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.

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.