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.