Development of a Cardiac Sarcomere Functional Genomics Platform to Enable Scalable Interrogation of Human TNNT2 Variants
| العنوان: | Development of a Cardiac Sarcomere Functional Genomics Platform to Enable Scalable Interrogation of Human TNNT2 Variants |
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| المؤلفون: | Robert Romano, Ketan Thakar, David J. Mellert, Anthony M. Pettinato, J. Travis Hinson, Nicholas Legere, Rachel Cohn, Yu-Sheng Chen, Feria A. Ladha |
| المصدر: | Circulation |
| بيانات النشر: | Ovid Technologies (Wolters Kluwer Health), 2020. |
| سنة النشر: | 2020 |
| مصطلحات موضوعية: | Male, Sarcomeres, TNNT2, Induced Pluripotent Stem Cells, Genomics, Computational biology, 030204 cardiovascular system & hematology, Sarcomere, Article, 03 medical and health sciences, 0302 clinical medicine, Troponin T, Physiology (medical), medicine, Humans, Myocytes, Cardiac, Induced pluripotent stem cell, 030304 developmental biology, 0303 health sciences, business.industry, Genetic Variation, medicine.disease, Heart failure, Female, Cardiology and Cardiovascular Medicine, business, Functional genomics |
| الوصف: | Background: Pathogenic TNNT2 variants are a cause of hypertrophic and dilated cardiomyopathies, which promote heart failure by incompletely understood mechanisms. The precise functional significance for 87% of TNNT2 variants remains undetermined, in part, because of a lack of functional genomics studies. The knowledge of which and how TNNT2 variants cause hypertrophic and dilated cardiomyopathies could improve heart failure risk determination, treatment efficacy, and therapeutic discovery, and provide new insights into cardiomyopathy pathogenesis, as well. Methods: We created a toolkit of human induced pluripotent stem cell models and functional assays using CRISPR/Cas9 to study TNNT2 variant pathogenicity and pathophysiology. Using human induced pluripotent stem cell–derived cardiomyocytes in cardiac microtissue and single-cell assays, we functionally interrogated 51 TNNT2 variants, including 30 pathogenic/likely pathogenic variants and 21 variants of uncertain significance. We used RNA sequencing to determine the transcriptomic consequences of pathogenic TNNT2 variants and adapted CRISPR/Cas9 to engineer a transcriptional reporter assay to assist prediction of TNNT2 variant pathogenicity. We also studied variant-specific pathophysiology using a thin filament–directed calcium reporter to monitor changes in myofilament calcium affinity. Results: Hypertrophic cardiomyopathy–associated TNNT2 variants caused increased cardiac microtissue contraction, whereas dilated cardiomyopathy–associated variants decreased contraction. TNNT2 variant–dependent changes in sarcomere contractile function induced graded regulation of 101 gene transcripts, including MAPK (mitogen-activated protein kinase) signaling targets, HOPX , and NPPB . We distinguished pathogenic TNNT2 variants from wildtype controls using a sarcomere functional reporter engineered by inserting tdTomato into the endogenous NPPB locus. On the basis of a combination of NPPB reporter activity and cardiac microtissue contraction, our study provides experimental support for the reclassification of 2 pathogenic/likely pathogenic variants and 2 variants of uncertain significance. Conclusions: Our study found that hypertrophic cardiomyopathy–associated TNNT2 variants increased cardiac microtissue contraction, whereas dilated cardiomyopathy–associated variants decreased contraction, both of which paralleled changes in myofilament calcium affinity. Transcriptomic changes, including NPPB levels, directly correlated with sarcomere function and can be used to predict TNNT2 variant pathogenicity. |
| تدمد: | 1524-4539 0009-7322 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::2617806be21e2987e10562992f2b0500 https://doi.org/10.1161/circulationaha.120.047999 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....2617806be21e2987e10562992f2b0500 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 15244539 00097322 |
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