Abstract 12625: Troponin I3 A157V Homozygous Mutation Induces Diastolic Dysfunction

Abstract

Introduction: Previous studies describe a morbid restrictive cardiomyopathy in patients with substitution of alanine with valine at amino acid 157 (A157V) within the calcium-binding domain of the inhibitory subunit of cardiac troponin (TNNI3); yet the precise mechanism of disease remains unknown. Hypothesis: A murine model of A157V TNNI3 would recapitulate key features of disease and serve as a model of cardiac restriction. Methods: TNNI3 A157V mice were generated using a cloning-free CRISPR/Cas-9 system to introduce a point mutation (c.470c>T) resulting in missense substitution of valine for alanine at amino acid 157 in Exon 7 of TNNI3 . Heterozygous and homozygous mice were generated in a C57BL/6 background. At one year of age animals had echocardiography and electrocardiogram (ECG) performed, followed by invasive hemodynamics. Mice were then sacrificed for cardiac morphometry and hearts were fixed and embedded for histology. TNNI3 A157V adeno-associated virus-9 (AAV-9) construct was generated and injected retro-orbitally into 8-12 week old wild-type mice. Results: Serial echocardiography at 2, 4, 6 and 12 months of age demonstrated normal wall thickness, ejection fraction and strain. No differences were noted in cardiac arrhythmias assessed using ECG. Hemodynamics performed at 15 months of age demonstrated significantly reduced minimum derivative of pressure/time (dP/dT) and Tau in response to dobutamine in TNNI3 A157V mice v. controls. No significant differences between genotypes were detected in cardiac morphometry (heart weight and heart weight normalized to body weight or tibia length), myocyte cross sectional area measured on trichrome-stained heart sections, or myocardial oxygen consumption rate measured by oroboros. Mice injected with TNNI3 A157V AAV-9 demonstrated expression of the mutant TNNI3 construct on western analysis and immunohistochemistry. Conclusions: We have developed a novel mouse model in which substitution of valine for alanine at amino acid 157 in TNNI3 induces diastolic dysfunction despite the absence of cardiac hypertrophy that recapitulates key features of human disease and can serve as a platform for further mechanistic and translational studies.