Heart Care Info - Heart Disease Prevention & Treatment

Generation and Functional Characterization of Knock-in Mice Harboring the Cardiac Troponin I-R21C Mutation Associated with Hypertrophic Cardiomyopathy

Y Wang, JR Pinto, RS Solis, D Dweck... - Journal of Biological ..., 2012 - ASBMB↵1 To whom correspondence should be addressed: 1600 NW 10th Ave. (R-189), Miami, FL 33136. Fax: 305-243-4555; E-mail: jdpotter{at}. ... Background: The R21C substitution in cardiac troponin I (c Tn I) is associated with hypertrophic cardiomyopathy in man. [HTML]

The R21C substitution in cardiac troponin I (c Tn I) is the only identified mutation within its unique N-terminal extension that is associated with hypertrophic cardiomyopathy (HCM) in man. Particularly, this mutation is located in the consensus sequence for β-adrenergic-activated protein kinase A (PKA)-mediated phosphorylation. The mechanisms by which this mutation leads to heart disease are still unclear. Therefore, we generated c Tn I knock-in mouse models carrying an R21C mutation to evaluate the resultant functional consequences. Measuring the in vivo levels of incorporated mutant and WT c Tn I, and their basal phosphorylation levels by top-down mass spectrometry demonstrated: 1) a dominant-negative effect such that, the R21C+/− hearts incorporated 24.9% of the mutant c Tn I within the myofilament; and 2) the R21C mutation abolished the in vivo phosphorylation of Ser23/Ser24 in the mutant c Tn I. Adult heterozygous (R21C+/−) and homozygous (R21C+/+) mutant mice activated the fetal gene program and developed a remarkable degree of cardiac hypertrophy and fibrosis. Investigation of cardiac skinned fibers isolated from WT and heterozygous mice revealed that the WT c Tn I was completely phosphorylated at Ser23/Ser24 unless the mice were pre-treated with propranolol. After propranolol treatment (−PKA), the p Ca-tension relationships of all three mice (i.e. WT, R21C+/−, and R21C+/+) were essentially the same. However, after treatment with propranolol and PKA, the R21C c Tn I mutation reduced (R21C+/−) or abolished (R21C+/+) the well known decrease in the Ca2+ sensitivity of tension that accompanies Ser23/Ser24 c Tn I phosphorylation. Altogether, the combined effects of the R21C mutation appear to contribute toward the development of HCM and suggest that another physiological role for the phosphorylation of Ser23/Ser24 in c Tn I is to prevent cardiac hypertrophy.