We studied Ca(2+) dependence of tension and actomyosin ATPase rate in detergent extracted fiber bundles isolated from transgenic mice (TG), in which cardiac troponin I (cTnI) serines 43 and 45 were mutated to alanines (cTnI S43A/S45A). Basal phosphorylation levels of cTnI were lower in TG than in wild-type (WT) mice, but phosphorylation of cardiac troponin T was increased. Compared with WT, TG fiber bundles showed a 13% decrease in maximum tension and a 20% increase in maximum MgATPase activity, yielding an increase in tension cost. Protein kinase C (PKC) activation with endothelin (ET) or phenylephrine plus propranolol (PP) before detergent extraction induced a decrease in maximum tension and MgATPase activity in WT fibers, whereas ET or PP increased maximum tension and stiffness in TG fibers. TG MgATPase activity was unchanged by ET but increased by PP. Measurement of protein phosphorylation revealed differential effects of agonists between WT and TG myofilaments and within the TG myofilaments. Our results demonstrate the importance of PKC-mediated phosphorylation of cTnI S43/S45 in the control of myofilament activation and cross-bridge cycling rate.