Effect of troponin I Ser23/24 phosphorylation on Ca2+-sensitivity in human myocardium depends on the phosphorylation background

Protein kinase A (PKA)-mediated phosphorylation of Ser23/24 of cardiac troponin I (cTnI) causes a reduction in Ca2+-sensitivity of force development. This study aimed to determine whether the PKA-induced modulation of the Ca2+-sensitivity is solely due to cTnI phosphorylation or depends on the phosphorylation status of other sarcomeric proteins. Endogenous troponin (cTn) complex in donor cardiomyocytes was partially exchanged (up to 66 ± 1%) with recombinant unphosphorylated human cTn and in failing cells similar exchange was achieved using PKA-(bis)phosphorylated cTn complex. Cardiomyocytes immersed in exchange solution without complex added served as controls. Partial exchange of unphosphorylated cTn complex in donor tissue significantly increased Ca2+-sensitivity (pCa50) to 5.50 ± 0.02 relative to the donor control value (pCa50 = 5.43 ± 0.04). Exchange in failing tissue with PKA-phosphorylated cTn complex did not change Ca2+-sensitivity relative to the failing control (pCa50 = 5.60 ± 0.02). Subsequent treatment of the cardiomyocytes with the catalytic subunit of PKA significantly decreased Ca2+-sensitivity in donor and failing tissue. Analysis of phosphorylated cTnI species revealed the same distribution of un-, mono- and bis-phosphorylated cTnI in donor control and in failing tissue exchanged with PKA-phosphorylated cTn complex. Phosphorylation of myosin-binding protein-C in failing tissue was significantly lower compared to donor tissue. These differences in Ca2+-sensitivity in donor and failing cells, despite similar distribution of cTnI species, could be abolished by subsequent PKA-treatment and indicate that other targets of PKA are involved the reduction of Ca2+-sensitivity. Our findings suggest that the sarcomeric phosphorylation background, which is altered in cardiac disease, influences the impact of cTnI Ser23/24 phosphorylation by PKA on Ca2+-sensitivity.