Abstract 13908: Protein Kinase Cα-mediated Phosphorylation of Cardiac Troponin Reduces Maximal Force and Increases Ca2+-Sensitivity in Human Cardiomyocytes
Rationale: Alpha-adrenergic receptor activated protein kinase C (PKC) is able to modify cardiac function via phosphorylation of several thin and thick myofilament proteins. The isoform PKCα has been implicated in heart failure; however, the exact consequences for the contractile function of the human myocardium are unclear.
Objective: This study aimed to identify the origin and the impact of PKCα-mediated phosphorylation of cardiac troponin (cTn) on myofilament function in human end-stage failing cardiomyocytes.
Methods and results: Endogenous cTn from left ventricular (LV) permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn(DD + PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out cross-phosphorylation of the PKA sites on cTnI by PKCα. Isometric force was measured at various [Ca2+] after exchange. Cardiomyocytes exchanged with cTn(DD + PKCα) showed an increased Ca2+-sensitivity of force and a significant reduction (35.5%) in the maximal force (Fmax) compared to cTn(DD) group. In contrast, subsequent PKCα incubation of the cells exchanged with cTn(DD + PKCα) reduced pCa50, but did not restore Fmax. Using mass spectrometry (MS), two novel phosphorylated residues were identified: Ser198 on cTnI and Ser179 on cTnT and with a MS based-multiple reaction monitoring method, the extent of phosphorylation at each known and novel site were quantified before and after treatment with PKCα.
Conclusion: PKCα-mediated phosphorylation of cTn increases myofilament Ca2+-sensitivity and decreases Fmax. The net result showed a depression of force at all Ca2+-concentrations, suggesting a detrimental effect on LV myocardial contractile function. All known PKC sites as well as the “novel” sites are phosphorylated in the human cTn complex treated with PKCα. However, the results showed a high degree of selectivity for Thr143 indicating an important role for this site on contractile function.
- © 2011 by American Heart Association, Inc.