Phosphorylation of cardiac troponin I (cTnI) by protein kinase C (PKC) is implicated in cardiac dysfunction. Recently, Serine 199 (Ser199) was identified as a target for PKC phosphorylation and increased Ser199 phosphorylation occurs in end-stage failing compared with non-failing human myocardium. The functional consequences of cTnI-Ser199 phosphotylation in the heart are unknown. Therefore, we investigated the impact of phosphorylation of cTnI-Ser199 on myofilament function in human cardiac tissue and the susceptibility of cTnI to proteolysis. cTnI-Serl 99 was replaced by aspartic acid (1990) or alanine (199A) to mimic phosphorylation and dephosphorylation, respectively, with recombinant wild-type (Wt) cTn as a negative control. Force development was measured at various [Ca2+] and at sarcomere lengths of 1.8 and 2.2 mu m in demembranated cardiomyocytes in which endogenous cTn complex was exchanged with the recombinant human cTn complexes. In idiopathic dilated cardiomyopathy samples, myofilament Ca2+-sensitivity (pCa(50)) at 2.2 pm was significantly higher in 199D (pCa(50) = 5.79 +/- 0.01) compared to 199A (pCa(50) = 5.65 +/- 0.01) and Wt (pCa(50) = 5.66 +/- 0.02) at -63% cTn exchange. Myofilament Ca2+-sensitivity was significantly higher even with only 5.9 +/- 2.5% 199D exchange compared to 199A, and saturated at 123 +/- 2.6%199D exchange. Ser199 pseudo-phosphorylation decreased cTnI binding to both actin and actin-tropomyosin. Moreover, altered susceptibility of cTnI to proteolysis by calpain I was found when Ser199 was pseudo-phosphorylated. Our data demonstrate that low levels of cTnI-Ser199 pseudo-phosphorylation (similar to 6%) increase myofilament Ca2+-sensitivity in human cardiomyocytes, most likely by decreasing the binding affinity of cTnI for actin-tropomyosin. In addition, cTnI-Ser199 pseudophosphorylation or mutation regulates calpain I mediated proteolysis of cTnI. (C) 2015 Elsevier Ltd. All rights reserved.
Wijnker, P. J. M., Li, Y., Zhang, P., Foster, D. B., dos Remedios, C., van Eyk, J. E., ... van der Velden, J. (2015). A novel phosphorylation site, Serine 199, in the C-terminus of cardiac troponin I regulates calcium sensitivity and susceptibility to calpain-induced proteolysis. Journal of Molecular and Cellular Cardiology, 82, 93-103. https://doi.org/10.1016/j.yjmcc.2015.03.006