Hypothermia is a very powerful temperature-dependent method to protect against clinical cardiac ischemia and reperfusion (I/R) injury. Hypothermia decreases energy utilization and preserves mechanisms to rapidly generate ATP on reperfusion. But hypothermia does not provide complete protection and has deleterious effects; cold perfusion can result in elevated cytosolic (cyt) Ca2+ and cause hyper-contracture and reduced compliance. Formation of reactive oxygen species, (ROS) and, mitochondrial (m) Ca2+ loading are major factors that cause reperfusion stunning and permanent damage after normothermic ischemia. The roles of mROS formation, m Ca2+ loading, mNADH, and other indices of mitochondrial bioenergetics during hypothermic perfusion and I/R have not been studied. Increased cellular sodium-hydrogen exchange (NHE) activity observed during I/R is an attractive candidate to link increased ROS production and m Ca2+ overload. These studies will assess hypothermic ischemia -induced effects on cyt Na+ and m Ca2+ loading, formation of ROS, KAav channels, and mitochondrial bioenergetics (NADH, ATP synthesis, O2 consumption) to determine the mechanism of protection by hypothermia and inhibition of NHE.
Major aims are: 1) To Assess the deleterious effects of hypothermia on mitochondrial function and to evaluate the cardio protective role of cardioplegia, NHE inhibition, ROS scavengers, and Kxav channel opening during and after hypothermic ischemia. 2) To determine the mechanisms of cold perfusion and I/R -induced Na+ and m Ca2+ loading on mitochondrial and myocardial dysfunction using alkalosis, ROS generators and mKAav blockers alone and together with NIIE inhibition. We will use unique on-line fluorescence techniques to assess ROS formation, redox balance, cyt [Na+], and m [Ca2+] at 37 and 27 degrees C in intact guinea pig hearts and ROS release in coronary effluent. Protection will be evidenced in intact hearts by reduced global infarct size, enzyme release, and better mechanical and metabolic function. The mechanism of hypothermic protection will be further investigated in isolated mitochondria measured for NADH, membrane potential, ATP synthesis, and 02 consumption. Emphasis will be placed on the effects of drug-induced blockade of mitochondrial ROS formation, NHE activity and KAav channel opening on cellular cation balance and mitochondrial bioenergetics.
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