolating murine cardiac Purkinje cells (PCs), to characterize major ionic currents and to use the data to simulate action potentials (APs) recorded from PCs., Methods: Light microscopy was used to isolate and identify PCs from apical and septal cells. Current and voltage clamp techniques were used to record APs and whole cell currents. We then simulated a PC AP on the basis of our experimental data., Results: APs recorded from PCs were significantly longer than those recorded from ventricular cells. The prominent plateau phase of the PC AP was very negative (≈-40 mV). Spontaneous activity was observed only in PCs. The inward rectifier current demonstrated no significant differences compared to ventricular myocytes (VMs). However, sodium current density was larger, and the voltage-gated potassium current density was significantly less in PCs compared with myocytes. T-type Ca(2+) currents (I(Ca,T)) were present in PCs but not VMs. Computer simulations suggest that I(Ca,T) and cytosolic calcium diffusion significantly modulate AP profile recorded in PCs, as compared to VMs., Conclusions: Our study provides the first comprehensive ionic profile of murine PCs. The data show unique features of PC ionic mechanisms that govern its excitation process. Experimental data and numerical modeling results suggest that a smaller voltage-gated potassium current and the presence of I(Ca,T) are important determinants of the longer and relatively negative plateau phase of the APs., (Copyright © 2013 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Background: Inherited arrhythmias can be caused by mutations in the cardiac ryanodine receptor (RyR2). The cellular source of these arrhythmias is unknown. Isolated RyR2(R4496C) mouse ventricular myocytes display arrhythmogenic activity related to spontaneous Ca(2+) release during diastole. On the other hand, recent whole-heart epicardial and endocardial optical mapping data demonstrate that ventricular arrhythmias in the RyR2(R4496C) mouse model of catecholaminergic polymorphic ventricular tachycardia (CPVT) originate in the His-Purkinje system, suggesting that Purkinje cells, and not ventricular myocytes, may be the cellular source of arrhythmogenic activity. The relative effect of the RyR2(R4496C) mutation on calcium homeostasis in ventricular myocytes versus Purkinje cells is unknown., Objective: This study sought to determine which cardiac cell type is more severely affected, in terms of calcium handling, by expression of the RyR2(R4496C) mutant channel: the ventricular myocytes or the Purkinje cells., Methods and Results: To discriminate Purkinje cells from ventricular myocytes, we crossed the RyR2(R4496C) mouse model of CPVT with the Cx40(EGFP/+) transgenic mouse. This genetic cross yields Purkinje cells that express eGFP, and therefore fluoresce green when excited by the appropriate wavelength; ventricular myocytes, which do not express connexin 40, are not green. Intracellular calcium was measured in each cell type using calcium-sensitive probes. Purkinje cells of the RyR2(R4496C) mouse model of CPVT show an approximately 2x greater rate (P < .05) and approximately 2x to 3x greater amplitude (P < .000001) of spontaneous calcium release events than ventricular myocytes isolated from the same heart., Conclusion: These results demonstrate that focally activated arrhythmias originate in the specialized electrical conducting cells of the His-Purkinje system in the RyR2(R4496C) mouse model of CPVT., (Copyright 2010 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)