ACTIONS OF ADENOSINE IN NORMAL AND ISCHEMIC MYOCARDIUM

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The broad, long-term objectives of the proposed research are to further our understanding of the mechanisms underlying the pathophysiology of myocardial ischemia-reperfusion injury and to develop therapeutic regimens to treat the ischemic heart. The proposed experiments are designed to determine the effects of reversible myocardial ischemic injury (termed myocardial stunning) on excitation-contraction coupling, and to determine the mechanism by which the purine nucleoside adenosine exerts its well- recognized beneficial effect on postischemic ventricular function. Altered excitation-contraction coupling in stunned myocardium is thought to be due to altered myocardial energetics, contractile protein dysfunction, and/or altered intracellular calcium handling as a result of transient calcium overload. Myocardial stunning has been documented clinically in patients following coronary artery bypass surgery and thrombolysis after acute myocardial infarction. The proposed experiments thus are clinically relevant and of potential therapeutic relevance for humans with ischemic heart disease. The effects of myocardial stunning and its attenuation with adenosine treatment will be addressed in an in vivo porcine regional myocardial ischemia model and in myocytes isolated from normal and stunned myocardium. The specific aims of the proposed research are; (1) To determine the effects of in vivo myocardial stunning and transient calcium overload on the relationship between myocardial energetics and basal and stimulated myocardial contractility. The effects of pretreatment with adenosine or with adenosine A1 receptor agonists and antagonists on myocardial phosphorylation potential and inotropic reserve will be assessed. (2) To determine whether adenosine-induced attenuation of myocardial stunning is associated with a normalization of myofibrillar contractile properties. Assessments of tension-pCa relationships and maximum velocity of shortening (Vmax) will be determined in skinned preparations isolated from in vivo stunned and normal porcine myocardium. Studies will be performed on skinned preparations after in vivo pretreatment with adenosine or with adenosine A1 receptor agonists or antagonists. Specific mechanisms of adenosine action at the level of the myofilaments will be investigated by assessing phosphorylation states of thick (c-proteins, myosin LC2) and thin (TnT, TnT, Tm) filament proteins under various experimental conditions. (3) To determine whether myocardial stunning is associated with altered myocyte intracellular Ca2+ homeostasis. Basal and stimulated Ca2+ current and free intracellular (Ca2+)i will be determined in myocytes isolated from normal and stunned pig myocardium. Our main interest here is to elucidate the effects of adenosine treatment on Ca2+ homeostasis in normal and stunned myocardium.