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