Abstract 2728: Cardiac Purkinje Cells of the Ryr2R4496C Mutant Mouse Model of CPVT Have a Higher Frequency and Amplitude of Spontaneous Calcium Release Than Ventricular Myocytes
Introduction: Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is associated with mutations of the ryanodine receptor (RyR2). Ventricular cardiac myocytes from knock-in mice carrying the RyR2R4496C mutation have a lower threshold for spontaneous calcium release from the sarcoplasmic reticulum and thus a lower threshold for triggered activity than wild-type myocytes. Importantly, in this mouse model ventricular arrhythmias, including bidirectional tachycardia, are the result of triggered activity in the Purkinje fiber network. The effects of the RyR2R4496C mutation on Purkinje cell calcium homeostasis, however, are unknown.
Hypothesis: Cardiac Purkinje cells from the RyR2R4496C mouse have a higher incidence and amplitude of spontaneous calcium release than ventricular myocytes.
Methods: We isolated Purkinje cells and ventricular myocytes from RyR2R4496C mice. Myocyte and Purkinje cells were loaded with calcium sensitive fluorescent probes to monitor intracellular calcium concentrations by epi-fluorescent microscopy. To measure spontaneous calcium release (SCR), each cell type was paced at 6Hz for 30s, after which time the field stimulation was switched off and intracellular calcium concentrations were continuously recorded.
Results: Diastolic calcium concentration was not different between Purkinje cells and ventricular myocytes (245.1±1.94 nmol/L, n = 8 vs. 243.9±4.6 nmol/L, n = 6). The amplitude of spontaneous calcium release events was ~2X larger in Purkinje cells than in ventricular myocytes (85.4±5.1 nmol/L, n = 57 vs. 41.8±4.9nmol/L, n = 40; P < 0.00001). The frequency of spontaneous calcium release events was also greater in Purkinje cells (0.15±0.03 events/s, n = 8 cells) than in ventricular myocytes (0.07±0.01 events/s, n = 6 cells; P < 0.05). In addition the latency from the cessation of electrical stimulation to the first SCR event was shorter in Purkinje cells (4.17±0.93s, n = 7) than in myocytes (8.54±1.7s, n = 5, P < 0.05).
Conclusion: Calcium dysregulation is more apparent in the Purkinje cells than in the ventricular myocytes of the RyR2R4496C knock-in mouse. The results provide definite proof that the Purkinje cells are more likely to be the source of focally activated arrhythmias than cardiac myocytes.