Ischemia/reperfusion (I/R) injury has been recognized to be an important pathologic process; this is manifest clinically as reperfusion induced arrhythmias, the phenomenon of myocardial stunning and an increase in the rate of cell necrosis. Oxygen-derived free radicals have been implicated as important mediators of this injury. The overall goals of the present proposal are (a) to investigate if singlet oxygen is generated following I/R, (b) to develop novel strategies to reduce this injury based on singlet oxygen scavenging and (c) to study the underlying biochemical, physiological and morphological mechanisms of injury. Accordingly, the specific aims of the project are to test the following hypotheses. The first hypothesis is that singlet oxygen is generated as a result of I/R in the isolated perfused rat heart. Using electron paramagnetic resonance (EPR), chemiluminescence and HPLC techniques we will measure single oxygen generation. Specific singlet oxygen spin trap, 2,2,6,6- tetramethylpiperidine (TEMP) will be used in EPR and HPLC measurements. Specificity of singlet oxygen will be confirmed by scavengers such as histidine, tryptophan and B-carotene. We will also monitor contractility, arrhythmia, myocardial metabolism, cell membrane hyperpermeability sarcolemmal and sarcoplasmic reticulum functions and ultrastructural morphology. The second hypothesis is that singlet oxygen inactivates Ca2+-ATPase enzyme of sarcoplasmic reticulum by aggregation and fragmentation or by modification of amino acid residues. We shall study the molecular structure of Ca2+-ATPase following exposure to singlet oxygen. Using SDS-PAGE and HPLC techniques, we shall assess the damage to the 97 K dalton monomer protein of Ca2+-ATPase and compare with O2, H2O2 and OH radical. Protective effects of histidine, alpha- tocopherol B-carotene and ascorbic acid will also be evaluated. The third hypothesis is that singlet oxygen and activate neutrophils produce significant mechanical and hemodynamic dysfunction, similar to I/R in the isolated heart preparation. We will perfuse irradiated rose bengal and activated neutrophils in the isolated perfused heart and monitor isovolumetric left ventricular pressure, + and - dP/dt max, ECG and coronary flow and measure sarcoplasmic reticulum function, sarcolemmal Na+K+-ATPase activity, thiol groups and lipid peroxidation. The efficacy of histidine beta-carotene, ascorbic acid, alpha-tocopherol (for singlet oxygen), SOD, catalase, deferoxamine (O2, H2O2, OH radical human platelets (which possess antioxidative activity) will be tested in this model. the fourth hypothesis is that singlet oxygen itself is a direct, negative inotropic species and exerts its negative inotropic action by disrupting the excitation-contraction coupling system of cardiac muscle. Using isolated papillary muscle isometric contraction model we shall study the mechanical dysfunction associated with singlet oxygen injury by monitoring alterations in contraction of the papillary muscle. The effects of potential scavengers (protective, ameliorating effect and recovery) will also be studied. This work will initiate one of the first studies aimed at (1) measuring the generation of singlet oxygen following I/R (2) demonstrating the physiological, biochemical and morphological characterization of free radicals/neutrophils mediated injury. We anticipate that our findings will foster a precise understanding of the role and mechanism(s) of singlet oxygen mediated physiological and biochemical events.
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