Hypothermia improves resistance to ischemia in the cardioplegia-arrested heart. This adaptive process produces changes in specific signaling pathways for mitochondrial proteins and heat-shock response. To further test for hypothermic modulation of other signaling pathways such as apoptosis, we used various molecular techniques, including cDNA arrays. Isolated rabbit hearts were perfused and exposed to ischemic cardioplegic arrest for 2 h at 34 degrees C [ischemic group (I); n = 13] or at 30 degrees C before and during ischemia [hypothermic group (H); n = 12]. Developed pressure, the maximum first derivative of left ventricular pressure, oxygen consumption, and pressure-rate product (P < 0.05) recovery were superior in H compared with in I during reperfusion. mRNA expression for the mitochondrial proteins, adenine translocase and the beta-subunit of F1-ATPase, was preserved by hypothermia. cDNA arrays revealed that ischemia altered expression of 13 genes. Hypothermia modified this response to ischemia for eight genes, six related to apoptosis. A marked, near fivefold increase in transformation-related protein 53 in I was virtually abrogated in H. Hypothermia also increased expression for the anti-apoptotic Bcl-2 homologue Bcl-x relative to I but decreased expression for the proapoptotic Bcl-2 homologue bak. These data imply that hypothermia modifies signaling pathways for apoptosis and suggest possible mechanisms for hypothermia-induced myocardial protection.

IntroductionThis study investigates the relation between myocardial oxygen consumption (MV 02), function, and high energy phos- Severe hypoxia ultimately leads to contractile failure, which is attributed to an imbalance between myocardial oxygen supply and demand (1)(2)(3)(4). Sudden extreme oxygen deprivation causes rapid depletion of energy stores in the form of the high energy phosphates, phosphocreatine, and ATP, as well as accumulation of ATP hydrolysis products. In isolated myocytes (5) and buffer-perfused hearts (5, 6) anoxia or hypoxia results in a reduced oxidative phosphorylation rate. Myocardial respiration wanes despite increasing ADP and intracellular phosphate (Pi),' which both stimulate mitochondrial ATP production under aerobic conditions. The intact animal generally maintains or increases myocardial oxygen consumption during early or moderate hypoxia (7-9). Small decreases in myocardial phosphocreatine content with no depletion of the cytosolic ATP pool have been documented during moderate hypoxic conditions (10, 11). However, myocardial oxygen consumption rates have not been directly measured during periods of rapid high energy phosphate depletion and repletion in the intact animal. The purpose of this study was to examine the relation between myocardial oxygen consumption, function, and high energy phosphates during severe hypoxia and reoxygenation in vivo. Additionally, rephosphorylation parameters were measured to determine if evidence of respiratory uncoupling or mitochondrial damage occurs during reoxygenation in vivo. Graded hypoxia was performed in an effort to gradually reduce arterial oxygen tension to attain the P02 at which high energy phosphate stores rapidly decreased. Highly time-resolved 31P nuclear magnetic resonance (NMR) spectroscopy enabled monitoring of myocardial phosphates throughout hypoxia and recovery with simultaneous measurement of oxygen consumption. Consequently, these measurements enhanced analysis of mitochondrial function in vivo during reoxygenation. MethodsAnimal preparation. Animals used in this study were handled in accordance with institutional and National Institutes of Health animal care and use guideline. Sheep between 30 and 70 d old (mean 47 d±6.8) were sedated with an intramuscular injection of 10 mg/kg ketamine, and 0.2-0.4 mg/kg xylazine, intubated, and then ventilated (C-900 pediatric ventilator; Siemens Corp., Schaumberg, IL) with room air and oxygen, followed by an intravenous dose of alpha-chloralose (40 mg/ kg). Femoral arterial cannulation was performed for monitoring systemic blood pressure and sampling blood. Arterial pH was maintained between 7.35 and 7.45 by adjustment of ventilatory tidal volume and 1. Abbreviations used in this paper: FIo2, fractional inspiratory oxygen

Studies focusing on the functional border zone have been performed largely with anesthetized, open-chest preparations. Therefore, we instrumented 14 dogs at sterile surgery with sonomicrometers arrayed to measure systolic wall thickening across the perfusion boundary produced by circumflex coronary occlusion. We fitted sigmoid curves to the data to model the distribution of wall thickening impairment as a function of distance from the perfusion boundary, which was delineated with myocardial blood flow (15 micron diameter microspheres) maps. Using this approach, we defined the functional border zone as the distance from the perfusion boundary to 97.5% of the sigmoid curve's nonischemic asymptote. The lateral extent of the functional border zone, measured 10 min and 3 hr after occlusion, was 32 and 28 degrees of circumference, respectively. To evaluate the severity of nonischemic dysfunction, we measured average systolic wall thickening within the functional border zone. It was reduced from 3.69 +/- 1.10 (mean +/- SD) mm to 2.98 +/- 1.07 mm (p less than .01) and 2.74 +/- 1.12 mm (p less than .01) early and late after coronary occlusion. Thus, a narrow functional border zone was evident during circumflex coronary occlusion in conscious dogs. Its lateral extent was limited to approximately 30 degrees (similar to findings in open-chest, anesthetized dogs), severe dysfunction was restricted to the immediate vicinity of the perfusion boundary, and the average severity of nonischemic dysfunction within the functional border zone was mild.

The role of creatine kinase in regulation of myocardial respiration was studied in vivo as a function of maturation. Unidirectional creatine kinase flux (JCK), phosphocreatine to gamma-ATP, was measured in newborn lambs (age 3-9 days, n = 8) and mature sheep (age 30-60 days, n = 6) using 31P saturation transfer techniques, and total creatine kinase activity was measured using standard methods. Myocardial oxygen consumption (MVO2) was measured simultaneously via an extracorporeal shunt from the coronary sinus as cardiac work was increased via epinephrine (1-3 micrograms.kg-1.min-1). Findings were as follows: 1) baseline newborn JCK was markedly lower than in mature sheep despite higher levels of MVO2, and this could be related to a decrease in total creatine kinase activity; 2) JCK was substantially higher than the rate of ATP synthesis in both groups at baseline rates of oxygen consumption; and 3) JCK decreased significantly in newborns during increases in MVO2, whereas there was no change in flux rate in the mature sheep during even larger relative changes in work and oxygen consumption. These data imply that creatine kinase does not limit oxidative phosphorylation. However, this enzyme system probably maintains at least an indirect role in respiratory control that is a function of the myocardial developmental state.