Impact of site-specific phosphorylation of protein kinase A sites Ser23 and Ser24 of cardiac troponin I in human cardiomyocytes

Wijnker PJ, Foster DB, Tsao AL, Frazier AH, dos Remedios CG, Murphy AM, Stienen GJ, van der Velden J. Impact of site-specific phosphorylation of protein kinase A sites Ser(23) and Ser(24) of cardiac troponin I in human cardiomyocytes. Am J Physiol Heart Circ Physiol 304: H260-H268, 2013. First published November 9, 2012; doi:10.1152/ajpheart.00498.2012.-PKA-mediated phosphorylation of contractile proteins upon beta-adrenergic stimulation plays an important role in the regulation of cardiac performance. Phosphorylation of the PKA sites (Ser(23)/Ser(24)) of cardiac troponin (cTn)I results in a decrease in myofilament Ca2(+) sensitivity and an increase in the rate of relaxation. However, the relation between the level of phosphorylation of the sites and the functional effects in the human myocardium is unknown. Therefore, site-directed mutagenesis was used to study the effects of phosphorylation at Ser(23) and Ser(24) of cTnI on myofilament function in human cardiac tissue. Serines were replaced by aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. cTnI-DD mimics both sites phosphorylated, cTnI-AD mimics Ser(23) unphosphorylated and Ser(24) phosphorylated, cTnI-DA mimics Ser(23) phosphorylated and Ser(24) unphosphorylated, and cTnI-AA mimics both sites unphosphorylated. Force development was measured at various Ca2(+) concentrations in permeabilized cardiomyocytes in which the endogenous troponin complex was exchanged with these recombinant human troponin complexes. In donor cardiomyocytes, myofilament Ca2(+) sensitivity (pCa(50)) was significantly lower in cTnI-DD (pCa(50): 5.39 +/- 0.01) compared with cTnI-AA (pCa(50): 5.50 +/- 0.01), cTnI-AD (pCa(50): 5.48 +/- 0.01), and cTnI-DA (pCa(50): 5.51 +/- 0.01) at similar to 70% cTn exchange. No effects were observed on the rate of tension redevelopment. In cardiomyocytes from idiopathic dilated cardiomyopathic tissue, a linear decline in pCa(50) with cTnI-DD content was observed, saturating at similar to 55% bisphosphorylation. Our data suggest that in the human myocardium, phosphorylation of both PKA sites on cTnI is required to reduce myofilament Ca2(+) sensitivity, which is maximal at similar to 55% bisphosphorylated cTnI. The implications for in vivo cardiac function in health and disease are detailed in the DISCUSSION in this article.