Protein kinase A (PKA)-mediated phosphorylation of contractile proteins upon activation of β-adrenergic receptors decreases myofilament Ca2+-sensitivity and accelerates relaxation of the heart. Phosphorylation of the PKA sites (Ser23/24) of cardiac troponin I (cTnI) is decreased in end-stage failing compared to non-failing human hearts. However, the site-specific functional consequences of phosphorylation at these sites in human myocardium are unknown. Therefore, we studied the effect of phosphorylation of cTnI-Ser23/24 on myofilament function in explanted human heart tissue.Myofilament force development was measured at various [Ca2+] in permeabilized cardiomyocytes in which endogenous troponin complex was partially exchanged (69±2%) with recombinant (Myc-tag labeled) whole human troponin complexes (1 mg/ml). Site-directed mutations were used to mimic phosphorylation of both Ser23 and Ser24 (cTnI-DD), Ser24 only (cTnI-AD), Ser23 only (cTnI-DA) or dephosphorylation of both sites (cTnI-AA).In donor cardiomyocytes,myofilament Ca2+-sensitivity (pCa50) was significantly reduced in cTnI-DD (pCa50=5.39±0.01) compared to cTnI-AA (pCa50=5.50±0.01), cTnI-AD (pCa50=5.48±0.01) and cTnI-DA (pCa50=5.51±0.01). The maximal rate of tension redevelopment (ktr) was decreased by 15.1-19.8% in cTnI-AD compared to cTnI-AA, cTnI-DA and cTnI-DD. Pseudo-phosphorylation of Ser23/24 had no effect on maximal or passive force nor on the steepness of the force-pCa relation. In end-stage failing cardiomyocytes, using cTnI-DD concentrations between 0 and 1 mg/ml in order to vary the degree of exchange, a linear decline in the pCa50 vs cTnI-DD content by 0.13±0.03 units was observed, saturating at 52±7% of cTnI-DD.Our data indicate that (1) pseudo-phosphorylation of both PKA-sites on cTnI is required to reduce myofilament Ca2+-sensitivity, (2) pseudo-phosphorylation of Ser24 on cTnI results in a slowing of crossbridge kinetics and (3) the maximal reduction in pCa50 is reached at ∼50% bis-phosphorylated cTnI.