Abstract
Introduction: Troponin inhibitor3 ( TNNI3 ) is a thin-filament protein that regulates contraction of thick filaments. The role of the switch domain of TNNI3 (aa147-163), which interacts with the calcium-binding pocket of troponin C, is poorly defined. Pathogenic mutations to the switch domain cause restrictive cardiomyopathy in humans, but no therapies exist that address the underlying problem of this mutation at the sarcomeric level. Further, few models of genetic restrictive cardiomyopathy exist to aid in development of new therapies. Hypothesis: Substitution of alanine with valine at position 157 (A157V) in the switch domain of TNNI3 causes restrictive cardiomyopathy by blunting response to adrenergic stimulus. Methods: A known pathogenic mutation to the TNNI3 switch domain (A157V) was identified in a family of patients with cardiomyopathy and restrictive features. A mutant knock-in mouse homozygous for this mutation (A157V) was generated using CRISPR-Cas9 and used to elucidate the function of the switch domain. Results: Compared to wild type controls (WT), mutant A157V mice demonstrate significant restrictive features on invasive hemodynamics that worsen with age but do not show evidence of systolic dysfunction or hypertrophy on echocardiography. Heart size and myocyte cross-sectional area were significantly smaller in mutant A157V mice compared to WT controls. Molecular dynamics simulations revealed reduced TNNI3 activation in response to PKA-mediated phosphorylation at serine23/24. Isolated myocytes from A157V mice demonstrated impaired relaxation, lower peak systolic calcium and delayed reuptake of calcium into the sarcoplasmic reticulum compared to WT controls. Conclusions: The A157V mutation to the switch domain of TNNI3 , a critical regulatory domain that interacts with the calcium binding pocket of troponin C, causes diastolic dysfunction by impairing responsiveness to PKA-mediated phosphorylation of S23/24. This mouse model recapitulates the key restrictive features of human disease and could be used as a platform to study future targeted therapeutics for thin filament cardiomyopathy.
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