Cardiac hypertrophy and arrhythmia in mice induced by a mutation in ryanodine receptor 2
Francisco J. Alvarado, J. Martijn Bos, Zhiguang Yuchi, Carmen R. Valdivia, Jonathan J. Hernández, Yan-Ting Zhao, Dawn S. Henderlong, Yan Chen, Talia R. Booher, Cherisse A. Marcou, Filip Van Petegem, Michael J. Ackerman, Héctor H. Valdivia
Francisco J. Alvarado, J. Martijn Bos, Zhiguang Yuchi, Carmen R. Valdivia, Jonathan J. Hernández, Yan-Ting Zhao, Dawn S. Henderlong, Yan Chen, Talia R. Booher, Cherisse A. Marcou, Filip Van Petegem, Michael J. Ackerman, Héctor H. Valdivia
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Research Article Cardiology

Cardiac hypertrophy and arrhythmia in mice induced by a mutation in ryanodine receptor 2

Abstract

Hypertrophic cardiomyopathy (HCM) is triggered mainly by mutations in genes encoding sarcomeric proteins, but a significant proportion of patients lack a genetic diagnosis. We identified a potentially novel mutation in ryanodine receptor 2, RyR2-P1124L, in a patient from a genotype-negative HCM cohort. The aim of this study was to determine whether RyR2-P1124L triggers functional and structural alterations in isolated RyR2 channels and whole hearts. We found that P1124L induces significant conformational changes in the SPRY2 domain of RyR2. Recombinant RyR2-P1124L channels displayed a cytosolic loss-of-function phenotype, which contrasted with a higher sensitivity to luminal [Ca2+], indicating a luminal gain of function. Homozygous mice for RyR2-P1124L showed mild cardiac hypertrophy, similar to the human patient. This phenotype, evident at 1 year of age, was accompanied by an increase in the expression of calmodulin (CaM). P1124L mice also showed higher susceptibility to arrhythmia at 8 months of age, before the onset of hypertrophy. RyR2-P1124L has a distinct cytosolic loss-of-function and a luminal gain-of-function phenotype. This bifunctionally divergent behavior triggers arrhythmias and structural cardiac remodeling, and it involves overexpression of CaM as a potential hypertrophic mediator. This study is relevant to continue elucidating the possible causes of genotype-negative HCM and the role of RyR2 in cardiac hypertrophy.

Authors

Francisco J. Alvarado, J. Martijn Bos, Zhiguang Yuchi, Carmen R. Valdivia, Jonathan J. Hernández, Yan-Ting Zhao, Dawn S. Henderlong, Yan Chen, Talia R. Booher, Cherisse A. Marcou, Filip Van Petegem, Michael J. Ackerman, Héctor H. Valdivia

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Figure 2

Molecular phenotype of RyR2-P1124L.

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Molecular phenotype of RyR2-P1124L. (A) Ca2+-dependent [3H]ryanodine bin...
(A) Ca2+-dependent [3H]ryanodine binding curve corrected for ryanodine receptor 2 (RyR2) expression. The mouse Ryr2 cDNA was transiently transfected into HEK293 cells. Cells were lysed 48 hours after transfection. Recombinant RyR2 was then incubated with [3H]ryanodine in the presence of increasing concentrations of [Ca2+] to activate the channel. [3H]ryanodine was determined by liquid scintillation. (B) Maximum binding (BMax) calculated from the curves in A using Hill’s equation. (C) Ca2+-dependent [3H]ryanodine binding curves (same as in A) normalized to 10 μM [Ca2+]. (D) EC50 for [Ca2+]-dependent [3H]ryanodine binding (A–D, n = 7 curves from at least 4 independent transfections. *P < 0.05, **P < 0.01, rank sum test). (E) Representative single RyR2 channel recordings from cardiac microsomes prepared WT and homozygous (Homo) P1124L hearts. Channels were fused into artificial planar lipid bilayers. Single channel current and open probability (PO) were recorded at nominally free [Ca2+] in a 300/50 mM Cs+ gradient. (F) Overlapped histograms of normalized current calculated from 2 representative channels. (G) Average PO of WT and P1124L channels. (H) Average single channel current calculated from recordings in a 300/50 mM Cs+ gradient (G and H, n = 3 WT, 5–6 P1124L channels. *P < 0.05, 2-tailed t test).