The reversible mechanical dysfunction that persists during reperfusion following brief periods of myocardial ischemia has been termed postischemic myocardial stunning. The objective of this proposal is to investigate possible mechanisms of this clinically important phenomenon. The main hypothesis to be tested is that decreased myofilament Ca2+ sensitivity is an important cause of depressed function in postischemic stunned myocardium. The approach that will be employed combines an in vivo reperfusion model of stunned myocardium with physiological and biochemical measurements on an in vitro preparation of permeabilized myocardium. Stunning will be induced in the LAD bed of an open-chest pig heart preparation in which regional myocardial function will be assessed from the load independent relationship between end-systolic pressure and wall thickness. Single cell-sized preparations of skinned myocardium will be obtained by mechanical disruption of small needle biopsies from the LAD perfusion bed of pig myocardium. Tension-pCa relationships assessed in these preparations will provide a direct measure of myofilament Ca2+ sensitivity. Preliminary data from our laboratory using this approach show a significant decrease in myofilament Ca2+ sensitivity in stunned myocardium. This proposal has four specific aims: (1) To further characterize the relationship between in vivo measures of postischemic mechanical dysfunction and in vitro measure of myofilament Ca2+ sensitivity from the same heart. A positive relation would suggest that reduced sensitivity to Ca2+ is a direct mechanism of ventricular dysfunction. (2) To determine whether decreases in myofilament Ca2+ sensitivity occur during ischemia, reperfusion, or both. Serial measures of myofilament Ca2+ sensitivity from control, ischemic, and postischemic stunned myocardium obtained from the same hearts should indicate whether reperfusion injury occurs and is an independent cause of stunning. The role of extracellular calcium in reperfusion injury will be tested both in vivo and in vitro. (3) To determine whether decreased myofilament Ca2+ sensitivity is secondary to abnormalities in the regulatory protein troponin and its subunits, troponin-C,-I, and-T. Tension-pCa relationships from stunned myocardium will be compared after extraction of endogenous troponin and replacement with control troponin. (4) To assess a possible role of phosphorylation of troponin I as a mechanism of postischemic stunning. Because phosphorylation of troponin I is a well characterized cause of decreased myofilament Ca2+ sensitivity in normal myocardium, the effect of treatment with phosphatase on tension- pCa relationships from control and stunned myocardium will be measured and compared. Studies proposed in this application will allow direct assessment of decreased sensitivity of myofilaments to Ca2+ as a mechanism of myocardial stunning, and will provide tests of possible mechanisms of stunning at the levels of the myofilaments and regulatory proteins.