Impact of site-specific phosphorylation of protein kinase A sites Ser~(23) and Ser~(24) of cardiac troponin I in human cardiomyocytes

摘要

PKA-mediated phosphorylation of contractile proteins upon (3-adrenergic stimulation plays an important role in the regulation of cardiac performance. Phosphorylation of the PKA sites (Ser23/Ser24) of cardiac troponin (cTn)I results in a decrease in myofilament Ca~(2+) 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 Ser23 and Ser24 of cTnl on myofilament function in human cardiac tissue. Serines were replaced by aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. cTnl-DD mimics both sites phosphorylated, cTnl-AD mimics Ser23 unphosphorylated and Ser24 phosphorylated, cTnl-DA mimics Ser23 phosphorylated and Ser24 unphosphorylated, and cTnl-AA mimics both sites unphosphorylated. Force development was measured at various Ca~(2+) concentrations in permeabilized cardiomyocytes in which the endogenous troponin complex was exchanged with these recombinant human troponin complexes. In donor cardiomyocytes, myofilament Ca~(2+) sensitivity (pCa_(50)) was significantly lower in cTnl-DD (pCa_(50): 5.39 +- 0.01) compared with cTnl-AA (pCa_(50): 5.50 +- 0.01), cTnl-AD (pCa_(50): 5.48 +-0.01), and cTnl-DA (pCa_(50): 5.51 +- 0.01) at -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 cTnl-DD content was observed, saturating at -55% bisphosphorylation. Our data suggest that in the human myocardium, phosphorylation of both PKA sites on cTnl is required to reduce myofilament Ca~(2+) sensitivity, which is maximal at ~55% bisphosphorylated cTnl. The implications for in vivo cardiac function in health and disease are detailed in the discussion in this article.myofilament function; protein phosphorylation; cardiomyocyte; troponin I