Abstract 4370053: Unique Insights into the Pathobiology of TNNI3 Cardiomyopathy using Novel Murine and Porcine Models of Disease

Abstract

Background: Cardiac troponin I (cTnI, encoded by TNNI3 ) inhibits contraction by preventing actin-myosin interaction. Pathogenic TNNI3 variants can cause cardiomyopathy, but no targeted therapies exist. We identified a family with restrictive cardiomyopathy carrying the cTnI A157V variant. Previously, we generated a homozygous mouse model (cTnI A158V, murine equivalent) that showed impaired cardiac relaxation on invasive hemodynamics but had normal lifespan and no cardiac hypertrophy or fibrosis. Aims: It is known that murine models of cardiomyopathies often exhibit milder phenotypes than humans, limiting their translational utility. Hence, to better model human disease, we generated a homozygous cTnI A158V pig model using CRISPR-Cas9. We aimed to characterize phenotypic and molecular differences between the mouse and pig models to gain insight into disease mechanisms. Methods: Heart tissue was collected from wild-type (WT) and homozygous A158V mice (9-10 months) and from WT and homozygous A158V pigs (2 months). Bulk RNA sequencing (RNA-seq) was performed on RNA extracted from A158V pig hearts (n=5) and compared to publicly available data from age-matched WT pigs (n=10) supplemented by one in-house WT control. Protein lysates were analyzed by Western blot. Isolated myofibrils were measured using the fast solution switching method. Results: A158V pigs had 100% mortality by 2 months (n=7), with gross hypertrophy and fibrosis. Myofibril studies showed significant prolongation of the linear relaxation phase in both A158V pigs (p<0.01) and mice (p<0.01) compared to respective WTs, with no significant differences in active tension in both models. Both models showed reduced phosphorylation of cTnI at serine 23/24, reaching significance in pigs (p=0.046) but not in mice (p=0.064). Unsupervised clustering of bulk-RNAseq data separated A158V from WT, with genotype explaining 44% and 53% of transcriptional variation in mice and pigs, respectively. Gene set enrichment analysis revealed significant upregulation of inflammatory pathways in A158V pigs only. Conclusions: Both A158V mouse and pig models recapitulate impaired cardiac relaxation seen in affected patients as shown by myofibril studies. However, the pig model more closely mirrors the human phenotype, including early mortality, hypertrophy, and fibrosis. Inflammatory pathway upregulation was observed only in A158V pigs, suggesting that inflammation may play a role in the severity or progression of disease.