Abstract Body: The heart regulates its contractile and relaxation functions on a beat-to-beat basis by kinase mediated serine/threonine phosphorylation. Key to this regulation is the well described phosphorylation of the cardiac myofilament regulatory troponin inhibitory subunit (cTnI). Tyrosine phosphorylation is also present in the heart, however its effect to modulate myofilament contractile function is largely unknown. We previously demonstrated cTnI undergoes Src-mediated phosphorylation at tyrosine 26 (Y26) resulting in decreased myocyte calcium sensitivity and accelerated cardiac relaxation in vivo. These findings are the first to demonstrate tyrosine phosphorylation of a myofilament protein directly modulates cardiac contractile function. In addition to Y26, cTnI contains 2 other tyrosine residues. To determine if these other cTnI tyrosine residues are phosphorylated, we subjected human tissue to mass spectrometry. Results demonstrate cTnI is also phosphorylated at tyrosine 29 (Y29) and 112 (Y112) in the human heart. Based upon these findings we hypothesize that all three cTnI phosphorylations are significant to the modulation of cardiac contractile function by tyrosine kinase signaling pathways. To determine the effects of cTnI Y29 and Y112 phosphorylation on cardiac contractile function we measured calcium regulated force production in skinned rat trabeculae following exchange with recombinant human cardiac troponin (cTn) containing WT unphosphorylated (cTn WT), Y29E (cTn Y29E) or Y112E (cTn Y112E) pseudo-phosphorylated cTnI. Fibers exchanged with cTn Y29E and cTn Y112E demonstrated decreased calcium sensitivity compared to cTn WT exchange (pCa50: cTn WT=5.78; cTn Y29E=5.66; cTn Y112E=5.69; ANOVA p<0.05) without differences in maximal force production or Hill coefficient. To determine the effects of cTnI tyrosine phosphorylation on the kinetic rate of thin filament deactivation, current efforts are focused on determining the rate of calcium dissociation from reconstituted thin filaments. These results demonstrate cTnI tyrosine phosphorylation is a novel integrator of tyrosine kinase signaling to modulate cardiac function and a potential target to increase cardiac relaxation.