title: 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.
authors.affiliation: Department of Physiology, Center for Muscle Biology, University of Kentucky, Lexington, KY 40536, USA.
authors.forename: Liliana
authors.initials: L
authors.lastname: Sfichi-Duke
authors.forename: Mary L
authors.initials: ML
authors.lastname: Garcia-Cazarin
authors.forename: C Amelia
authors.initials: CA
authors.lastname: Sumandea
authors.forename: Gail A
authors.initials: GA
authors.lastname: Sievert
authors.forename: C William
authors.initials: CW
authors.lastname: Balke
authors.forename: Dong-Yun
authors.initials: DY
authors.lastname: Zhan
authors.forename: Sachio
authors.initials: S
authors.lastname: Morimoto
authors.forename: Marius P
authors.initials: MP
authors.lastname: Sumandea
mesh_terms: D000818:Animals; D009202:Cardiomyopathies / Q000235:genetics* / Q000503:physiopathology*; D002352:Carrier Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009210:Myofibrils / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics / Q000502:physiology; D051571:Protein Kinase C-alpha / Q000378:metabolism; D012518:Sarcomeres / Q000378:metabolism*; D019210:Troponin I / Q000378:metabolism; D020107:Troponin T / Q000235:genetics*; D020798:Two-Hybrid System Techniques; D000099163:Myosin Binding Protein C
chemical_list: D002352:Carrier Proteins; D051571:Protein Kinase C-alpha; D019210:Troponin I; D020107:Troponin T; D000099163:Myosin Binding Protein C