Effect of Troponin I Ser23/24 Bis-Phosphorylation on Ca2+-Sensitivity is Dependent on PKA Phosphorylation of Other Contractile Proteins

Abstract

Upon β-adrenergic stimulation, protein kinase A (PKA) enhances cardiac Troponin I (cTnI) phosphorylation at ser23/24. PKA treatment leads to a decrease in myofilament Ca2+-sensitivity. However, the specific effect of PKA-mediated phosphorylation of cTnI in human myocardium is unclear since PKA phosphorylates a broader set of contractile proteins, in particular myosin binding protein C (cMyBP-C).To address this issue, a selective exchange procedure was used in which 50% and 70% of the endogenous cTn complex in permeabilized human cardiomyocytes was exchanged with recombinant unphosphorylated human cTn. Cardiomyocytes isolated from healthy donor hearts showed almost saturated phosphorylation levels at the ser23/24 of cTnI. Endogenous phosphorylated cTn of donor cardiomyocytes (pCa50= 5.45±0.03) was exchanged with 0.5 and 1.0 mg/ml unphosphorylated recombinant human cTn (cTn-U), which resulted in an increase in Ca2+-sensitivity (ΔpCa50=0.08). Subsequent incubation of the cells with PKA reversed Ca2+-sensitivity to baseline levels (pCa50= 5.46±0.03).To study if the effect of PKA-mediated phosphorylation on cTnI ser23/24 depends on phosphorylation of other contractile proteins, failing human cardiac tissue was used in which phosphorylation of cTnI and cMyBP-C is depressed. Cells from failing tissue showed increased Ca2+-sensitivity (pCa50 5.56±0.03) compared to donor cells. Endogenous cTn of failing cardiomyocytes was exchanged with 0.5 and 1.0 mg/ml cTn pre-treated with PKA to fully saturate ser23/24 (cTn-bisP). However, upon exchange with the cTn-bisP complex, Ca2+-sensitivity did not decrease. Subsequent PKA incubation reduced pCa50 back to the level observed in donor myocardium. This indicates that the effect of cTnI ser23/24 bis-phosphorylation on Ca2+-sensitivity is dependent on PKA-mediated phosphorylation of other contractile protein(s). Preliminary protein phosphorylation data point towards the involvement of cMyBP-C.