Generation and functional characterization of knock-in mice harboring the cardiac troponin I-R21C mutation associated with hypertrophic cardiomyopathy

Yingcai Wang, Jose Renato Pinto, Raquel Sancho Solis, David Dweck, Jingsheng Liang, Zoraida Diaz-Perez, Ying Ge, Jeffery W. Walker, James D. Potter

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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.

Original languageEnglish
Pages (from-to)2156-2167
Number of pages12
JournalJournal of Biological Chemistry
Volume287
Issue number3
DOIs
StatePublished - Jan 13 2012

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Troponin I
Hypertrophic Cardiomyopathy
Phosphorylation
Mutation
Cyclic AMP-Dependent Protein Kinases
Propranolol
Cardiomegaly
Myofibrils
Consensus Sequence
Adrenergic Agents
Mass spectrometry
Heart Diseases
Mass Spectrometry
Fibrosis
Substitution reactions
Genes
Fibers

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Generation and functional characterization of knock-in mice harboring the cardiac troponin I-R21C mutation associated with hypertrophic cardiomyopathy. / Wang, Yingcai; Pinto, Jose Renato; Solis, Raquel Sancho; Dweck, David; Liang, Jingsheng; Diaz-Perez, Zoraida; Ge, Ying; Walker, Jeffery W.; Potter, James D.

In: Journal of Biological Chemistry, Vol. 287, No. 3, 13.01.2012, p. 2156-2167.

Research output: Contribution to journalArticle

Wang, Yingcai ; Pinto, Jose Renato ; Solis, Raquel Sancho ; Dweck, David ; Liang, Jingsheng ; Diaz-Perez, Zoraida ; Ge, Ying ; Walker, Jeffery W. ; Potter, James D. / Generation and functional characterization of knock-in mice harboring the cardiac troponin I-R21C mutation associated with hypertrophic cardiomyopathy. In: Journal of Biological Chemistry. 2012 ; Vol. 287, No. 3. pp. 2156-2167.
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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.",
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AU - Wang, Yingcai

AU - Pinto, Jose Renato

AU - Solis, Raquel Sancho

AU - Dweck, David

AU - Liang, Jingsheng

AU - Diaz-Perez, Zoraida

AU - Ge, Ying

AU - Walker, Jeffery W.

AU - Potter, James D.

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