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Ryanodine Receptor Mutations Associated With Stress-Induced Ventricular Tachycardia Mediate Increased Calcium Release in Stimulated Cardiomyocytes

From the Wales Heart Research Institute, Department of Cardiology, University of Wales College of Medicine, Cardiff, UK.

Correspondence to Dr Christopher H. George, Wales Heart Research Institute, University of Wales College of Medicine, Heath Park, Cardiff, UK CF14 4XN. E-mail georgech{at}cf.ac.uk

Ca²⁺ release from the sarcoplasmic reticulum mediated by the cardiac ryanodine receptor (RyR2) is a fundamental event in cardiac muscle contraction. RyR2 mutations suggested to cause defective Ca²⁺ channel function have recently been identified in catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia (ARVD) affected individuals. We report expression of three CPVT-linked human RyR2 (hRyR2) mutations (S²²⁴⁶L, N⁴¹⁰⁴K, and R⁴⁴⁹⁷C) in HL-1 cardiomyocytes displaying correct targeting to the endoplasmic reticulum. N⁴¹⁰⁴K also localized to the Golgi apparatus. Phenotypic characteristics including intracellular Ca²⁺ handling, proliferation, viability, RyR2:FKBP12.6 interaction, and beat rate in resting HL-1 cells expressing mutant hRyR2 were indistinguishable from wild-type (WT) hRyR2. However, Ca²⁺ release was augmented in cells expressing mutant hRyR2 after RyR activation (caffeine and 4-chloro-m-cresol) or ß-adrenergic stimulation (isoproterenol). RyR2:FKBP12.6 interaction remained intact after caffeine or 4-CMC activation, but was dramatically disrupted by isoproterenol or forskolin, an activator of adenylate cyclase. Isoproterenol and forskolin elevated cyclic-AMP to similar magnitudes in all cells and were associated with equivalent hyperphosphorylation of mutant and WT hRyR2. CPVT-linked mutations in hRyR2 did not alter resting cardiomyocyte phenotype but mediated augmented Ca²⁺ release on RyR-agonist or ß-AR stimulation. Furthermore, equivalent interaction between mutant and WT hRyR2 and FKBP12.6 was demonstrated.

Key Words: ryanodine receptors • mutations • ventricular tachycardia • stress • cardiomyocytes

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