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 3

Abnormal termination of spontaneous Ca2+ release in HEK293 cells expressing RyR2.

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Abnormal termination of spontaneous Ca2+ release in HEK293 cells express...
(A) Representative traces of the intra-ER FRET signal using the intra-ER Ca2+ sensor D1ER in cells expressing RyR2. Stable HEK293 cells with inducible expression of the mouse RyR2 were transfected with D1ER. RyR2 expression was induced with 1 μg/ml tetracycline 18–24 hours after transfection. Time-lapse imaging of D1ER FRET was performed after 18–24 hours of RyR2 induction. Images were collected for 3 minutes at 0 [Ca2+], 3 minutes at 1 mM [Ca2+], 5 minutes at 2 mM [Ca2+], 5 minutes in the presence of 1 mM tetracycline to inhibit RyR2 and measure maximum ER Ca2+ capacity (FMax), and about 3 minutes in the presence of 20 mM caffeine to empty the ER and measure the residual FRET signal (FMin). (B–D) Activation (B) and termination (C) thresholds and fraction of the ER Ca2+ content released (D) during spontaneous oscillations (*P < 0.05, **P < 0.01, rank sum test [B], 2-tailed t test [C–D]). (E) ER store capacity determined as (FMax–FMin) and expressed as percentage of WT (rank sum test). (F) Percentage of cells showing oscillations in the store [Ca2+] (z test). (G) Average number of [Ca2+] oscillations per cell (**P < 0.01, rank sum test) (n = 40 [B–D], 49 [E], 87 [F], and 39 [G] WT cells; 44 [B–D], 61 [E], 104 [F] and 43 [G] P1124L cells from 4 independent D1ER transfections and inductions of RyR2 expression).