Biophysical Defects Link Rare TNNC1 Variants to Cardiomyopathy

Cardiomyopathies are a heterogenous group of myocardial disorders that are frequently associated with a genetic etiology. The cardiac/slow skeletal troponin C gene (TNNC1) encodes a sarcomeric protein (TNNC1) that has a central role in the Ca2+-dependent regulation of myocardial contraction and is linked to cardiomyopathy in humans. Although the genetic basis of cardiomyopathy is well documented, the molecular mechanisms that underlie the pathophysiology remain poorly defined. The overall objective of my dissertation research was to test the hypothesis that biophysical defects at the molecular level underlie the pathogenesis of rare cardiomyopathic variants in TNNC1. Studying these rare variants provides a unique opportunity to uncover new regulatory properties of TNNC1 that are essential for cardiac physiology. The findings in this dissertation also suggest novel therapeutic targets to potentially augment contractile performance in the setting of heart failure, irrespective of etiology. In the first project, an integrative approach was leveraged to establish pathogenicity and an underlying molecular mechanism of a previously unreported heterozygous variant in TNNC1 identified in a newborn with sporadic cardiomyopathy. Trio whole exome sequencing revealed that this variant arose de novo and encoded an isoleucine to methionine substitution at residue 4 in the primary amino-acid sequence of TNNC1. The proband carrying the TNNC1-I4M variant displayed the clinical hallmarks of dilated cardiomyopathy (DCM). Reconstitution of this recombinantly produced human TNNC1 mutant (cTnC-I4M) in permeabilized cardiac muscle preparations (CMPs) decreased the magnitude and rate of isometric tension generation at physiological Ca2+ concentrations. Mathematical modeling indicated that cTnC-I4M decreased the rates of cross-bridge attachment and detachment. Further, a novel interaction between the carboxy terminus of recombinantly produced human cardiac troponin T (cTnT) and regulatory N-domain of cTnC-WT was identified and cTnC-I4M exhibited tighter binding to cTnT. Solution-state spectroscopy experiments suggested that altered binding between cTnC-I4M and cTnT could be explained by changes in the overall structure and backbone dynamics of cTnC-I4M. According to the American College of Medical Genetics and Genomics (ACMG) criteria, there is strong evidence of pathogenicity for TNNC1-I4M. Together, the observations led to the proposal that the cTnT carboxy terminus directly interacts with cTnC-WT to modulate myocardial force generation by controlling the dynamic equilibrium of the three states of thin filament activity. Thus, the increased binding affinity between cTnC-I4M and cTnT is expected to shift these thin filament equilibrium states to a conformation that favors contractile impairment. In the second project, a Tnnc1 knock-in (A8V) mouse model of hypertrophic cardiomyopathy (HCM) was used to determine whether TNNC1 has a direct role in regulating the kinetics of cross-bridge cycling through myofilament Ca2+ sensitivity. To address this question, permeabilized, osmotically compressed, CMPs were prepared from A8V mutant (HCM) and wild-type (Ctrl) mouse hearts and subjected to muscle mechanics experiments. Increased Ca2+ sensitivity of isometric contraction was observed for HCM CMPs, as well as WT CMPs treated with a TNNC1-targeting Ca2+sensitizer, bepridil, compared to untreated Ctrl CMPs. Furthermore, HCM CMPs and bepridil-treated Ctrl CMPs increased the rate of cross-bridge cycling and increased the magnitude of force per cross-bridge. Mathematical modeling of cross-bridge kinetics revealed that TNNC1-mediated myofilament Ca2+ sensitization, through bepridil or the A8V variant, accelerated cross-bridge cycling kinetics by promoting a faster rate of cross-bridge detachment. To ascertain the biophysical basis for such observations, small angle X-ray diffraction was used to simultaneously monitor inter-myofilament spacing and isometric tension in permeabilized, osmotically compressed, CMPs during Ca2+ activation. At submaximal Ca2+ activations, increased isometric tension was accompanied by increased inter-myofilament spacing. Together, these observations led to the proposal that the regulatory N-domain of TNNC1 controls isometric force production by modulating the rate of cross-bridge cycling and number of force-generating cross-bridges, which may in part be mediated by changes in the inter-myofilament distance. Furthermore, these findings also provide a potential structural explanation for myocardial hypercontractility and pathophysiology of HCM in this knock-in mouse model. In the final and third project, the same Tnnc1 knock-in (A8V) mouse model of HCM was used to address a different question. Although it is well documented that sarcomeric variants associated with cardiomyopathy, including TNNC1-A8V, typically perturb the biophysical properties of the sarcomere (i.e., contractile force generation), it has been unclear whether the physical properties of cardiomyocyte nuclei are also altered. To this end, measurements of cardiomyocyte nuclear morphology were carried out on myocardial tissue sections and freshly isolated, living cardiomyocytes from the HCM mice. Cardiomyocyte nuclei from HCM mice were significantly smaller in area and volume, as well as rounder in shape compared to control mice. Interestingly, the alterations in nuclear morphology could not be explained by differences in DNA content or size of the cardiomyocytes. The HCM cardiomyocytes also revealed evidence for defective nuclear mechanics and decreased nuclear localization of TNNC1. Together, these results suggest that a mouse model of sarcomeric cardiomyopathy is associated with marked abnormalities in cardiomyocyte nuclear structure and function and point to a new role for TNNC1.