PKCα-specific phosphorylation of the troponin complex in human myocardium: a functional and proteomics analysis.

Aims: Protein kinase Ca (PKCa) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCa is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKCa phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved. Methods and Results: Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (,69%) with PKCa-treated recombinant human cTn (cTn (DD+PKCa)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKCa. Isometric force was measured at various [Ca 2+ ] after exchange. The maximal force (Fmax) in the cTn (DD+PKCa) group (17.161.9 kN/m 2 ) was significantly reduced compared to the cTn (DD) group (26.161.9 kN/m 2 ). Exchange of endogenous cTn with cTn (DD+PKCa) increased Ca 2+ -sensitivity of force (pCa50=5.5960.02) compared to cTn (DD) (pCa50=5.5160.02). In contrast, subsequent PKCa treatment of the cells exchanged with cTn (DD+PKCa) reduced pCa50 to 5.4560.02. Two PKCa-phosphorylated residues were identified with mass spectrometry: Ser198 on cTnI and Ser179 on cTnT, although phosphorylation of Ser198 is very low. Using mass spectrometry based-multiple reaction monitoring, the extent of phosphorylation of the cTnI sites was quantified before and after treatment with PKCa and showed the highest phosphorylation increase on Thr143. Conclusion: PKCa-mediated phosphorylation of the cTn complex decreases Fmax and increases myofilament Ca 2+ -sensitivity, while subsequent treatment with PKCa in situ decreased myofilament Ca 2+ -sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCa are phosphorylated in human cTn complex treated with PKCa with a high degree of specificity for Thr143.