Preconditioning in Globally Ischemic Isolated Rat Hearts: Effect on Function and Metabolic Indices of Myocardial Damage

We assessed the effects of ischemic preconditioning on heart recovery and metabolic indices of damage following global ischemia and reperfusion, in relationship to post-ischemic lactate release. Three groups of Langendorff rat hearts were studied: (1) A control group of 40 min global ischemia and 45 min reperfusion; (2) preconditioning by 5 min global ischemia and 15 min reperfusion prior to sustained ischemia and reperfusion; (3) Preconditioning by three episodes of brief ischemia–reperfusion prior to sustained ischemia. Repetitive episodes of brief ischemia–reperfusion were associated with increased reactive hyperemia, decreased release of purines and prostaglandin 6-keto F_1α, lower tissue glycogen but no change in lactate washout. After 40 min ischemia, release of lactate was 173±17, 196±6 and 149±9μmol/g in groups 1, 2 and 3, respectively (P<0.01, group 2vgroup 3). Preconditioning had no effect on ischemic arrest but had divergent effects on the development and the magnitude of ischemic contracture: delay and attenuation in group 2 but enhanced onset in group 3. Preconditioning provided a dose-dependent protection from the increase in left ventricular end-diastolic pressure, reduced the reperfusion release of purine metabolites and of creatine kinase, but neither improved systolic function nor prevented arrhythmia. 6-Keto F_1αproduction was 87±13, 132±19 and 241±35 pmol/g in groups 1, 2, 3, respectively (P<0.01 group 1vgroup 3). We conclude that when subjected to prolonged global ischemia, preconditioned isolated rat hearts develop less post-ischemic contracture, lose less purine nucleosides and creatine kinase activity. In addition, preconditioning leads to increased production of prostacyclin. Differences among preconditioning protocols in lactate production seem to be related to the ischemic contracture development, but may not play an ultimate role in attenuation of myocardial damage or improvement of postischemic recovery.     

The Molecular Energetics of the Failing Heart from Animal Models—Large Animal Models

Large animal models of left ventricular hypertrophy (LVH) and cardiac failure are associated with alterations of myocardial high energy phosphate (HEP) content and abnormalities of oxidative phosphorylation regulation. Concentric LVH secondary to pressure overload can result in loss of myocardial ATP, a decrease of the phosphocreatine (PCr)/ATP ratio, and an increase of calculated free ADP; these changes are, at least in part, the result of alterations in the regulation of oxidative phosphorylation, but can be aggravated by impaired blood flow to the subendocardium during increased cardiac workloads. Eccentric LVH resulting from volume overload produces only modest reductions of the myocardial PCr/ATP ratio which are not worsened during increases of cardiac work. Post-infarction left ventricular remodeling is associated with a decrease of the myocardial PCr/ATP ratio that is most marked in animals that develop overt congestive heart failure. The depressed PCr/ATP ratio and ATP content in hypertrophied hearts are not the result of persistent myocardial hypoperfusion, since they are not corrected by pharmacologic coronary vasodilation. Furthermore, the additional decrease of myocardial PCr/ATP which occurs during high workloads in hypertrophied as well as in normal hearts occur without evidence of myoglobin desaturation, indicating that these changes cannot be ascribed to oxygen insufficiency. There is some evidence that impairment of long chain fatty acid uptake contributes to HEP abnormalities in hypertrophied or failing hearts. Furthermore, alterations in creatine kinase isoform expression in hypertrophied or failing hearts may result in higher levels of free ADP that could contribute to the observed HEP abnormalities.     

Comparison of the Effects of ORG 30029, Dobutamine and High Perfusate Calcium on Function and Metabolism in Rat Heart

Cardiac contractility may be enhanced via multiple cellular mechanisms resulting in varied effects on cardiac energetics. The mechanisms that account for the varied energetic responses are not well understood. The purpose of this investigation was to compare the effects of the calcium sensitizing agent ORG 30029 (N-hydroxy-5,6-dimethoxy-benzo[b]thiophene-2-carboximidamide hydrochloride, a calcium sensitizing agent which increases contractility without increasing calcium transients significantly), dobutamine and high perfusate calcium on contractility and energetics. Langendorff-perfused rat hearts were stimulated with ORG 30029, dobutamine and high perfusate calcium in graduated concentrations while myocardial oxygen consumption (MVO₂) and force-time integral were measured. ORG 30029, dobutamine and high perfusate calcium increased contractility in a dose-dependent manner. Despite an increase of 50% in systolic pressure and a 17% increase in force-time integral from control, ORG 30029 had no significant effect on MVO₂at the lower concentrations (n=6). However, dobutamine (n=4) and high perfusate calcium (n=4) caused a 65% increase in systolic pressure and a 17% increase in force-time integral and a 50% and 41% increase in MVO₂respectively (P<0.05). High energy phosphates (by³¹P NMR), and lactate production were unaltered by these agents, suggesting that metabolism was steady state. Basal metabolism tended to increase slightly with dobutamine but not with ORG 30029 or high perfusate calcium. ORG 30029, dobutamine, and high perfusate calcium increase contractility in perfused rat hearts with disparate effects on energetics. These differences may be accounted for, in part, by differences in energy expenditure for calcium handling.     

R56865, a Na- and Ca-Overload Inhibitor, Reduces Myocardial Ischemia-Reperfusion Injury in Blood-Perfused Rabbit Hearts

The cardioprotective effects of R56865 were studied in isolated rabbit hearts, blood-perfused with a support rabbit system. The effect on ischemic injury was evaluated by comparing myocardial contracture and contents of ATP catabolites and of lactate during 60 min of normothermic ischemia in untreated hearts (group I) and in hearts treated with 0.63 mg/kg of R56865 starting 20 min before ischemia (group II; n = 5 in each group). R56865 delayed the onset, and decreased the extent of ischemic contracture, but had no effect on the myocardial content of ATP, of its catabolites or of lactate. The effect on reperfusion injury was studied by monitoring left ventricular function during 80-min reperfusion after the 60-min ischemia in three groups (n = 6 in each): an untreated group (group I) and two groups treated with R56865 given either before (group II) or after ischemia (group III). Ultrastructural changes and cellular calcium distribution after reperfusion were also studied. R56865 improved the recovery of function and prevented contracture during reperfusion. Left ventricular end-diastolic pressure was 13.2 ± 2.8 mmHg in group II and 31.3 ± 8.1 mmHg in group III vs 45.0 ± 2.6 mmHg in group I (P < 0.0001 for II vs I; P > 0.05 for III vs I). Left ventricular developed pressure, maximum dP/dt and minimum dP/dt recovered to 71.0 ± 5.4%, 98.9 ± 6.1%, 85.3 ± 4.8% of baseline values, respectively, in group II, to 64.5 ± 3.0% (P > 0.05), 76.8 ± 3.0%, 70.2 ± 4.0% in group III, vs 52.0 ± 6.5%, 58.9 ± 6.9% and 53.6 ± 5.8% in untreated hearts (P < 0.05 for II or III vs I). Coronary flow was 24.5 ± 2.2 ml/min and 19.8 ± 1.8 ml/min in groups II and III vs 14.8 ± 0.7 ml/min (P < 0.05) in the untreated group. On histology the myocardium in hearts treated either before after ischemia was well protected and calcium distribution was almost normal after reperfusion, while in untreated hearts, most of the myocardium displayed irreversible damage accompanied by massive intracellular calcium accumulation. We conclude that R56865 could attenuate Ca²⁺-overload, thereby reducing myocardial ischemia-reperfusion injury after an extended period of ischemia.     

Altered Creatine Kinase Enzyme Kinetics in Diabetic Cardiomyopathy. AP NMR Magnetization Transfer Study of the Intact Beating Rat Heart

M. Spindler, K. W. Saupe, R. Tian, S. Ahmed, M. A. Matlib and J. S. Ingwall. Altered Creatine Kinase Enzyme Kinetics in Diabetic Cardiomyopathy. A³¹P NMR Magnetization Transfer Study of the Intact Beating Rat Heart.Journal of Molecular and Cellular Cardiology (1999) 31, 2175–2189. To determine whether the decreased contractile performance in diabetic hearts is associated with a reduced energy reserve due to decreased creatine kinase (CK) activity, we measured total CK activity (V_max) in vitro and CK reaction velocity in vivo using³¹P NMR spectroscopy in isolated perfused rat hearts after 4 and 6 weeks of diabetes. After 4 weeks of diabetes, V_maxdecreased by 22% with a larger decrease of CK MB than of CK MM and mitochondrial-CK isoenzymes. There was no further decrease in these parameters after 6 weeks of diabetes. Isovolumic contractile performance of 4 and 6 week diabetic hearts, estimated as rate-pressure product under identical perfusion and loading conditions (EDP set at 6–8 mmHg), was only 50% of that of control. ATP, PCr and total creatine concentrations were not different in control and 4 or 6 weeks diabetic rat hearts. After 4 weeks of diabetes, CK reaction velocity decreased by 22%. This was in proportion to the decline of V_maxand therefore predicted by the rate equation for the CK reaction. However, the further decline in the CK reaction velocity after 6 weeks of diabetes (45%) was greater than that predicted from the CK rate equation (17% decrease), and cannot be explained by substrate control of the enzyme. When hearts were inotropically stimulated by increasing perfusate calcium concentration, CK reaction velocity increased slightly (15%) in both control and diabetic hearts, thereby maintaining a constant ATP concentration. We conclude that in the diabetic myocardium, the CK reaction velocity decreases but does not limit the availability of high-energy phosphates for contraction over the range of workloads studied. We also conclude that a mechanism(s) in addition to substrate control regulates CK reaction velocity in the 6 week diabetic hearts.     

Substrate selection in the isolated working rat heart: effects of reperfusion,afterload, and concentration

A study of substrate selection in the isolated heart was made using¹³C NMR isotopomer analysis, a method that unequivocally identifies relative substrate utilization. This technique has several advantages over conventional approaches used to study this problem. It detects the labeling of metabolic end-products present in tissue, as opposed to more indirect methods such as measurement of respiratory quotient, arteriovenous differences, or specific activity changes in the added substrate. It also has advantages over methods such as¹⁴CO₂ release, which may involve dilution of label with unlabeled pools before CO₂ release. Furthermore, it can measure the relative oxidation of up to four substrates in a single experiment, which other labeling techniques cannot conveniently achieve. Substrates selection was considered in light of its effects on myocardial efficiency and recovery from ischemia. A mixture of four substrates (acetoacetate, glucose, lactate, and a mixture of long chain fatty acids), present at physiological concentration (0.17, 5.5, 1.2, and 0.35 mM, respectively), was examined. This is the first use of such a mixture in the study of substrate selection in an isolated organ preparation. At these concentrations, it was found that fatty acids supplied the majority of the acetyl-CoA (49%), and a substantial contribution was also provided by acetoacetate (23%). This suggests that the ketone bodies are a more important substrate than generally considered. Indeed, normalizing the relative utilizations on the basis of acetyl-CoA equivalents, ketone bodies were by far the preferred substrate. The relative lactate oxidation was only 15%, and glucose oxidation could not be detected. No change in utilization was detected after 15 min of ischemia followed by 40 min of reperfusion. The change in substrate selection with afterload was examined, to mimic the stress-related changes in workload found with ischemia. Only minor changes were found. Substrate selection from the same group of substrates, but employing concentrations observed during starvation, was also assessed. This represents the state during which most clinical treatments and evaluations are performed. In this case, acetoacetate was the most used substrate (78%), with small and equal contributions from fatty acids and endogenous substrates; the oxidation of lactate was suppressed.     

The Protective Effect of L-Cysteine and Glutathione on the Adult and Aged Rat Brain (Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>)-ATPase and Mg<Superscript>2+</Superscript>-ATPase Activities in Galactosemia In Vitro

The aim of this study was to evaluate whether the addition of the antioxidants L-cysteine (Cys) or the reduced glutathione (GSH) could reverse the alterations of brain total antioxidant status (TAS) and the modulated activities of the enzymes (Na⁺,K⁺)-ATPase, and Mg²⁺-ATPase in adult or aged rat brain homogenates induced by galactosemia in vitro. Mixture A [mix. A: galactose-1-phosphate (Gal-1-P, 2 mM) plus galactitol (Galtol, 2 mM) plus galactose (Gal, 4 mM) = classical galactosemia] or mixture B [mix. B: Galtol (2 mM) plus Gal (1 mM) = galactokinase deficiency galactosemia] were preincubated in the presence or absence of Cys (0.83 mM) or GSH (0.83 mM) with adult or aged brain homogenates at 37C for 1 h. TAS and the enzyme activities were determined spectrophotometrically. Mix. A or mix. B preincubation with the adult brain resulted in a significant (Na⁺,K⁺)-ATPase inhibition (–30%) and a Mg²⁺-ATPase stimulation (+300% and +33%, respectively), whereas lower modifications of the enzyme activities (p < 0.001) were found in the aged brain. Gal mixtures decreased TAS by 40% (p < 0.001) and by 20% (p < 0.01) in adult and aged samples, respectively. The antioxidants significantly increased TAS resulting in the reversion of (Na⁺,K⁺)-ATPase inhibition and Mg²⁺-ATPase stimulation by mix. B only. The inhibitory effect of Gal and its derivatives on brain (Na⁺,K⁺)-ATPase and their stimulatory effect on Mg²⁺-ATPase are being decreased with age, probably due to the producion of free radicals. Cys and GSH increased TAS resulting in a reversion of the inhibited (Na⁺,K⁺)-ATPase in both models of the in vitro galactosemia and the stimulated Mg²⁺-ATPase in galactokinase deficiency galactosemia only.     

Decreased energy and phosphorylation status in the liver of lung cancer patients with weight loss

BACKGROUND/AIMS: Altered energy status has been reported in the liver of tumour-bearing animals, but data on energy status in humans are scarce. Therefore, bioenergetics in tumour-free liver of lung cancer patients were monitored using 31P magnetic resonance spectroscopy (MRS) with infusion of L-alanine as a gluconeogenic challenge. METHODS: Twenty-one overnight-fasted lung cancer patients without liver metastases, with (CaWL) or without weight loss (CaWS), and 12 healthy control subjects (C) were studied. Hepatic energy status was monitored before and during an i.v. L-alanine infusion of 1.4-2.8 mmol/kg + 2.8 mmol x kg(-1) x h(-1) for 90 min by 31p MR spectroscopy. RESULTS: Baseline levels of ATP in WL lung cancer patients, expressed relative to total MR-detectable phosphate, were reduced (CaWL, 9.5+/-0.9% vs. CaWS, 12.6+/-0.8% and C, 12.4+/-0.8%; p<0.05) and inversely correlated with the degree of weight loss in lung cancer patients (r=-0.46, p=0.03). Pi/ATP ratios were increased (p<0.05), indicating reduced liver phosphorylation status. During L-alanine infusion, ATP levels decreased in all groups (p<0.05); in CaWL, ATP levels were lower at all time-points between 0-90 min as compared to both CaWS and C (p<0.05). Pi/ATP ratios were significantly higher after 70-90 min of L-alanine infusion in CaWL compared to CaWS and C (p<0.05). CONCLUSIONS: Hepatic ATP and phosphorylation status are reduced in WL lung cancer patients, in contrast to WS patients and healthy subjects, and continue to decrease during infusion of a gluconeogenic substrate, suggesting impaired energy regenerating capacity in these patients.