Cardiac function is regulated by environmental cues that activate intracellular protein kinases through calcium, cyclic nucleotides and lipid second messengers. The contractile apparatus and associated regulatory proteins represent major targets of protein kinases in the heart. Phosphorylation serves to regulate myofilament properties such as calcium affinity of the regulatory switch, rates of transition through the cross-bridge cycle, power output versus efficiency and rate of relaxation during diastole. One of the major phosphoproteins on cardiac myofilaments is troponin I, the inhibitory subunit of the troponin-tropomyosin complex. Transgenic mouse models in which five previously identified phosphorylation sites on troponin I were mutated to alanine or to aspartate, namely serines^23/24/43/45 and threonine¹⁴⁴, have been especially informative. These ‘non-phosphorylatable’ or ‘pseudophosphorylated’ troponin I constructs rescue the lethal phenotype of cardiac troponin I null mice, and are therefore able to support near normal cardiac function. Similarly, a construct with only two of five sites mutated to alanine, namely serines^23/24, supports normal cardiac function and permits the remaining three sites for protein kinase C (PKC) to be investigated without cross-talk with cyclic AMP dependent protein kinase (PKA) sites. This presentation will describe recent investigations of the function and regulatory properties of these rescued transgenic mice, which in effect harbor troponin I with defined phosphorylation status at several sites and no confounding endogenous troponin I. Intact hearts and isolated myocytes from rescued cTnI-Ala₂, cTnI-Ala₅ and cTnI-Asp₅ mice responded nearly normally to agonists of G-protein coupled receptors. The positive inotropic response to the β-adrenergic agonist isoproterenol was largely unaltered, but twitch narrowing was blunted if troponin I could not be phosphorylated on PKA sites serines^23/24. Similarly, rescued myocytes showed normal positive inotropic responses to endothelin-1, but twitch broadening was blunted if troponin I could not be phosphorylated on PKC sites. Studies in skinned myocardial preparations from rescued mouse lines generally reinforced these observations. Desesensitization of myofilaments to calcium was blunted if troponin I could not be phosphorylated by PKA, whereas sensitization to calcium was blunted if troponin I could not be phosphorylated by PKC. These observations suggest dynamic phosphorylation of troponin I by PKA and PKC reciprocally that can alter myofilament calcium sensitivity and in turn impact twitch duration. In related studies, treatment of skinned myocytes with the βII isoform of PKC caused a pronounced increase in calcium sensitivity in cTnI-Ala₂ myocytes, but not in cTnI-Ala₅, cTnI-Asp₅ or WT mouse myocytes. Several lines of evidence indicate that this sensitizing effect of PKC does not involve phosphorylation of myosin regulatory light chains. The sensitizing effect of PKC may have escaped detection in past investigations if PKA sites on troponin I were cross-phosphorylated by the PKC treatment. These observations indicate that one or more of the three PKC sites on cardiac troponin I may be responsible for the enhanced myofilament calcium sensitivity observed in failing hearts, as a consequence of up-regulation of PKC-βII.