Rationale: The Purkinje fiber network has been proposed as the source of arrhythmogenic Ca2+ release events in catecholaminergic polymorphic ventricular tachycardia (CPVT), yet evidence supporting this mechanism at the cellular level is lacking. Objective: We sought to determine the frequency and severity of spontaneous Ca2+ release events and the response to the antiarrhythmic agent flecainide in Purkinje cells and ventricular myocytes from RyR24496C/+ CPVT mutant mice and littermate controls. Methods and Results: We crossed RyR24496C/+ knock-in mice with the newly described Cntn2-EGFP BAC transgenic mice, which express a fluorescent reporter gene in cells of the cardiac conduction system, including the distal Purkinje fiber network. Isolated ventricular myocytes (EGFP) and Purkinje cells (EGFP) from wild-type hearts and mutant hearts were distinguished by epifluorescence and intracellular Ca dynamics recorded by microfluorimetry. Both wild-type and RyR24496C/+ mutant Purkinje cells displayed significantly slower kinetics of activation and relaxation compared to ventricular myocytes of the same genotype, and τdecay in the mutant Purkinje cells was significantly slower than that observed in wild-type Purkinje cells. Of the 4 groups studied, RyR24496C/+ mutant Purkinje cells were also most likely to develop spontaneous Ca2+ release events, and the number of events per cell was also significantly greater. Furthermore, with isoproterenol treatment, although all 4 groups showed increases in the frequency of arrhythmogenic Ca2+ events, the RyR24496C/+ Purkinje cells responded with the most profound abnormalities in intracellular Ca2+ handling, including a significant increase in the frequency of unstimulated Ca 2+ events and the development of alternans, as well as isolated and sustained runs of triggered beats. Both Purkinje cells and ventricular myocytes from wild-type mice showed suppression of spontaneous Ca2+ release events with flecainide, whereas in RyR24496C/+ mice, the Purkinje cells were preferentially responsive to drug. In contrast, the Ry R24496C/+ blocker tetracaine was equally efficacious in mutant Purkinje cells and ventricular myocytes. Conclusions: Purkinje cells display a greater propensity to develop abnormalities in intracellular Ca handling than ventricular myocytes. This proarrhythmic behavior is enhanced by disease-causing mutations in the RyR24496C/+ Ca2+ release channel and greatly exacerbated by catecholaminergic stimulation, with the development of arrhythmogenic triggered beats. These data support the concept that Purkinje cells are critical contributors to arrhythmic triggers in animal models and humans with CPVT and suggest a broader role for the Purkinje fiber network in the genesis of ventricular arrhythmias.
- Purkinje fiber
- triggered beat
ASJC Scopus subject areas
- Cardiology and Cardiovascular Medicine