Functional heterogeneity of ryanodine receptor mutations associated with sudden cardiac death.
OBJECTIVE
Point mutations in the cardiac ryanodine receptor (RyR2) mediate abnormal intracellular Ca(2+) release and are associated with stress-induced ventricular tachycardia (VT), leading to sudden cardiac death (SCD). Although the precise molecular basis of RyR2 dysfunction in SCD remains controversial, there is consensus that the mutations characterised to date all exhibit gain-of-function Ca(2+) release properties following cell stimulation. We investigated the functional impact of a distinct set of SCD-linked RyR2 mutations (L(433)P, N(2386)I, R(176)Q/T(2504)M) on intracellular Ca(2+) handling.


METHODS
We expressed full-length recombinant human wild-type (WT) and SCD-linked RyR2 mutations in human embryonic kidney (HEK) cells, and profiled the spatial and amplitude characteristics of caffeine-evoked Ca(2+) release through homo-tetrameric channels in living cells using rapid confocal laser scanning microscopy.


RESULTS
Analysis of the precise mode of Ca(2+) release in HEK cells expressing RyR2 mutants demonstrated profound differences when compared with WT channels. The SCD-linked RyR2 mutations characterised in this study exhibited heterogeneous Ca(2+) release profiles, including the novel observation that one of the mutants, (L(433)P), exhibited a marked reduction in sensitivity to channel activation. However, all SCD-linked RyR2 mutations characterised in this study resulted in an increased duration of elevated cytoplasmic Ca(2+) levels following channel activation.


CONCLUSIONS
Our live cell-based data demonstrates functional heterogeneity of Ca(2+) release through SCD-linked RyR2 mutants, suggesting that the mechanistic basis of RyR2 dysfunction in SCD may be more complex than previously anticipated. These findings may have profound consequences for the therapeutic modulation of RyR2 in stress-induced VT and SCD.
Thomas
N Lowri
NL
Department of Cardiology, Wales Heart Research Institute, University of Wales College of Medicine, Heath Park, Cardiff, Wales CF14 4XN, UK.
George
Christopher H
CH
Lai
F Anthony
FA
D001145:Arrhythmias, Cardiac / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D016757:Death, Sudden, Cardiac / Q000209:etiology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D017354:Point Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D000697:Central Nervous System Stimulants; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D002118:Calcium