The influence of lactate,pyruvate and glucose as exogenous substrates on free radical defense mechanisms in isolated rat hearts during ischaemia and reperfusion

Previous studies have shown that exogenous lactate impairs mechanical function of reperfused ischaemic hearts, while pyruvate improves post-ischaemic recovery. The aim of this study was to investigate whether the diverging influence of exogenous lactate and pyruvate on functional recovery can be explained by an effect of the exogenous substrates on endogenous protecting mechanisms against oxygen-derived free radicals. Isolated working rat hearts were perfused by a Krebs-Henseleit bicarbonate buffer containing glucose (5 mM) as basal substrate and either lactate (5 mM) or pyruvate (5 mM) as cosubstrate. In hearts perfused with glucose as sole substrate the activity of glutathione reductase was decreased by 32% during 30 min of ischaemia (p<0.10 versus control value), while the activity of superoxide dismutase and catalase was reduced by 27 and 35%, respectively, during 5 min of reperfusion (p<0.10 versus control value). The GSH level in the glucose group was reduced by 29% following 30 min of ischaemia and 35 min of reperfusion (p<0.10). In lactate- and pyruvateperfused hearts there were no significant decreases of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activity during 30 min of ischaemia, 5 min of reperfusion or 35 min of reperfusion. In pyruvate-perfused hearts the glutathione peroxidase activity was even increased by 43% during 30 min of ischaemia (p<0.05). Glutathione levels (reduced and oxidized) did not markedly change in the lactate and pyruvate groups. Thus, the endogenous defense mechanism against oxygen-derived free radicals is compromised at the onset of reperfusion when glucose as sole substrate is present, while addition of lactate or pyruvate prevents reduction of the endogenous capacity to scavenge oxygen-derived free radicals. The equivocal relationship between endogenous scavenging enzyme activity and haemodynamic recovery indicates that involvement of the endogenous antioxidants, if any, in functional recovery of the post-ischaemic heart is complex. Pyruvate may exert protective effects on mechanical function after mild ischaemia by functioning as exogenous scavenger in itself, as pyruvate is able to react with hydrogen peroxide.     

Oxfenicine diverts rat muscle metabolism from fatty acid to carbohydrate oxidation and protects the ischaemic rat heart

In isolated diaphragms from rats fed on a high-fat diet, oxfenicine (S-4-hydroxyphenylglycine) stimulated the depressed rates of pyruvate decarboxylation (2-fold) and glucose oxidation (5-fold). In diaphragms from normal-fed rats, oxfenicine had no effect on pyruvate decarboxylation but doubled the rate of glucose oxidation and inhibited the oxidation of palmitate. Treatment of fat-fed rats with oxfenicine restored the proportion of myocardial pyruvate dehydrogenase in the active form to that observed in normal-fed rats. In rat hearts perfused in the presence of glucose, insulin and palmitate, oxfenicine increased carbohydrate oxidation and stimulated cardiac performance with no increase in oxygen consumption - i.e. improved myocardial efficiency. Working rat hearts perfused with glucose, insulin and palmitate and subjected to 10 min global ischaemia recovered to 81% of their pre-ischaemic cardiac output after 30 min reperfusion, and released large amounts of lactate dehydrogenase into the perfusate. Hearts perfused with oxfenicine had slightly higher pre-ischaemic cardiac outputs and, on reperfusion, recovered more completely (to 96% of the pre-ischaemic value). Oxfenicine reduced the amount of lactate dehydrogenase released by 73%. We conclude that, in rat hearts with high rates of fatty acid oxidation, a relative increase in carbohydrate oxidation will improve myocardial efficiency, and preserve mechanical function and cellular integrity during acute ischaemia.     

31P-NMR study of high-energy phosphorylated compounds metabolism and intracellular pH in the perfused rat heart

Using 31P-NMR and haemodynamical measurements, this work assesses different aspects of myocardial preservation improvement during a global ischaemia, based on a simultaneous and correlated study of high-energy phosphorylated compounds, intracellular pH and left ventricular function. Isolated perfused working rat hearts were subjected to 2 or 3 h of hypothermic ischaemia followed by 30 or 45 min of reperfusion. A study of the influence of pH and buffer used in cardioplegic solutions has demonstrated a better preservation of high-energy phosphates and an improved functional recovery when using a pH 7.0, glutamate - containing solution. Protection provided by cardioplegia can be enhanced by the appropriate use of a fluorocarbon-oxygenated cardioplegic reperfusate. The use of nifedipine, a calcium antagonist, in the cardioplegic solutions, does not provide any additional protection under hypothermic conditions.     

An increase in the redox state during reperfusion contributes to the cardioprotective effect of GIK solution

This study aimed at determining whether glucose-insulin-potassium (GIK) solutions modify the NADH/NAD(+) ratio during postischemic reperfusion and whether their cardioprotective effect can be attributed to this change in part through reduction of the mitochondrial reactive oxygen species (ROS) production. The hearts of 72 rats were perfused with a buffer containing glucose (5.5 mM) and hexanoate (0.5 mM). They were maintained in normoxia for 30 min and then subjected to low-flow ischemia (0.5% of the preischemic coronary flow for 20 min) followed by reperfusion (45 min). From the beginning of ischemia, the perfusate was subjected to various changes: enrichment with GIK solution, enrichment with lactate (2 mM), enrichment with pyruvate (2 mM), enrichment with pyruvate (2 mM) plus ethanol (2 mM), or no change for the control group. Left ventricular developed pressure, heart rate, coronary flow, and oxygen consumption were monitored throughout. The lactate/pyruvate ratio of the coronary effluent, known to reflect the cytosolic NADH/NAD(+) ratio and the fructose-6-phosphate/dihydroxyacetone-phosphate (F6P/DHAP) ratio of the reperfused myocardium, were evaluated. Mitochondrial ROS production was also estimated. The GIK solution improved the recovery of mechanical function during reperfusion. This was associated with an enhanced cytosolic NADH/NAD(+) ratio and reduced mitochondrial ROS production. The cardioprotection was also observed when the hearts were perfused with fluids known to increase the cytosolic NADH/NAD(+) ratio (lactate, pyruvate plus ethanol) compared with the other fluids (control and pyruvate groups). The hearts with a high mechanical recovery also displayed a low F6P/DHAP ratio, suggesting that an accelerated glycolysis rate may be responsible for increased cytosolic NADH production. In conclusion, the cardioprotection induced by GIK solutions could occur through an increase in the cytosolic NADH/NAD(+) ratio, leading to a decrease in mitochondrial ROS production.     

5-3H]glucose overestimates glycolytic flux in isolated working rat heart: role of the pentose phosphate pathway

We set out to study the pentose phosphate pathway (PPP) in isolated rat hearts perfused with [5-3H]glucose and [1-14C]glucose or [6-14C]glucose (crossover study with 1- then 6- or 6- then 1-14C-labeled glucose). To model a physiological state, hearts were perfused under working conditions with Krebs-Henseleit buffer containing 5 mM glucose, 40 microU/ml insulin, 0.5 mM lactate, 0.05 mM pyruvate, and 0.4 mM oleate/3% albumin. The steady-state C1/C6 ratio (i.e., the ratio from [1-14C]glucose to [6-14C]glucose) of metabolites released by the heart, an index of oxidative PPP, was not different from 1 (1.06 +/- 0.19 for 14CO2, and 1.00 +/- 0.01 for [14C]lactate + [14C]pyruvate, mean +/- SE, n = 8). Hearts exhibited contractile, metabolic, and 14C-isotopic steady state for glucose oxidation (14CO2 production). Net glycolytic flux (net release of lactate + pyruvate) and efflux of [14C]lactate + [14C]pyruvate were the same and also exhibited steady state. In contrast, flux based on 3H2O production from [5-3H]glucose increased progressively, reaching 260% of the other measures of glycolysis after 30 min. The 3H/14C ratio of glycogen (relative to extracellular glucose) and sugar phosphates (representing the glycogen precursor pool of hexose phosphates) was not different from each other and was <1 (0.36 +/- 0.01 and 0.43 +/- 0.05 respectively, n = 8, P < 0.05 vs. 1). We conclude that both transaldolase and the L-type PPP permit hexose detritiation in the absence of net glycolytic flux by allowing interconversion of glycolytic hexose and triose phosphates. Thus apparent glycolytic flux obtained by 3H2O production from [5-3H]glucose overestimates the true glycolytic flux in rat heart.     

Metabolic basis for contractile dysfunction following chronic partial bladder outlet obstruction in rabbits

Our study is designed to correlate nitrite concentration, an index of nitric oxide (NO) release with mast cell peroxidase (MPO), a marker of cardiac mast cell degranulation and cardioprotective effect of ischaemic preconditioning in isolated perfused rat heart subjected to 30 min of global ischaemia and 30 min of reperfusion. Ischaemic preconditioning, comprised of four episodes of 5 min global ischaemia and 5 min of reperfusion, markedly reduced the release of lactate dehydrogenase (LDH) and creatine kinase (CK) in coronary effluent and incidence of ventricular premature beats (VPBs) and ventricular tachycardia and fibrillation (VT/VF) during reperfusion phase. Ischaemia-reperfusion induced release of MPO was markedly reduced in ischaemic preconditioned hearts. Increased release of nitrite was noted during reperfusion phase after sustained ischaemia in preconditioned hearts as compared to control hearts. No alterations in the release of nitrite was observed immediately after ischaemic preconditioning. However, ischaemic preconditioning markedly increased the release of MPO prior to global ischaemia. It is proposed that cardioprotective and antiarrhythmic effect of ischaemic preconditioning may be ascribed to degranulation of cardiac mast cells. Depletion of cytotoxic mediators during ischaemic preconditioning and consequent decreased release of these mediators during sustained ischaemia-reperfusion may be associated with preservation of structures in isolated rat heart responsible for NO release.     

Evidence for a lactate transport system in the sarcolemmal membrane of the perfused rabbit heart: kinetics of unidirectional influx, carrier specificity and effects of glucagon

The kinetics and specificity of L-lactate transport into cardiac muscle were studied during a single transit through the isolated perfused rabbit heart using a rapid (15 s) paired-tracer dilution technique. Kinetic experiments revealed that lactate influx was highly stereospecific and saturable with an apparent Kt = 19 +/- 6 mM and a Vmax = 8.4 +/- 1.5 mumol/min per g (mean +/- S.E., n = 14 hearts). At high perfusate concentrations (10 mM), the inhibitors alpha-cyano-4-hydroxycinnamate (Ki = 7.3 mM), pyruvate (Ki = 6.5 mM), acetate (Ki = 19.4 mM) and chloroacetate (Ki = 28 mM) reduced L-lactate influx, and Ki values were estimated assuming a purely competitive interaction of the inhibitors with the monocarboxylate carrier. The monocarboxylic acids [14C]pyruvate and [3H]acetate were themselves transported, and sarcolemmal uptakes of respectively 38 +/- 1% and 70 +/- 8% were measured relative to D-mannitol. Perfusion of hearts for 10-30 min with 0.15 or 1.5 microM glucagon increased myocardial lactate production and simultaneously inhibited tracer uptake of lactate, pyruvate and acetate. It is concluded that a stereospecific lactate transporter exhibiting an affinity for other substituted monocarboxylic acids is operative in the sarcolemmal plasma membrane of the rabbit myocardium.     

Substrate-dependent proton load and recovery of stunned hearts during pyruvate dehydrogenase stimulation

Stimulation of pyruvate dehydrogenase (PDH) improves functional recovery of postischemic hearts. This study examined the potential for a mechanism mediated by substrate-dependent proton production and intracellular pH. After 20 min of ischemia, isolated rabbit hearts were reperfused with or without 5 mM dichloroacetate (DCA) in the presence of either 5 mM glucose, 5 mM glucose + 2.5 mM lactate, or 5 mM glucose + 2.5 mM pyruvate. DCA inhibits PDH kinase, increasing the proportion of dephosphorylated, active PDH. Unlike pyruvate or glucose alone, lactate + glucose did not support the effects of DCA on the recovery of rate-pressure product (RPP) (without DCA, RPP = 14,000 +/- 1,200, n = 6; with DCA, RPP = 13,700 +/- 1,800, n = 9). Intracellular pH, from (31)P nuclear magnetic resonance spectra, returned to normal within 2.1 min of reperfusion with all substrates except for lactate + glucose + DCA or lactate + DCA, which delayed pH recovery for up to 12 min (at 2.1 min pH = 6. 00 +/- 0.08, lactate + glucose + DCA; pH = 6.27 +/- 0.34, for lactate + DCA). Hearts were also reperfused after 10 min of ischemia with 0.5 mM palmitate + 5 mM DCA and either 2.5 mM pyruvate or 2.5 mM lactate. Again, intracellular pH recovery was delayed in the presence of lactate. PDH activation in the presence of lactate also decreased coupling of oxidative metabolism to mechanical work. These findings have implications for therapeutic use of stimulated carbohydrate oxidation in stunned hearts.     

Studies of acidosis in the ischaemic heart by phosphorus nuclear magnetic resonance.

1. Phosphorus-nuclear-magnetic-resonance measurements were made on perfused rat hearts at 37 degrees C. 2. With the improved sensitivity obtained by using a wide-bore 4.3 T superconducting magnet, spectra could be recorded in 1 min. 3. The concentrations of ATP, phosphocreatine and Pi and, from the position of the Pi resonance, the intracellular pH (pHi) were measured under a variety of conditions. 4. In a normal perfused heart pHi = 7.05 +/- 0.02 (mean +/- S.E.M. for seven hearts). 5. During global ischaemia pHi drops to 6.2 +/- 0.06 (mean +/- S.E.M.) in 13 min in a pseudoexponential decay with a rate constant of 0.25 min-1. 6. The relation between glycogen content and acidosis in ischaemia is studied in glycogen-depleted hearts. 7. Perfusion of hearts with a buffer containing 100 mM-Hepes before ischaemia gives a significant protective effect on the ischaemic myocardium. Intracellular pH and ATP and phosphocreatine concentrations decline more slowly under these conditions and metabolic recovery is observed on reperfusion after 30min of ischaemia at 37 degrees C. 8. The relation between acidosis and the export of protons is discussed and the significance of glycogenolysis in ischaemic acid production is evaluated.     

Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart

The bioenergetic basis by which the Krebs cycle substrate pyruvate increased cardiac contractile function over that observed with the Embden-Meyerhof substrate glucose was investigated in the isovolumic guinea pig heart. Alterations in the content of the high energy phosphate metabolites and the rate of high energy phosphate turnover were measured by 31P NMR. These were correlated to the changes in contractile function and rates of myocardial oxygen consumption. Maximum left ventricular developed pressure (LVDP) and high energy phosphates were observed with 16 mM glucose or 10 mM pyruvate. In hearts perfused with 16 mM glucose, the intracellular phosphocreatine (PCr) concentration was 15.2 +/- 0.6 mM with a PCr/Pi ratio of 10.3 +/- 0.9. The O2 consumption was 5.35 mumol/g wet weight/min, and these hearts exhibited a LVDP of 97 +/- 3.7 mm Hg at a constant paced rate of 200 beats/min. In contrast, when hearts were switched to 10 mM pyruvate, the PCr concentration was 18.3 +/- 0.4 mM, the PCr/Pi ratio was 30.4 +/- 2.2, the O2 consumption was 6.67 mumol/g wet weight/min, and the LDVP increased to 125 +/- 3.3 mm Hg. From NMR saturation transfer experiments, the steady-state flux of ATP synthesis from PCr was 4.9 mumol/s/g of cell water during glucose perfusion and 6.67 mumol/s/g of cell water during pyruvate perfusion. The flux of ATP synthesis from ADP was measured to be 0.99 mumol/s/g of cell water with glucose and calculated to be 1.33 mumol/s/g of cell water with pyruvate. These results suggest that pyruvate quite favorably alters myocardial metabolism in concert with the increased contractile performance. Thus, as a mechanism to augment myocardial performance, pyruvate appears to be unique.     

Pyruvate-fortified cardioplegia suppresses oxidative stress and enhances phosphorylation potential of arrested myocardium

Cardioplegic arrest for bypass surgery imposes global ischemia on the myocardium, which generates oxyradicals and depletes myocardial high-energy phosphates. The glycolytic metabolite pyruvate, but not its reduced congener lactate, increases phosphorylation potential and detoxifies oxyradicals in ischemic and postischemic myocardium. This study tested the hypothesis that pyruvate mitigates oxidative stress and preserves the energy state in cardioplegically arrested myocardium. In situ swine hearts were arrested for 60 min with a 4:1 mixture of blood and crystalloid cardioplegia solution containing 188 mM glucose alone (control) or with additional 23.8 mM lactate or 23.8 mM pyruvate and then reperfused for 3 min with cardioplegia-free blood. Glutathione (GSH), glutathione disulfide (GSSG), and energy metabolites [phosphocreatine (PCr), creatine (Cr), P(i)] were measured in myocardium, which was snap frozen at 45 min arrest and 3 min reperfusion to determine antioxidant GSH redox state (GSH/GSSG) and PCr phosphorylation potential {[PCr]/([Cr][P(i)])}. Coronary sinus 8-isoprostane indexed oxidative stress. Pyruvate cardioplegia lowered 8-isoprostane release approximately 40% during arrest versus control and lactate cardioplegia. Lactate and pyruvate cardioplegia dampened (P < 0.05 vs. control) the surge of 8-isoprostane release following reperfusion. Pyruvate doubled GSH/GSSG versus lactate cardioplegia during arrest, but GSH/GSSG fell in all three groups after reperfusion. Myocardial [PCr]/([Cr][P(i)]) was maintained in all three groups during arrest. Pyruvate cardioplegia doubled [PCr]/([Cr][P(i)]) versus control and lactate cardioplegia after reperfusion. Pyruvate cardioplegia mitigates oxidative stress during cardioplegic arrest and enhances myocardial energy state on reperfusion.     

Differential modulation of glucose,lactate, and pyruvate oxidation by insulin and dichloroacetate in the rat heart

Despite the fact that lactate and pyruvate are potential substrates for energy production in vivo, our understanding of the control and regulation of carbohydrate metabolism is based principally on studies where glucose is the only available carbohydrate. Therefore, the purpose of this study was to determine the contributions of lactate, pyruvate, and glucose to energy production in the isolated, perfused rat heart over a range of insulin concentrations and after activation of pyruvate dehydrogenase with dichloroacetate (DCA). Hearts were perfused with physiological concentrations of [1-13C]glucose, [U-13C]lactate, [2-13C]pyruvate, and unlabeled palmitate for 45 min. Hearts were freeze clamped, and 13C NMR glutamate isotopomer analysis was performed on tissue extracts. Glucose, lactate, and pyruvate all contributed significantly to myocardial energy production; however, in the absence of insulin, glucose contributed only 25-30% of total pyruvate oxidation. Even under conditions where carbohydrates represented >95% of substrate entering the tricarboxylic acid (TCA) cycle, we found that glucose contributed at most 50-60% of total carbohydrate oxidation. Despite being present at only 0.1 mM, pyruvate contributed between approximately 10% and 30% of total acetyl-CoA entry into the TCA cycle. We also found that insulin and DCA not only increased glucose oxidation but also exogenous pyruvate oxidation; however, lactate oxidation was not increased. The differential effects of insulin and DCA on pyruvate and lactate oxidation provide further evidence for compartmentation of cardiac carbohydrate metabolism. These results may have important implications for understanding the mechanisms underlying the beneficial effects of increasing cardiac carbohydrate metabolism.     

Adenine nucleotide synthesis from inosine during normoxia and after ischaemia in the isolated perfused rat heart

[14C]inosine in a range of concentrations of 20 microM to 1 mM was administered to the isolated perfused rat heart for 30 min. The incorporation of the nucleoside into myocardial adenine nucleotides increased for extracellular concentrations of the precursor up to 50 microM, reaching a plateau at 60 nmol . g-1 X 30 min-1 with concentrations ranging between 50 and 200 microM. The supply of 500 microM and 1 mM of inosine induced a further increase in cardiac adenine nucleotide synthesis to about 200 nmol . g-1 X 30 min-1. When supplied during low flow ischaemia (0.5 mL . min-1, 30 min.), 1 mM of inosine protected the heart against ATP degradation, while 100 microM of inosine was inefficacious. In the presence of 1 mM of inosine on reperfusion the adenine nucleotide content of the heart was similar to that observed in the absence of the nucleoside. The incorporation of [14C]inosine into adenine nucleotides was, in this last condition, below the value measured before ischaemia. Inosine administration was effective in protecting the heart against ischaemic breakdown of glycogen and favoured postischaemic restoration of glycogen stores.     

The influence of exaprolol upon the ischaemic rat heart and its interaction with sarcolemmal (Na+ + K+)-ATPase

The capability of cyclohexylphenol exaprolol of protecting the ischaemic myocardium during ischaemic cardiac arrest was assessed in the isolated working rat heart. Exaprolol added to the perfusion medium in a dose of 10(-7) mol.l-1 only minimally influenced the left ventricular function (reduced the stroke volume by 18.84% and cardiac output by 14.63%). The hearts were subjected to global ischaemia for 75 min at 26 degrees C and subsequently reperfused for 60 min at 37 degrees C. The recovery of left ventricular function following reperfusion, expressed as a percentage of preischaemic functional performance was used as an indicator of the ischaemic tolerance of the heart. The effect of exaprolol on sarcolemmal (Na+ + K+)-, Mg2+- and Ca2+-ATPase activities was also examined. Exaprolol-pretreated hearts revealed better postischaemic recovery of the left ventricular dP/dt max and stroke volume as well as improved efficiency in the transformation of chemical energy to mechanical work. Exaprolol in 10(-4) mol.l-1 concentration significantly stimulated the specific activity of sarcolemmal (Na+ + K+)-ATPase. Possible mechanisms of the salutary effect of exaprolol on the ischaemic heart are discussed.     

Effects of insulin on the metabolism of the isolated working rat heart perfused with undiluted rat blood

Working rat hearts were perfused with either buffer or with defibrinated, undiluted rat blood dialyzed to remove vasoconstrictor factors. With precautions taken for sterility in the preparation of the perfusate and the apparatus, hearts were obtained which were stable as judged by stroke rate and cardiac output. In these hearts, cardiac output and coronary flow averaged 46.0 and 1.7 ml/g heart per min, respectively. Perfusion with erythrocyte-free buffer depressed cardiac output by 30%, while coronary flow averaged 8.8 ml/g of heart per min. The mean stroke rate of blood-perfused hearts was 300 beats/min but only 240 beats/min during buffer perfusion. In blood-perfused hearts, insulin did not alter stroke rate but significantly lowered coronary flow. The hormone caused a transient increase in cardiac output in hearts perfused with buffer. Insulin did not alter glucose uptake in buffer-perfused hearts but increased lactate release in perfusions with blood. Both serum fatty acids and triacylglycerol fatty acids were significant metabolic fuels in hearts perfused with undiluted blood. The preparation described would appear to be potentially useful for the study of myocardial metabolism in vitro.     

The role of inhibition of NO formation in the metabolic recovery of ischemic rat heart by apelin-12

The effects of apelin-12, a 12 amino acid peptide (H-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-OH, A-12), on recovery of energy metabolism and cardiac function have been studied in isolated working rat hearts perfused with Krebs buffer (KB) containing 11 mM glucose and subjected to global ischemia and reperfusion. Infusion of 140 μM A-12 before ischemia enhanced myocardial ATP, the total pool of adenine nucleotides (ΣAN = ATP+ADP+AMP) and the energy charge of cardiomyocytes ((ATP + 0.5ADP)/ΣAN) at the end of reperfusion compared with control (KB infusion) and decreased lactate content and lactate/pyruvate ratio in the reperfused myocardium up to the initial values. This was accompanied by improved recovery of coronary flow and cardiac function. Co-administration of A-12 and 100 μM L-NAME (an inhibitor of NO synthases) significantly attenuated the A-12 effects on metabolic and functional recovery of reperfused hearts. These results indicate involvement of NO in mechanisms of cardioprotection that are tightly associated with recovery of energy metabolism in the postischemic heart.     

Ischemic preconditioning preserves proton leakage from mitochondrial membranes but not oxidative phosphorylation during heart reperfusion

The aim of this study was to evaluate the role of mitochondria in the recovery of cardiac energetics induced by ischaemic preconditioning at reperfusion. Isolated rat hearts were aerobically perfused (control), subjected to global ischaemia and reperfusion (reperfusion), or subjected to 3 brief cycles of ischaemia/reperfusion and then to the protocol of reperfusion (preconditioning). At the end of the perfusion, antimycin A was delivered to the heart for 25 min, to inhibit mitochondrial respiration and stimulate glycolysis. The increased amount of lactate released in the coronary effluent was correlated with the number of viable cells producing this end-product of glycolysis. Preconditioned hearts released 18% more lactate than reperfused hearts (p < 0.05). This result indicates that preconditioning partially preserved cell viability, as was also evidenced by the MTT assay performed on cardiac biopsies. The difference between antimycin A-stimulated and basal lactate concentration, representing the contribution of mitochondria to the overall energetics of cardiac tissue, was also 18% more elevated in the preconditioned hearts than in the reperfused hearts (p < 0.01). The study of the respiratory function of mitochondria isolated at the end of perfusion, showed that preconditioning did not improve the oxygen-dependent production of ATP (state 3 respiration, ADP/O). On the contrary, state 4 respiration, which is related to proton leakage, was 35.0% lower in the preconditioned group than reperfusion group (p < 0.05). Thus, preconditioning ameliorates cardiac energetics by preserving cell death, but without affecting mitochondrial oxidative phosphorylation. Mitochondria can contribute to cell survival by the attenuation of proton leak from inner membrane.     

The effect of ischaemia and reperfusion on sarcolemmal inositol phospholipid and cytosolic inositol phosphate metabolism in the isolated perfused rat heart

In this study the mass of polyphosphoinositides as well as the turnover of [³H]inositol phospholipids and [³H]inositol phosphates during ischaemia and short periods of reperfusion were studied in the isolated perfused rat heart. Since the phosphoinositides located within the sarcolemma are precursors for release of inositoltrisphosphate (InsP₃) and diacylglycerol, sarcolemmal membranes (rather than whole tissue) isolated at the end of the experimental procedure, were used. Hearts were prelabelled with [³H]inositol and subsequently perfused with 10 mM LiCI to block the phosphatidylinositol (PI) pathway. The results showed that 20 min of global ischaemia depressed the amount of [³H]inositol present in both sarcolemmal phosphatidylinositol-4-phosphate (PI-4-P) and phosphatidylinositol-4,5-bisphosphate (PI-4,5-P₂), as well as in the cytosolic [³H]inositol phosphates, [³H]InsP₂ and [³H]InsP₃. The mass of the sarcolemmal inositol phospholipids remained unchanged during ischaemia. Reperfusion caused an immediate (within 30 sec) increase in the amount of [³H]inositol in sarcolemmal PI, PI-4-P and PI-4,5-P₂. PI-4-P levels showed a transient increase after 30 seconds postischaemic reperfusion, while the mass of the other sarcolemmal inositol phospholipids, PI and PI-4,5-P₂, remained unchanged. [³H]Insp, [³H]InsP₂ and [³H]InsP₃ also increased significantly in comparison to ischaemic hearts after only 30 sec postischaemic reperfusion.In summary, the results obtained indicate inhibition of the PI pathway during ischaemia with an immediate significant stimulation upon reperfusion. In view of the capacity of InsP3 to mobilize Ca²⁺ the possibility exists that stimulation of this pathway during reperfusion may play a role in the intracellular Ca²⁺ overload, characteristic of postischaemic reperfusion.     

Lipid-mediated impairment of normal energy metabolism in the isolated perfused diabetic rat heart studied by phosphorus-31 NMR and chemical extraction

The relationship between extracellular palmitate and the accumulation of long-chain fatty-acyl coenzyme A with that of high-energy phosphate metabolism was investigated in the isolated perfused diabetic rat heart. Hearts were perfused with a glucose/albumin buffer supplemented with 0, 0.5, 1.2 or 2.0 mM palmitate. ³¹P-NMR was used to analyze phosphocreatine and ATP metabolism during 1 h of constant-flow recirculation perfusion. At the end of perfusion, frozen samples were taken for chemical analysis of high-energy phosphates and the free and acylated fractions of coenzyme A and carnitine. Perfusion of diabetic hearts with palmitate, unlike control hearts, caused a time-dependent and concentration-dependent reduction in ATP, despite normal and constant phosphocreatine. Concentrations of acid-soluble coenzyme A, long-chain-acyl coenzyme A and total tissue coenzyme A were elevated in palmitate-perfused diabetic hearts, while the total tissue carnitine pool was decreased. Increases in long-chain-acyl coenzyme A correlated with the reduction in myocardial ATP. This reduction in ATP could not be adequately explained by alterations in heart rate, perfusion pressure or vascular resistance.     

The relative contribution of glucose and fatty acids to ATP production in hearts reperfused following ischemia

High levels of fatty acids decrease the extent of mechanical recovery of hearts reperfused following a transient period of severe ischemia. Glucose oxidation rates during reperfusion are low under these conditions, which can result in a decreased recovery of mechanical function. Stimulation of glucose oxidation with the carnitine palmitoyl transferase I inhibitor, Etomoxir, or by directly stimulating pyruvate dehydrogenase activity with dichloroacetate (DCA) results in an improvement in mechanical function during reperfusion of previously ischemic hearts. Addition of DCA (1 mM) to hearts perfused with 11 mM glucose and 1.2 mM palmitate results in an increase in contribution of glucose oxidation to overall ATP production from 6 to 23%, with a parallel decrease in that of fatty acid oxidation from 90 to 69%. In aerobic hearts, endogenous myocardial triglycerides are an important source of fatty acids for -oxidation. Using hearts in which the myocardial triglycerides were pre-labeled, the contribution of both endogenous and exogenous fatty acid oxidation to myocardial ATP production was determined in hearts perfused with 11 mM glucose, 1.2 mM palmitate and 500 µU/ml insulin. In hearts reperfused following a 30 min period of global no flow ischemia, 91.9% of ATP production was derived from endogenous and exogenous fatty acid oxidation, compared to 87.7% in aerobic hearts. This demonstrates that fatty acid oxidation quickly recovers following a transient period of severe ischemia. Furthermore, therapy aimed at overcoming fatty acid inhibition of glucose oxidation during reperfusion of ischemic hearts appears to be beneficial to recovery of mechanical function.