In Vivo Analysis of Troponin C Knock-In (A8V) Mice

Abstract

Background Mutations in thin-filament proteins have been linked to hypertrophic cardiomyopathy (HCM), but it has never been demonstrated that variants identified in the TNNC1 (gene encoding troponin C) can evoke cardiac remodeling in-vivo. The goal of this study was to determine whether TNNC1 can be categorized as an HCM susceptibility gene, such that a mouse model can recapitulate the clinical presentation of the proband. Methods and Results The TNNC1-A8V proband diagnosed with severe obstructive HCM at 34-years of age exhibited mild-to-moderate thickening in left and right ventricular walls, decreased left-ventricular dimensions, left-atrial enlargement and hyperdynamic left-ventricular systolic function. Genetically-engineered knock-in mice containing the A8V mutation (heterozygote=KI-TnC-A8V+/−; homozygote=KI-TnC-A8V+/+) were characterized by echocardiography and pressure-volume studies. Three-month-old, KI-TnC-A8V+/+ mice displayed decreased ventricular dimensions, mild diastolic dysfunction, and enhanced systolic function, while KI-TnC-A8V+/− mice displayed cardiac restriction at 14-months of age. KI hearts exhibited atrial enlargement, papillary-muscle hypertrophy and fibrosis. Liquid chromatography-mass spectroscopy was used to determine incorporation of mutant cTnC (~21%) into the KI-TnC-A8V+/− cardiac myofilament. Reduced diastolic sarcomeric length, increased shortening and prolonged Ca2+ and contractile transients were recorded in intact KI-TnC-A8V+/− and KI-TnC-A8V+/+ cardiomyocytes. Ca2+-sensitivity of contraction in skinned fibers increased with mutant gene dose: KI-TnC-A8V+/+ > KI-TnC-A8V+/− > WT, while KI-TnC-A8V+/+ relaxed more slowly upon flash-photolysis of diazo-2. Conclusions The TNNC1-A8V mutant increases the Ca2+-binding affinity of the thin filament, and elicits changes in Ca2+ homeostasis and cellular remodeling, which leads to diastolic dysfunction. These in-vivo alterations further implicate the role of TNNC1 mutations in the development of cardiomyopathy.

Enhanced troponin I binding explains the functional changes produced by the hypertrophic cardiomyopathy mutation A8V of cardiac troponin C

“…The functional consequences of the A8V substitution in TnC were evaluated in a knock-in (KI) mouse (45). Intact cardiomyocytes isolated from both heterozygous and homozygous KI mice had slower sarcomere length relaxation time and Ca 2+ decay at different frequencies of stimulation.…”

“…Intact cardiomyocytes isolated from both heterozygous and homozygous KI mice had slower sarcomere length relaxation time and Ca 2+ decay at different frequencies of stimulation. Furthermore, homozygous mice showed signs of diastolic dysfunction and a hypercontractile phenotype, e.g ., increased ejection fraction (45). The in vivo data can be correlated with the myofibrillar ATPase findings since higher myofibrillar ATPase activation can indicate a greater degree of muscle activation.…”

Pathogenic troponin T mutants with opposing effects on myofilament Ca2+ sensitivity attenuate cardiomyopathy phenotypes in mice

“…Skinned cardiac preparations were prepared according to established protocols [10, 19]. Papillary muscle preparations were isolated from the left ventricles of 15–36 week old male and female mice.…”

Stabilization of the cardiac sarcolemma by sarcospan rescues DMD-associated cardiomyopathy

“…Left ventricular size, mass, wall thickness, ventricular and valve function, and Doppler blood flows were obtained using previously described methods (54,55). Briefly, mice were anesthetized with isoflurane (1%-3%) and body temperature maintained at 37°C.…”