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Cardiomyopathy-causing deletion K210 in cardiac troponin T alters phosphorylation propensity of sarcomeric proteins.

ABSTRACT: Ca(2+) desensitization of myofilaments is indicated as a primary mechanism for the pathogenesis of familial dilated cardiomyopathy (DCM) associated with the deletion of lysine 210 (DeltaK210) in cardiac troponin T (cTnT). DeltaK210 knock-in mice closely recapitulate the clinical phenotypes documented in patients with this mutation. Considerable evidence supports the proposition that phosphorylation of cardiac sarcomeric proteins is a key modulator of function and may exacerbate the effect of the deletion. In this study we investigate the impact of K210 deletion on phosphorylation propensity of sarcomeric proteins. Analysis of cardiac myofibrils isolated from DeltaK210 hearts identified a decrease in phosphorylation of cTnI (46%), cTnT (30%) and MyBP-C (32%) compared with wild-type controls. Interestingly, immunoblot analyses with phospho-specific antibodies show augmented phosphorylation of cTnT-Thr(203) (28%) and decreased phosphorylation of cTnI-Ser(23/24) (41%) in mutant myocardium. In vitro kinase assays indicate that DeltaK210 increases phosphorylation propensity of cTnT-Thr(203) three-fold, without changing cTnI-Ser(23/24) phosphorylation. Molecular modeling of cTnT-DeltaK210 structure reveals changes in the electrostatic environment of cTnT helix (residues 203-224) that lead to a more basic environment around Thr(203), which may explain the enhanced PKC-dependent phosphorylation. In addition, yeast two-hybrid assays indicate that cTnT-DeltaK210 binds stronger to cTnI compared with cTnT-wt. Collectively, our observations suggest that cardiomyopathy-causing DeltaK210 has far-reaching effects influencing cTnI-cTnT binding and posttranslational modifications of key sarcomeric proteins.

SUBMITTER: Sfichi-Duke L

PROVIDER: S-EPMC2854196 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Cardiomyopathy-causing deletion K210 in cardiac troponin T alters phosphorylation propensity of sarcomeric proteins.

Sfichi-Duke Liliana L Garcia-Cazarin Mary L ML Sumandea C Amelia CA Sievert Gail A GA Balke C William CW Zhan Dong-Yun DY Morimoto Sachio S Sumandea Marius P MP

Journal of molecular and cellular cardiology 20100115 5

Ca(2+) desensitization of myofilaments is indicated as a primary mechanism for the pathogenesis of familial dilated cardiomyopathy (DCM) associated with the deletion of lysine 210 (DeltaK210) in cardiac troponin T (cTnT). DeltaK210 knock-in mice closely recapitulate the clinical phenotypes documented in patients with this mutation. Considerable evidence supports the proposition that phosphorylation of cardiac sarcomeric proteins is a key modulator of function and may exacerbate the effect of the ...[more]

PMID: 20079745

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Project description:Phosphorylation of troponin I by protein kinase A (PKA) reduces Ca(2+) sensitivity and increases the rate of Ca(2+) release from troponin C and the rate of relaxation in cardiac muscle. In vitro experiments indicate that mutations that cause dilated cardiomyopathy (DCM) uncouple this modulation, but this has not been demonstrated in an intact contractile system. Using a Ca(2+)-jump protocol, we measured the effect of the DCM-causing mutation ACTC E361G on the equilibrium and kinetic parameters of Ca(2+) regulation of contractility in single transgenic mouse heart myofibrils. We used propranolol treatment of mice to reduce the level of troponin I and myosin binding protein C (MyBP-C) phosphorylation in their hearts before isolating the myofibrils. In nontransgenic mouse myofibrils, the Ca(2+) sensitivity of force was increased, the fast relaxation phase rate constant, kREL, was reduced, and the length of the slow linear phase, tLIN, was increased when the troponin I phosphorylation level was reduced from 1.02 to 0.3 molPi/TnI (EC50 P/unP = 1.8 ± 0.2, p < 0.001). Native myofibrils from ACTC E361G transgenic mice had a 2.4-fold higher Ca(2+) sensitivity than nontransgenic mouse myofibrils. Strikingly, the Ca(2+) sensitivity and relaxation parameters of ACTC E361G myofibrils did not depend on the troponin I phosphorylation level (EC50 P/unP = 0.88 ± 0.17, p = 0.39). Nevertheless, modulation of the Ca(2+) sensitivity of ACTC E361G myofibrils by sarcomere length or EMD57033 was indistinguishable from that of nontransgenic myofibrils. Overall, EC50 measured in different conditions varied over a 7-fold range. The time course of relaxation, as defined by tLIN and kREL, was correlated with EC50 but varied by just 2.7- and 3.3-fold, respectively. Our results confirm that troponin I phosphorylation specifically alters the Ca(2+) sensitivity of isometric tension and the time course of relaxation in cardiac muscle myofibrils. Moreover, the DCM-causing mutation ACTC E361G blunts this phosphorylation-dependent response without affecting other parameters of contraction, including length-dependent activation and the response to EMD57033.

Dataset Information

Cardiomyopathy-causing deletion K210 in cardiac troponin T alters phosphorylation propensity of sarcomeric proteins.

Publications

Cardiomyopathy-causing deletion K210 in cardiac troponin T alters phosphorylation propensity of sarcomeric proteins.

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