title: Troponin phosphorylation and regulatory function in human heart muscle: dephosphorylation of Ser23/24 on troponin I could account for the contractile defect in end-stage heart failure.
abstract: We made quantitative measurements of phosphorylation in troponin isolated from 6 non-failing donor hearts and 6 explanted hearts with end-stage heart failure in SDS-PAGE gels using Pro-Q Diamond phosphoprotein stain. The troponin T phosphorylation level was the same in troponin from failing and non-failing heart (3.1 mol Pi/mol). However, troponin I phosphorylation was significantly lower in failing (0.37+/-0.18 mol Pi/mol) compared with non-failing heart troponin (2.25+/-0.36 mol Pi/mol). Levels of troponin I PKA-dependent phosphorylation, measured with a phosphoserine 23/24-specific antibody, were also significantly lower in failing heart troponin (0.19+/-0.06 mol Pi/mol) compared to non-failing troponin (1.14+/-0.09 mol Pi/mol). We calculate that there is phosphorylation in addition to serine 23/24 of 1.11+/-0.34 mol Pi/mol in non-failing reduced to 0.18+/-0.17 mol Pi/mol in failing heart troponin, attributed to phosphorylation on the PKC sites. To test for the functional role of troponin I phosphorylation, the native troponin I from either non-failing or failing heart troponin was exchanged for a recombinant (unphosphorylated) human cardiac troponin I. Thin filament Ca(2+)-regulatory function was studied with the quantitative in vitro motility assay: thin filaments containing the replaced troponin I resulted in a failing phenotype of a 17-26% reduced sliding speed and an increased Ca(2+)-sensitivity relative to non-failing troponin (EC(50) TnI-exchanged/non-failing=0.57, p<0.001). When exchanged with troponin I phosphorylated with PKA motility parameters reverted to a pattern indistinguishable from non-failing troponin (p=0.35-0.75). We suggest that changes in troponin function can account for the contractile abnormality in failing heart muscle and that the functional changes in troponin are due to reduced phosphorylation of troponin I at the PKA sites.
authors.affiliation: National Heart and Lung Institute, Imperial College London, Dovehouse Street, London SW3 6LY, UK.
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Messer
authors.forename: Adam M
authors.initials: AM
authors.lastname: Jacques
authors.forename: Steven B
authors.initials: SB
authors.lastname: Marston
mesh_terms: D000328:Adult; D002118:Calcium / Q000378:metabolism; D005260:Female; D006333:Heart Failure / Q000378:metabolism* / Q000503:physiopathology*; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008875:Middle Aged; D009200:Myocardial Contraction / Q000502:physiology*; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D019210:Troponin I / Q000737:chemistry / Q000378:metabolism*; D020107:Troponin T / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D019210:Troponin I; D020107:Troponin T; D012694:Serine; D002118:Calcium