Development of a Cardiac Sarcomere Functional Genomics Platform to Enable Scalable Interrogation of Human <i>TNNT2</i> Variants.

Abstract

Pathogenic <i>TNNT2</i> variants are a cause of hypertrophic and dilated cardiomyopathies, which promote heart failure by incompletely understood mechanisms. The precise functional significance for 87% of <i>TNNT2</i> variants remains undetermined, in part, because of a lack of functional genomics studies. The knowledge of which and how <i>TNNT2</i> 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. We created a toolkit of human induced pluripotent stem cell models and functional assays using CRISPR/Cas9 to study <i>TNNT2</i> variant pathogenicity and pathophysiology. Using human induced pluripotent stem cell-derived cardiomyocytes in cardiac microtissue and single-cell assays, we functionally interrogated 51 <i>TNNT2</i> variants, including 30 pathogenic/likely pathogenic variants and 21 variants of uncertain significance. We used RNA sequencing to determine the transcriptomic consequences of pathogenic <i>TNNT2</i> variants and adapted CRISPR/Cas9 to engineer a transcriptional reporter assay to assist prediction of <i>TNNT2</i> variant pathogenicity. We also studied variant-specific pathophysiology using a thin filament-directed calcium reporter to monitor changes in myofilament calcium affinity. Hypertrophic cardiomyopathy-associated <i>TNNT2</i> variants caused increased cardiac microtissue contraction, whereas dilated cardiomyopathy-associated variants decreased contraction. <i>TNNT2</i> variant-dependent changes in sarcomere contractile function induced graded regulation of 101 gene transcripts, including MAPK (mitogen-activated protein kinase) signaling targets, <i>HOPX</i>, and <i>NPPB</i>. We distinguished pathogenic <i>TNNT2</i> variants from wildtype controls using a sarcomere functional reporter engineered by inserting tdTomato into the endogenous <i>NPPB</i> locus. On the basis of a combination of <i>NPPB</i> 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. Our study found that hypertrophic cardiomyopathy-associated <i>TNNT2</i> variants increased cardiac microtissue contraction, whereas dilated cardiomyopathy-associated variants decreased contraction, both of which paralleled changes in myofilament calcium affinity. Transcriptomic changes, including <i>NPPB</i> levels, directly correlated with sarcomere function and can be used to predict <i>TNNT2</i> variant pathogenicity.