P110Multiphoton Ca2+ imaging in acute myocardial slices of CPVT hearts

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Rationale: Alterations in cardiomyocyte (CM) Ca2+ handling play a role in initiating and sustaining inherited arrhythmias (e.g. CPVT). Most of the current data on Ca2+ dynamics in CPVT have been obtained in isolated cells. Aim: We aimed to establish an intact cardiac tissue model to investigate Ca2+ dynamics in WT and CPVT cardiomyocytes. Results: Acute thick (450 μm) ventricular slices were obtained from WT and CPVT mice. Slices were loaded with the Ca2+-sensitive indicator Fluo4-AM and imaged with a multiphoton microscope (BioRad-Radiance2100) to optically monitor intracellular Ca2+ fluctuations during electrical pacing with or without the addition of drugs including β-adrenergic agonists. As previously reported in isolated cells, upon b-adrenergic stimulation CMs within the CPVT heart slice had increased propensity to develop diastolic Ca2+ releases (DCRs), than cells in the WT slice. When the pacing frequency was increased from 1 to 5 Hz, DCRs were substituted by Ca2+ alternans, that are a pathophysiologic mechanism associated to arrhythmia triggering. Moreover, after cessation of rapid pacing, CM in the CPVT myocardium developed whole-cell traveling Ca2+ waves with a significantly shorter latency compared to WT cells, which resulted in synchronization of DCR throughout the cells in the slice. Conclusions: We developed a powerful model for myocardial Ca2+ imaging in acute heart slices, that allows simultaneous detection of Ca2+ dynamics in several connected cells in their physiologic tissue environment. This experimental setting expands the notions on arrhythmogenesis accrued in single cell, and enables the study of the mechanisms underlying DCRs synchronization throughout several cardiomyocytes in the myocardial network, a well-accepted requirement for arrhythmic beat triggering.

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