Abstract
The physiological consequences of aberrant Ca2+ binding and exchange with cardiac myofilaments are not clearly understood. In order to examine the effect of decreasing Ca2+ sensitivity of cTnC on cardiac function, we generated knock-in mice carrying a D73N mutation (not known to be associated with heart disease in human patients) in cTnC. The D73N mutation was engineered into the regulatory N-domain of cTnC in order to reduce Ca2+ sensitivity of reconstituted thin filaments by increasing the rate of Ca2+ dissociation. In addition, the D73N mutation drastically blunted the extent of Ca2+ desensitization of reconstituted thin filaments induced by cTnI pseudo-phosphorylation. Compared to wild-type mice, heterozygous knock-in mice carrying the D73N mutation exhibited a substantially decreased Ca2+ sensitivity of force development in skinned ventricular trabeculae. Kaplan-Meier survival analysis revealed that median survival time for knock-in mice was 12 weeks. Echocardiographic analysis revealed that knock-in mice exhibited increased left ventricular dimensions with thinner walls. Echocardiographic analysis also revealed that measures of systolic function, such as ejection fraction (EF) and fractional shortening (FS), were dramatically reduced in knock-in mice. In addition, knock-in mice displayed electrophysiological abnormalities, namely prolonged QRS and QT intervals. Furthermore, ventricular myocytes isolated from knock-in mice did not respond to β-adrenergic stimulation. Thus, knock-in mice developed pathological features similar to those observed in human patients with dilated cardiomyopathy (DCM). In conclusion, our results suggest that decreasing Ca2+ sensitivity of the regulatory N-domain of cTnC is sufficient to trigger the development of DCM.
Highlights
Dilated cardiomyopathy (DCM) is a progressive disease of the myocardium characterized by dilation and impaired systolic function of left or both ventricles
Effect of the D73N Mutation on the Ca2+ Sensitivity and the Rate of Ca2+ Dissociation from Reconstituted Thin Filaments in the Absence and Presence of cardiac troponin I (cTnI) Pseudo-phosphorylation Previously, we demonstrated that Ca2+ binding and exchange with an ejection fraction (EF)-hand within a Ca2+ binding protein can be altered by mutations of the chelating loop residues (Black et al, 2000; Liu et al, 2012a)
The D73N mutation led to substantial Ca2+ desensitization of reconstituted thin filaments, without affecting Ca2+ binding cooperativity
Summary
Dilated cardiomyopathy (DCM) is a progressive disease of the myocardium characterized by dilation and impaired systolic function of left or both ventricles (for review Wexler et al, 2009; Jefferies and Towbin, 2010). While DCM can be caused by a variety of factors, mutations in over 40 genes account for ∼25–30% of cases (for review Towbin, 2014; McNally et al, 2015). An estimated 35–40% of genetic DCM cases are linked to mutations in genes encoding sarcomeric proteins, including all three subunits of the cardiac troponin (cTn) complex Chang and Potter, 2005; McNally et al, 2015) Understanding how these mutations trigger pathogenesis of DCM is lacking, DCM-linked mutations of sarcomeric proteins tend to desensitize myofilaments to Ca2+ (for review Kimura, 2010; Willott et al, 2010; Lu et al, 2013; Spudich, 2014)
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