A novel phosphorylation site, Serine 199, in the C-terminus of cardiac troponin I regulates calcium sensitivity and susceptibility to calpain-induced proteolysis

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.