The cTnI R21C mutation exerted a dominant-negative effect with 24.9% mutant incorporation, abolishing Ser23/Ser24 phosphorylation and leading to cardiac hypertrophy and fibrosis in knock-in mice.
The R21C mutation in cardiac troponin I abolishes PKA-mediated phosphorylation at Ser(23)/Ser(24), leading to altered calcium sensitivity and the development of cardiac hypertrophy and fibrosis in mice.
The R21C substitution in cardiac troponin I (cTnI) 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 cTnI knock-in mouse models carrying an R21C mutation to evaluate the resultant functional consequences. Measuring the in vivo levels of incorporated mutant and WT cTnI, 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 cTnI within the myofilament; and 2) the R21C mutation abolished the in vivo phosphorylation of Ser(23)/Ser(24) in the mutant cTnI. 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 cTnI was completely phosphorylated at Ser(23)/Ser(24) unless the mice were pre-treated with propranolol. After propranolol treatment (-PKA), the pCa-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 cTnI mutation reduced (R21C+/-) or abolished (R21C+/+) the well known decrease in the Ca(2+) sensitivity of tension that accompanies Ser(23)/Ser(24) cTnI 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 Ser(23)/Ser(24) in cTnI is to prevent cardiac hypertrophy.
Wang et al. (Wed,) conducted a other in Hypertrophic cardiomyopathy. cTnI R21C mutation vs. Wild-type (WT) mice was evaluated on Cardiac hypertrophy, fibrosis, and Ca(2+) sensitivity of tension. The cTnI R21C mutation exerted a dominant-negative effect with 24.9% mutant incorporation, abolishing Ser23/Ser24 phosphorylation and leading to cardiac hypertrophy and fibrosis in knock-in mice.
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