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 Ca(2+) homeostasis in the heart. However, the mechanism of Ca(2+) 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 Ca(2+) dynamics in intact Langendorff-perfused hearts (ex vivo) from wild-type and RyR2(R4496C+/-) mice using laser scanning confocal microscopy. We found that myocytes from both wild-type and RyR2(R4496C+/-) hearts displayed uniform, synchronized Ca(2+) transients. Ca(2+) transients from beat to beat were comparable in amplitude with identical activation and decay kinetics in wild-type and RyR2(R4496C+/-) hearts, suggesting that excitation-contraction coupling between the sarcolemmal Ca(2+) channels and mutated RyR2(R4496C+/-) channels remains intact under baseline resting conditions. On adrenergic stimulation, RyR2(R4496C+/-) hearts exhibited a high degree of Ca(2+) release variability. The varied pattern of Ca(2+) 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 Ca(2+) release variability on intense adrenergic stimulation. Ca(2+) release variability is a Ca(2+) release abnormality, resulting from electric defects rather than the failure of the Ca(2+) release response to action potentials in mutated ventricular myocytes. Our data provide important insights into Ca(2+) release and electric dysfunction in an established model of catecholaminergic polymorphic ventricular tachycardia.