The volatile anesthetics halothane, enflurane, and isoflurane that are in wide clinical use depress myocardial contractility, which can be potentially life-threatening in patients with pre-existing cardiac disease. The overall goal of this project is to characterize the subcellular mechanisms that underly the myocardial depressant effects. There is evidence that these anesthetics interfere with intracellular Ca2+ homeostasis necessary for contraction by decreasing the intracellular availability of Ca2+ in ventricular myocardium, yet it is still uncertain to what extent possibly the utilization and response to intracellular Ca2+ are modified. In experimental models of different physiological complexity we will (1) test the hypothesis that halothane, enflurane, and isoflurane modify Ca2+-responsiveness (Ca2+- sensitivity) of the contractile apparatus of ventricular myocardium, and (2) assess the relative importance of this mechanism with respect to actions of volatile anesthetics on other steps in cardiac excitation-contraction coupling. Similar studies will be carried out to elucidate the inotropic effects of nitrous oxide and of the intravenous anesthetic ketamine. In intact cardiac muscle we will detect the effects of these anesthetics on (1) the intracellular Ca2+ transient, (2) the changes in stiffness that measure the intensity of actomyosin interactions, and (3) variables of contractility (force, shortening, velocity) simultaneously, in order to assess the relative contribution of the anesthetics' effects on sarcoplasmic reticulum Ca2+ uptake and release, myofibrillar Ca2+-sensitivity, and kinetics of cross-bridge interaction, and (4) myocardial relaxation in physiologically sequenced contractions that mimic the contraction-relaxation cycle of the ventricle. We will determine the effects of volatile anesthetics on Ca2+ binding characteristics and Ca2+-induced conformational changes of the regulatory protein troponin C (TnC) at various levels of organization: (1) isolated TnC in solution, (2) TnC reconstituted with troponin I (TnI), (3) whole reconstituted troponin (TnC + TnI + TnT), (4) in skinned cardiac fibers where native TnC was replaced by homologous fluorescently labeled TnC. The results will allow to quantify the effects of each anesthetic on each of the studied steps in excitation-contraction coupling, and provide a rational basis of knowledge on which to formulate pharmacologic action to reverse myocardial depressant effects in surgical patients.
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