In situ confocal imaging in intact heart reveals stress-induced ca2+ release variability in a murine catecholaminergic polymorphic ventricular tachycardia model of type 2 ryanodine receptorr4496c+/? mutation

Background-Catecholaminergic polymorphic ventricular tachycardia is directly linked to mutations in proteins (eg, type 2 ryanodine receptor [RyR2]R4496C) responsible for intracellular Ca2+ homeostasis in the heart. However, the mechanism of Ca2+ release dysfunction underlying catecholaminergic polymorphic ventricular tachycardia has only been investigated in isolated cells but not in the in situ undisrupted myocardium. Methods and Results-We investigated in situ myocyte Ca2+ dynamics in intact Langendorff-perfused hearts (ex vivo) from wild-type and RyR2R4496C+/? mice using laser scanning confocal microscopy. We found that myocytes from both wild-type and RyR2R4496C+/? hearts displayed uniform, synchronized Ca2+ transients. Ca2+ transients from beat to beat were comparable in amplitude with identical activation and decay kinetics in wild-type and RyR2R4496C+/? hearts, suggesting that excitation-contraction coupling between the sarcolemmal Ca2+ channels and mutated RyR2R4496C+/? channels remains intact under baseline resting conditions. On adrenergic stimulation, RyR2R4496C+/? hearts exhibited a high degree of Ca2+ release variability. The varied pattern of Ca2+ release was absent in single isolated myocytes, independent of cell cycle length, synchronized among neighboring myocytes, and correlated with catecholaminergic polymorphic ventricular tachycardia. A similar pattern of action potential variability, which was synchronized among neighboring myocytes, was also revealed under adrenergic stress in intact hearts but not in isolated myocytes. Conclusions-Our studies using an in situ confocal imaging approach suggest that mutated RyR2s are functionally normal at rest but display a high degree of Ca2+ release variability on intense adrenergic stimulation. Ca2+ release variability is a Ca2+ release abnormality, resulting from electric defects rather than the failure of the Ca2+ release response to action potentials in mutated ventricular myocytes. Our data provide important insights into Ca2+ release and electric dysfunction in an established model of catecholaminergic polymorphic ventricular tachycardia.