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

• Published in: The Journal of biological chemistry Vol. 287; no. 3; pp. 2156 - 2167
• Main Authors: Wang, Yingcai, Pinto, Jose Renato, Solis, Raquel Sancho, Dweck, David, Liang, Jingsheng, Diaz-Perez, Zoraida, Ge, Ying, Walker, Jeffery W., Potter, James D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13.01.2012; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Substitution; Animals; Anti-Arrhythmia Agents - pharmacology; Calcium - metabolism; Calcium-binding Proteins; Cardiac Hypertrophy; Cardiac Troponin I; Cardiomyopathy; Cardiomyopathy, Hypertrophic, Familial - genetics; Cardiomyopathy, Hypertrophic, Familial - metabolism; Cardiomyopathy, Hypertrophic, Familial - pathology; Cyclic AMP-Dependent Protein Kinases - genetics; Cyclic AMP-Dependent Protein Kinases - metabolism; Endomyocardial Fibrosis - genetics; Endomyocardial Fibrosis - metabolism; Gene Knock-In Techniques; Humans; Knock-in mice; Mice; Mice, Mutant Strains; Mutant; Mutation, Missense; Myocardium - metabolism; Myocardium - pathology; Myofibrils - genetics; Myofibrils - metabolism; Myofibrils - pathology; Phosphorylation - genetics; Propranolol - pharmacology; Protein Kinase A (PKA); Protein Structure and Folding; R21C Mutation; Troponin; Troponin I - genetics; Troponin I - metabolism; β-Adrenergic Pathway; Knock-in mice; Protein Kinase A (PKA); Troponin; Cardiomyopathy; Cardiac Troponin I; Calcium-binding Proteins; Mutant; R21C Mutation; Cardiac Hypertrophy; β-Adrenergic Pathway
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.294306

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 Ser23/Ser24 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 Ser23/Ser24 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 Ca2+ sensitivity of tension that accompanies Ser23/Ser24 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 Ser23/Ser24 in cTnI is to prevent cardiac hypertrophy. Background: The R21C substitution in cardiac troponin I (cTnI) is associated with hypertrophic cardiomyopathy in man. Results: The R21C mutation disrupts the consensus sequence for cTnI phosphorylation. Conclusion: The KI mouse model showed remarkable degree of cardiac hypertrophy and fibrosis after 12 months of age. Significance: One of the physiological roles for the phosphorylation of the cTnI N-terminal extension is to prevent cardiac hypertrophy.