title: Generation and functional characterization of knock-in mice harboring the cardiac troponin I-R21C mutation associated with hypertrophic cardiomyopathy.
abstract: 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.
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami, Miller School of Medicine, Miami, Florida 33136, USA.
authors.forename: Yingcai
authors.initials: Y
authors.lastname: Wang
authors.forename: Jose Renato
authors.initials: JR
authors.lastname: Pinto
authors.forename: Raquel Sancho
authors.initials: RS
authors.lastname: Solis
authors.forename: David
authors.initials: D
authors.lastname: Dweck
authors.forename: Jingsheng
authors.initials: J
authors.lastname: Liang
authors.forename: Zoraida
authors.initials: Z
authors.lastname: Diaz-Perez
authors.forename: Ying
authors.initials: Y
authors.lastname: Ge
authors.forename: Jeffery W
authors.initials: JW
authors.lastname: Walker
authors.forename: James D
authors.initials: JD
authors.lastname: Potter
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D024741:Cardiomyopathy, Hypertrophic, Familial / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D004719:Endomyocardial Fibrosis / Q000235:genetics / Q000378:metabolism; D055879:Gene Knock-In Techniques; D006801:Humans; D051379:Mice; D008817:Mice, Mutant Strains; D020125:Mutation, Missense; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D009210:Myofibrils / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation / Q000235:genetics; D011433:Propranolol / Q000494:pharmacology; D019210:Troponin I / Q000235:genetics / Q000378:metabolism*
chemical_list: D000889:Anti-Arrhythmia Agents; D019210:Troponin I; D011433:Propranolol; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium
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