Differential contribution of troponin I phosphorylation sites to the endothelin-modulated contractile response

Cardiac troponin I is a phosphorylation target for endothelin-activated protein kinase C. Earlier work in cardiac myocytes expressing nonphosphorylatable slow skeletal troponin I provided evidence that protein kinase C-mediated cardiac troponin I phosphorylation accelerates relaxation. However, replacement with the slow skeletal isoform also alters the myofilament pH response and the Ca2+ transient, which could influence endothelin-mediated relaxation. Here, differences in the Ca2+ transient could not explain the divergent relaxation response to endothelin in myocytes expressing cardiac versus slow skeletal troponin I nor could activation of Na+/H+ exchange. Three separate clusters within cardiac troponin I are phosphorylated by protein kinase C, and we set out to determine the contribution of the Thr(144) and Ser(23)/Ser(24) clusters to the endothelin-mediated contractile response. Myocyte replacement with a cardiac troponin I containing a Thr(144) substituted with the Pro residue found in slow skeletal troponin I resulted in prolonged relaxation in response to acute endothelin compared with control myocytes. Ser(23)/Ser(24) also is a target for protein kinase C phosphorylation of purified cardiac troponin I, and although this cluster was not acutely phosphorylated in intact myocytes, significant phosphorylation developed within 1 h after adding endothelin. Replacement of Ser(23)/Ser(24) with Ala indicated that this cluster contributes significantly to relaxation during more prolonged endothelin stimulation. Overall, results with these mutants provide evidence that Thr(144) plays an important role in the acute acceleration of relaxation, whereas Ser(23)/Ser(24) contributes to relaxation during more prolonged activation of protein kinase C by endothelin.