Dataset Information

RAD-Deficient Human Cardiomyocytes Develop Hypertrophic Cardiomyopathy Phenotypes Due to Calcium Dysregulation.

ABSTRACT: Ras associated with diabetes (RAD) is a membrane protein that acts as a calcium channel regulator by interacting with cardiac L-type Ca2 + channels (LTCC). RAD defects can disrupt intracellular calcium dynamics and lead to cardiac hypertrophy. However, due to the lack of reliable human disease models, the pathological mechanism of RAD deficiency leading to cardiac hypertrophy is not well understood. In this study, we created a RRAD -/- H9 cell line using CRISPR/Cas9 technology. RAD disruption did not affect the ability and efficiency of cardiomyocytes differentiation. However, RAD deficient hESC-CMs recapitulate hypertrophic phenotype in vitro. Further studies have shown that elevated intracellular calcium level and abnormal calcium regulation are the core mechanisms by which RAD deficiency leads to cardiac hypertrophy. More importantly, management of calcium dysregulation has been found to be an effective way to prevent the development of cardiac hypertrophy in vitro.

SUBMITTER: Li Y

PROVIDER: S-EPMC7642210 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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RAD-Deficient Human Cardiomyocytes Develop Hypertrophic Cardiomyopathy Phenotypes Due to Calcium Dysregulation.

Li Ya'nan Y Chang Yun Y Li Xiaolei X Li Xiaowei X Gao Jian J Zhou Yafei Y Wu Fujian F Bai Rui R Dong Tao T Ma Shuhong S Zhang Siyao S Lu Wen-Jing WJ Tan Xiaoqiu X Wang Yongming Y Lan Feng F

Frontiers in cell and developmental biology 20201022

Ras associated with diabetes (RAD) is a membrane protein that acts as a calcium channel regulator by interacting with cardiac L-type Ca2 + channels (LTCC). RAD defects can disrupt intracellular calcium dynamics and lead to cardiac hypertrophy. However, due to the lack of reliable human disease models, the pathological mechanism of RAD deficiency leading to cardiac hypertrophy is not well understood. In this study, we created a RRAD -/- H9 cell line using CRISPR/Cas9 ...[more]

PMID: 33195237

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Project description:BACKGROUND:Aberrant calcium signaling may contribute to arrhythmias and adverse remodeling in hypertrophic cardiomyopathy (HCM). Mutations in sarcomere genes may distinctly alter calcium handling pathways. METHODS:We analyzed gene expression, protein levels, and functional assays for calcium regulatory pathways in human HCM surgical samples with (n=25) and without (n=10) sarcomere mutations compared with control hearts (n=8). RESULTS:Gene expression and protein levels for calsequestrin, L-type calcium channel, sodium-calcium exchanger, phospholamban, calcineurin, and calcium/calmodulin-dependent protein kinase type II (CaMKII) were similar in HCM samples compared with controls. CaMKII protein abundance was increased only in sarcomere-mutation HCM (P<0.001). The CaMKII target pT17-phospholamban was 5.5-fold increased only in sarcomere-mutation HCM (P=0.01), as was autophosphorylated CaMKII (P<0.01), suggestive of constitutive activation. Calcineurin (PPP3CB) mRNA was not increased, nor was RCAN1 mRNA level, indicating a lack of calcineurin activation. Furthermore, myocyte enhancer factor 2 and nuclear factor of activated T cell transcription factor activity was not increased in HCM, suggesting that calcineurin pathway activation is not an upstream cause of increased CAMKII protein abundance or activation. SERCA2A mRNA transcript levels were reduced in HCM regardless of genotype, as was sarcoplasmic endoplasmic reticular calcium ATPase 2/phospholamban protein ratio (45% reduced; P=0.03). 45Ca sarcoplasmic endoplasmic reticular calcium ATPaseuptake assay showed reduced uptake velocity in HCM regardless of genotype (P=0.01). The cardiac ryanodine receptor was not altered in transcript, protein, or phosphorylated (pS2808, pS2814) protein abundance, and [3H]ryanodine binding was not different in HCM, consistent with no major modification of the ryanodine receptor. CONCLUSIONS:Human HCM demonstrates calcium mishandling through both genotype-specific and common pathways. Posttranslational activation of the CaMKII pathway is specific to sarcomere mutation-positive HCM, whereas sarcoplasmic endoplasmic reticular calcium ATPase 2 abundance and sarcoplasmic reticulum Ca uptake are depressed in both sarcomere mutation-positive and -negative HCM.

Project description:RATIONALE:Subcellular Ca2+ indicators have yet to be developed for the myofilament where disease mutation or small molecules may alter contractility through myofilament Ca2+ sensitivity. Here, we develop and characterize genetically encoded Ca2+ indicators restricted to the myofilament to directly visualize Ca2+ changes in the sarcomere. OBJECTIVE:To produce and validate myofilament-restricted Ca2+ imaging probes in an adenoviral transduction adult cardiomyocyte model using drugs that alter myofilament function (MYK-461, omecamtiv mecarbil, and levosimendan) or following cotransduction of 2 established hypertrophic cardiomyopathy disease-causing mutants (cTnT [Troponin T] R92Q and cTnI [Troponin I] R145G) that alter myofilament Ca2+ handling. METHODS AND RESULTS:When expressed in adult ventricular cardiomyocytes RGECO-TnT (Troponin T)/TnI (Troponin I) sensors localize correctly to the sarcomere without contractile impairment. Both sensors report cyclical changes in fluorescence in paced cardiomyocytes with reduced Ca2+ on and increased Ca2+ off rates compared with unconjugated RGECO. RGECO-TnT/TnI revealed changes to localized Ca2+ handling conferred by MYK-461 and levosimendan, including an increase in Ca2+ binding rates with both levosimendan and MYK-461 not detected by an unrestricted protein sensor. Coadenoviral transduction of RGECO-TnT/TnI with hypertrophic cardiomyopathy causing thin filament mutants showed that the mutations increase myofilament [Ca2+] in systole, lengthen time to peak systolic [Ca2+], and delay [Ca2+] release. This contrasts with the effect of the same mutations on cytoplasmic Ca2+, when measured using unrestricted RGECO where changes to peak systolic Ca2+ are inconsistent between the 2 mutations. These data contrast with previous findings using chemical dyes that show no alteration of [Ca2+] transient amplitude or time to peak Ca2+. CONCLUSIONS:RGECO-TnT/TnI are functionally equivalent. They visualize Ca2+ within the myofilament and reveal unrecognized aspects of small molecule and disease-associated mutations in living cells.

Dataset Information

RAD-Deficient Human Cardiomyocytes Develop Hypertrophic Cardiomyopathy Phenotypes Due to Calcium Dysregulation.

Publications

RAD-Deficient Human Cardiomyocytes Develop Hypertrophic Cardiomyopathy Phenotypes Due to Calcium Dysregulation.

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