title: Ryanodine receptor mutations associated with stress-induced ventricular tachycardia mediate increased calcium release in stimulated cardiomyocytes.
abstract: 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 beta-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 beta-AR stimulation. Furthermore, equivalent interaction between mutant and WT hRyR2 and FKBP12.6 was demonstrated.
authors.affiliation: Wales Heart Research Institute, University of Wales College of Medicine, Heath Park, Cardiff, UK. georgech@cf.ac.uk
authors.forename: Christopher H
authors.initials: CH
authors.lastname: George
authors.forename: Gemma V
authors.initials: GV
authors.lastname: Higgs
authors.forename: F Anthony
authors.initials: FA
authors.lastname: Lai
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003408:Cresols / Q000494:pharmacology; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D009154:Mutation; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D010641:Phenotype; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics* / Q000378:metabolism; D013312:Stress, Physiological / Q000150:complications; D017180:Tachycardia, Ventricular / Q000209:etiology / Q000235:genetics; D022021:Tacrolimus Binding Proteins / Q000378:metabolism
chemical_list: D000318:Adrenergic beta-Agonists; D003408:Cresols; D019837:Ryanodine Receptor Calcium Release Channel; C006984:chlorocresol; D002110:Caffeine; D000262:Adenylyl Cyclases; D022021:Tacrolimus Binding Proteins; C403484:tacrolimus binding protein 1B; D007545:Isoproterenol; D002118:Calcium