What question did this study set out to answer?

This research aims to investigate how RyR2 mutations affect calcium release in cardiomyocytes during stress conditions.

August 19, 2003Circulation Research250 citationsOpen Access

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

CGChristopher H. George GHGemma Higgs FLF. Anthony Lai

Key Points

This research aims to investigate how RyR2 mutations affect calcium release in cardiomyocytes during stress conditions.
Expressed three CPVT-linked hRyR2 mutations in HL-1 cardiomyocytes.
Assessed intracellular calcium handling and RyR2:FKBP12.6 interaction after stimulation.
Evaluated the effects of caffeine, 4-chloro-m-cresol, isoproterenol, and forskolin on calcium release and cardiomyocyte viability.
After stimulation, calcium release was significantly increased in cardiomyocytes expressing mutant hRyR2 compared to wild-type (WT).
RyR2:FKBP12.6 interactions remained stable under caffeine and 4-CMC, but were disrupted by isoproterenol and forskolin.
No significant differences in resting cell phenotype or viability were noted between mutant and WT hRyR2.

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.

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