[Role of restricted nitric oxide overproduction in the cardioprotective effect of adaptation to intermittent hypoxia].

Adaptation to intermittent normobaric hypoxia is cardioprotective and can stimulate nitric oxide (NO) synthesis. However the role of nitric oxide (NO) in prevention of ischemia-reperfusion (IR) injury of myocardium is controversial. This study was focused on evaluating the effect of adaptation to hypoxia and IR on NO production and development of nitrative stress in the myocardium. Adaptation to hypoxia tended to increase NO production, which was determined by the total level of plasma nitrite and nitrate, and prevented IR-induced NO overproduction. The IR-induced NO overproduction was associated with significant 3-nitrotyrosine (3-NT) accumulation in the left ventricle but not in septum or aorta. In hypoxia-adapted rats, 3-NT after IR was similar to that of control rats without IR. IHC induced marked accumulation of HIF-1alpha in the left ventricle. We suggest that HIF-1alpha contributes to NO-synthase expression during adaptation to hypoxia and thereby facilitates the increase in NO production. NO, in turn, may subsequently prevent NO overproduction during IR by a negative feedback mechanism.

[Protective and damaging effects of periodic cerebral hypoxia: the role of nitric oxide].

Low oxygen delivery to organs and tissues is one of the most life-threatening situations. Periodic hypoxic episodes may have not only damaging, but also protective effects on the organism depending on how long and intensive this factor is. In both cases an important role is played by changes in the synthesis and metabolism of NO. The direction of NO synthesis and, finally, the direction of periodic hypoxia effect is determined by the regimen of hypoxic impact. The effect of NO depends on its concentration. Both NO excess and deficit are very unfavorable to the organism. Sleep apnea syndrome and pulmonary hypertension are typical examples of NO-dependent damaging effects of periodical hypoxia. NO-dependent protective effects of adaptation to periodic hypoxia are underlied by moderate stimulation of NO synthesis, which provides both compensation for NO deficit and the limitation of its hyperproduction. In turn, NO may increase the expression of other protective factors, which makes adaptive protection more reliable and durable. Understanding the mechanisms of adaptation to hypoxia will help develop new approaches to the prevention of hypoxia and ischemic lesions and the improvement of adaptive abilities of the organism.

Energetic modulation of cardiac inotropism and sarcoplasmic reticular Ca2+ uptake.

Myocardial contractile performance is a function of sarcoplasmic reticular Ca2+ uptake and release. Ca2+ handling is ATP-dependent and can account for up to 40% of total myocardial energy expenditure. We tested the hypothesis that the thermodynamics of the cytosolic adenylate system can modulate sarcoplasmic reticular Ca2+ handling and hence function in intact heart. Cellular energy level was experimentally manipulated by perfusing isolated working guinea-pig hearts with substrate-free medium or media fortified with lactate and/or pyruvate as the main energy substrate. Left ventricular contractile function was judged by stroke work and intraventricular dP/dt. Cytosolic energy level was indexed by measured creatinine kinase reactants. Relative to 5 mM lactate, 5 mM pyruvate increased left ventricular stroke work, dP/dtmax, and dP/dtmin, while lowering left ventricular end-diastolic pressure at physiological left atrial and aortic pressures. Pyruvate also doubled cytosolic phosphorylation potentials and increased [ATP]/[ADP] ratio; this energetic enhancement distinguishes pyruvate from inotropic stimulation by catecholamines, which are known to decrease cytosolic energy level in perfused heart. Sarcoplasmic reticular Ca2+ handling was assessed in hearts prelabeled with 45Ca, subjected to 45Ca washout in the presence of different cytosolic energy levels, then stimulated with 10 mM caffeine to release residual sarcoplasmic reticular 45Ca. When ryanodine (1 microM) was applied to open Ca2+ channels and thereby released 45Ca from the sarcoplasmic reticulum during washout, caffeine-stimulated 45Ca release was decreased 96%, demonstrating that virtually the entire caffeine-sensitive 45Ca pool was located in the sarcoplasmic reticulum. In detailed comparisons of pyruvate-energized vs. substrate-free deenergized hearts, an inverse relationship between cytosolic energy level and caffeine-mobilized 45Ca pool size was observed. Thus, caffeine-induced 45Ca release was decreased 60% by pyruvate energization and increased 2.5-fold by substrate-free deenergization. Taken together, these results support the hypothesis that enhancement of myocardial inotropism by energy-yielding substrate is mediated by increased sarcoplasmic reticular Ca2+ loading/release. Thus we propose that the known control of sarcoplasmic reticular Ca2+ turnover by the protein kinase/phospholamban system can be modulated by cytosolic energy level.

Mitochondrial pyruvate transport in working guinea-pig heart. Work-related vs. carrier-mediated control of pyruvate oxidation.

Myocardial pyruvate oxidation is work- or calcium-load-related, but control of pyruvate dehydrogenase (PDH) by the specific mitochondrial pyruvate transporter has also been proposed. To test the transport hypothesis distribution of pyruvate across the cell membrane as well as rates of mitochondrial pyruvate net transport plus oxidation were examined in isolated perfused but stable and physiologically working guinea-pig hearts. 150 microM-1.2 mM alpha-cyanohydroxycinnamate proved to specifically block mitochondrial pyruvate uptake in these hearts. When perfusate glucose as cytosolic pyruvate precursor was supplied in combination with octanoate (0.2 or 0.5 mM) as diffusible alternative fatty acid substrate, alpha-cyanohydroxycinnamate produced up to 20- and 3-fold increases in pyruvate and lactate efflux, respectively. Cinnamates did not alter myocardial hemodynamics nor sarcolemmal pyruvate and lactate export. In contrast the tested concentrations of cinnamate produced reversible, dose-dependent decreases in 14CO2 production from [1-14C]pyruvate or [U-14C]glucose by inhibiting mitochondrial pyruvate uptake. Linear least-squares estimates of available cinnamate-sensitive total pyruvate transport potential yielded rates close to 110 mumol/min per g dry mass at S0.5 approximately 120 microM, which compared reasonably well with literature values from isolated cardiac mitochondria. This transport potential was severalfold larger than total extractable myocardial PDH activity of approximately 32 mumol/min per g dry mass at 37 degrees C. Even when cytosolic pyruvate levels were in the lower physiologic range of about 90 microM, pyruvate oxidation readily kept pace with mitochondrial respiration over a wide range of workload and inotropism. Furthermore, dichloroacetate, a selective activator of PDH, stimulated pyruvate oxidation without affecting myocardial O2 consumption, regardless of the metabolic or inotropic state of the hearts. Consequently, little or no regulatory function with regard to pyruvate oxidation could be assigned to the native mitochondrial pyruvate carrier of the working heart. Therefore, mitochondrial pyruvate-H+ symport was the normal, highly efficient (rather than controlling) mechanism for pyruvate entry into the mitochondria where PDH regulation controlled pyruvate oxidation.

Use of cytosolic metabolite patterns to estimate free magnesium in normoxic myocardium.

Cytosolic free magnesium (Mgf) is considered relatively constant. To test this concept, Mgf was estimated during hyperkalemic ventricular akinesis, normal and maximum adrenergic stimulation, and sulfate loading of the normoxic perfused guinea-pig heart. The Mgf estimates utilized a new sliding scale derived from the Mg(2+)-dependence of glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK). The pseudo constant K'GAPDH.K'PGK was measured as ([creatine phosphate][3-phosphoglycerate][lactate]KLDH)/([creatine][Pi] [glyceraldehyde 3-phosphate][pyruvate]KCK), which varied with magnesium due to KCK (CK, LDH = creatine kinase, lactate dehydrogenase). However, the correct magnesium dependencies of the true constants KGAPDH.KPGK and KCK were taken from the literature. The [Mg2+] at which pseudo K'GAPDH.K'PGK equalled true KGAPDH.KPGK was the best estimate of Mgf.Mgf fell to approximately 0.13 mM in hyperkalemic arrest from a control of approximately 0.6 mM, rising to approximately 0.85 mM only during maximum adrenergic stress. Mgf increased further to approximately 1.3 mM during sulfate loading which induced ATP catabolism. Mgf and ATP were reciprocally related. Thus; (1) myocardial free [Mg2+] judged from GADPH/PGK mass-action relations changed appreciably only under extreme physiological states; (2) ATP was a major chelator of Mg2+ in perfused myocardium, i.e., acute ATP pool size reduction may be associated with increments in Mgf.

Magnesium activated adenosine formation in intact perfused heart: predominance of ecto 5'-nucleotidase during hypermagnesemia.

Magnesium ion is an allosteric effector of 5'-nucleotidase and thus activates adenosine production from AMP. Two distinct 5'-nucleotidase systems, the membrane-bound ecto and the soluble cytosolic isoforms, exist in mammalian myocardium. The aim of this study was to delineate the contributions of the ecto vs. cytosolic isoforms to Mg2+-stimulated cardiac purine nucleoside formation and release. Isolated guinea pig hearts were retrogradely perfused at their physiological aortic pressure with Krebs-Henseleit bicarbonate buffer fortified with 10 mM glucose. AMP and the adenylate degradatives adenosine and inosine were measured in coronary venous effluent and in epicardial transudate, which was sampled to estimate concentrations of adenylate degradatives in the interstitium. When perfusate Mg2+ was increased from 0.6 to 6 mM, coronary vascular resistance and spontaneous heart rate fell, and steady-state coronary venous release of adenosine + inosine rose severalfold. Cytosolic free magnesium, as estimated by 31P-NMR after 15 min of perfusion with 6 mM Mg2+ or from chemically measured indicator metabolites after 30 min, rose 60 and 144% respectively (P < 0.05). Excess Mg2+ stimulated purine nucleoside release nearly threefold in coronary venous effluent and four- to sevenfold in epicardial transudate. 50 microM, alpha,beta-methylene adenosine 5'-diphosphate (AOPCP), a selective inhibitor of ecto 5'-nucleotidase, elevated interstitial AMP concentration tenfold, did not attenuate basal nucleoside release, but completely inhibited Mg2+-stimulated coronary venous purine nucleoside release and blunted Mg2+-stimulated interstitial purine nucleoside formation by 69%. During perfusion with exogenous 1 microM [8-14C]AMP, excess perfusate MgCl2 increased [14C]adenosine release by 63% in coronary effluent and 133% in epicardial transudate. AOPCP decreased baseline [14C]adenosine release in coronary effluent and epicardial transudate by 85-90%, caused equilibration of arterial and epicardial AMP, and attenuated MgCl2 activation of p[14C]adenosine formation by approx. 75%, in both the vascular and interstitial compartments. Intramyocytic concentrations of allosteric regulators of the cytosolic 5'-nucleotidases were evaluated in stop-frozen myocardium. Excess magnesium did not appreciably alter intracellular pH and ATP concentration, but lowered free cytosolic ADP and AMP concentrations by 50 and 70%, respectively. A simplified model of compartmentalized adenosine metabolism is proposed in which magnesium ion-activated cardiac purine release originates predominantly from the ecto 5'-nucleotidase; magnesium ion stimulation of metabolic flux through the cytosolic isoforms was constrained by concomitant reductions in intracellular AMP substrate and allosteric activator ADP. Magnesium ion-enhanced adenosine formation by 5'-nucleotidase could contribute to the known cardioprotective effects of this clinically used cation.

Mitochondrial metabolism of pyruvate is required for its enhancement of cardiac function and energetics.

UNLABELLED: Pyruvate augmentation of contractile function and cytosolic free energy of ATP hydrolysis in myocardium could result from pyruvate catabolism in the mitochondria or from increased ratio of the cytosolic NAD-/NADH redox couple via the lactate dehydrogenase equilibrium. OBJECTIVE: To test the hypothesis that cytosolic oxidation by pyruvate is sufficient to increase cardiac function and energetics. METHODS: Isolated working guinea-pig hearts received 0.2 mM octanoate +/- 2.5 mM pyruvate as fuels. alpha-Cyano-3-hydroxycinnamate (COHC, 0.6 mM) was administered to selectively inhibit mitochondrial pyruvate uptake without inhibiting pyruvate's cytosolic redox effects or octanoate oxidation. The effects of pyruvate and COHC on sarcoplasmic reticular- Ca2+ handling were examined in 45Ca-loaded hearts. RESULTS: Pyruvate increased left ventricular stroke work and power 40%, mechanical efficiency 29%, and cytosolic ATP phosphorylation potential nearly fourfold. 14CO2 formation from [1-14C]pyruvate was inhibited 65% by COHC, and octanoate oxidation, i.e. 14CO2 formation from [1-14C]octanoate, concomitantly increased threefold. COHC prevented pyruvate enhancement of left ventricular function, mechanical efficiency and cytosolic phosphorylation potential, but did not alter respective levels in pyruvate-free control hearts and augmented cytosolic oxidation by pyruvate. Pyruvate increased sarcoplasmic reticular Ca2+ turnover, i.e. Ca2+ uptake and release, as indicated by 62% decrease in caffeine-induced 45Ca release following 40 min 45Ca washout (P < 0.01). In presence of COHC, pyruvate did not lower caffeine-induced 45Ca release; thus. COHC abrogated pyruvate enhancement of Ca2+ turnover (P < 0.001). CONCLUSION: Pyruvate oxidation of cytosolic redox state is not sufficient to increase cardiac function, cytosolic energetics and sarcoplasmic reticular Ca2+ turnover when mitochondrial pyruvate transport is disabled; thus, mitochondrial metabolism of pyruvate is essential for its metabolic inotropism.

Hypoxic preconditioning of ischaemic canine myocardium.

OBJECTIVE: The aim was to test whether a brief period of non-ischaemic hypoxia can precondition myocardium. METHODS: 60 anaesthetised adult mongrel dogs of either sex underwent 60 min occlusion of the left anterior descending coronary artery, followed by 5 h reperfusion. In treated groups, hearts were either preconditioned with 5 min coronary perfusion with hypoxic blood [O2 content 9.2(SEM 0.6) ml.litre-1] or 5 min occlusion followed by a 10 min reperfusion period prior to 60 min occlusion. The effect of these treatments on myocardial infarct size and regional contractile function was assessed. RESULTS: Infarct size, determined by tetrazolium staining, as a percentage of anatomical area at risk was markedly decreased in hypoxia preconditioned hearts, at 7.2(1.8)% v 22.4(4.6)% in controls (p < 0.01), but did not differ from ischaemia preconditioned hearts [4.6(1.7)%; p < 0.01 v control]. Anatomical area at risk, expressed as a percentage of left ventricular mass, and collateral blood flow to the inner two thirds of the ischaemic wall did not differ among the groups. Regional contractile function was depressed following ischaemic preconditioning but not following hypoxic preconditioning. During reperfusion following 60 min occlusion, marked paradoxical systolic lengthening was evident in ischaemia preconditioned and control hearts but not in hypoxia preconditioned myocardium. CONCLUSIONS: Five minutes of hypoxic and ischaemic preconditioning were equipotent in preventing infarction, whereas ischaemic preconditioning caused a greater decrement in postischaemic contractile function.

Endogenous adenosine increases O2 utilisation efficiency in isoprenaline-stimulated canine myocardium.

OBJECTIVE: We have previously demonstrated that myocardium is capable of down-regulating its O2 requirements and thus avoiding ischaemia when O2 supply is limited. The present study tested the hypothesis that endogenous adenosine produced this protective response when O2 supply was decreased by moderate coronary hypoperfusion or moderate coronary hypoxaemia. METHODS: In anaesthetised dogs, hearts were exposed by left thoracotomy and instrumented for measuring intraventricular pressure and regional myocardial segment length. The left anterior descending coronary artery was isolated, cannulated, and extracorporeally perfused. Coronary O2 supply was moderately reduced by lowering coronary perfusion pressure from 100 to 60 mmHg or by lowering coronary arterial O2 content by 50%. Hearts were treated with intracoronary infusions of adenosine, adenosine deaminase to degrade endogenous adenosine or with erythro-9-(2-hydroxy-3-nonyl)-adenine x HCl (EHNA) to inhibit adenosine degradation by endogenous adenosine deaminase, during beta-adrenergic stimulation with isoprenaline. Cardiac power in the left anterior descending perfusion territory was indexed by the product of heart rate x left ventricular peak systolic pressure x percent systolic segment shortening. O2 utilisation efficiency was taken as the ratio of power index/myocardial O2 consumption. RESULTS: Prior to a reduction in O2 supply, isoprenaline did not alter O2 utilisation efficiency. Intracoronary adenosine increased O2 utilisation efficiency during isoprenaline stimulation by 23% (P < 0.05). EHNA slightly increased O2 utilisation efficiency during isoprenaline stimulation (10%; P < 0.05); adenosine deaminase was without effect. When coronary perfusion pressure was decreased, adenosine deaminase sharply lowered cardiac power and O2 utilisation efficiency during isoprenaline stimulation, whereas EHNA augmented isoprenaline-enhanced power and increased efficiency. During hypoxaemia, adenosine deaminase lowered regional power but not efficiency during isoprenaline infusion; EHNA did not affect power but lowered O2 consumption and increased efficiency. Myocardial lactate extraction and contractile function during isoprenaline stimulation were not attenuated by reduced O2 supply, indicating that myocardial ischaemia did not occur under these conditions. CONCLUSION: Endogenous adenosine increases myocardial O2 utilisation efficiency during beta-adrenergic stimulation, and thus helps avert ischaemia when myocardial O2 supply is reduced.

Hypoxic preconditioning attenuates stunning caused by repeated coronary artery occlusions in dog heart.

OBJECTIVE: The aim was to test whether a brief period of non-ischaemic hypoxia can attenuate cardiac contractile dysfunction, ie, "stunning", due to repeated coronary artery occlusions. METHODS: 20 anaesthetised dogs underwent six 5 min occlusions of the left anterior descending coronary artery with intervening 10 min reperfusions, prior to 90 min reperfusion. In the treated group (n = 9), hearts were preconditioned by 5 min extracorporeal left anterior perfusion with severely hypoxic blood [O2 content 14(SEM 3) ml.litre-1] followed by 10 min reperfusion, prior to the repeated coronary occlusions. Controls (n = 9) were sham preconditioned by 5 min extracorporeal perfusion with normoxic blood [O2 content 179(7) ml.litre-1]. Regional contractile function was assessed by systolic segmental shortening measured by microsonometry. Regional myocardial oxygen consumption, an index of ATP utilisation, was measured in these protocols to evaluate the hypothesis that reduction of myocardial energy demand could be a mechanism of hypoxic preconditioning. RESULTS: Hypoxic preconditioning slightly decreased systolic segmental shortening [64.1(9.5)% of baseline at 10 min reoxygenation v 85.5(6.5)% for control, p < 0.05]. In contrast, 5 min coronary occlusion in controls produced more marked cardiodepression [segmental shortening 33.5(10.1)% of baseline at 10 min reperfusion; p < 0.05 v 10 min reoxygenation in the hypoxic-preconditioned group]. Systolic shortening was preserved in hypoxic-preconditioned hearts as compared to controls during each 10 min reperfusion period. Furthermore, functional recovery at 90 min reperfusion after the last occlusion in hypoxic preconditioned hearts was more complete than in control hearts, at 40.8(13.1)% v -16.1(14.4)%; p < 0.05. However, myocardial oxygen consumption was not suppressed by hypoxic preconditioning. CONCLUSIONS: Five minutes of hypoxic preconditioning attenuated contractile dysfunction due to repeated brief ischaemia/reperfusion stress. This protective effect was not due to the suppression of myocardial energy demand.

Adenosine receptor blockade enhance glycolysis in hypoperfused guinea-pig myocardium.

OBJECTIVE: This study tested the hypothesis that endogenous adenosine depresses anaerobic glycolysis in preischaemic and moderately ischaemic myocardium. METHODS: Isolated, working guinea-pig hearts, perfused with glucose-fortified Krebs-Henseleit buffer, were subjected to 15 min mild hypoperfusion (coronary flow 60% of baseline) followed by 10 min ischaemia (coronary flow 20% of baseline). Adenosine A1 receptors were blocked with 8-p-sulfophenyl theophylline (8-SPT; 20 microM). Glucose oxidation and lactate production from exogenous glucose were assessed from 14CO2 and [14C]lactate formation, respectively, from [U-14C]glucose. Energy metabolites, glycolytic intermediates and glycogen were measured in extracts of stop-frozen preischaemic, mildly hypoperfused and ischaemic myocardium. RESULTS: Adenosine receptor blockade did not affect left ventricular function assessed from heart rate x pressure product and pressure x volume work although coronary flow was slightly reduced. Adenosine receptor blockade increased glucose uptake (P < 0.05) by 100% during preischaemia and by 74% during mild hypoperfusion, and increased lactate production from exogenous glucose (P < 0.05) by 89% during preischaemia and fourfold during mild hypoperfusion, but did not stimulate glucose oxidation under any condition. Glycogen degradation was not increased by adenosine receptor blockade during ischaemia. Crossover plots of glycolytic intermediates revealed that phosphofructokinase was activated by adenosine receptor blockade at all three levels of perfusion. CONCLUSION: Endogenous adenosine attenuates anaerobic glycolysis in normally perfused, hypoperfused and ischaemic myocardium by blunting phosphofructokinase activity; this effect is mediated by adenosine A1 receptors.

Does interstitial adenosine mediate acute hibernation of guinea pig myocardium?

OBJECTIVE: The aim was to test the role of interstitial adenosine in protective downregulation of myocardial energy demand during myocardial hibernation. METHODS: Isolated working guinea pig hearts, perfused with glucose fortified Krebs-Henseleit, were subjected to 60 min global low flow ischaemia followed by 30 min reperfusion. Left ventricular performance was assessed from heart rate-developed pressure product and pressure-volume work. Cytosolic energy level was indexed by creatine phosphate and ATP phosphorylation potentials measured in snap frozen myocardium. Lactate and purine nucleosides (adenosine, inosine) were measured in venous effluent. RESULTS: When coronary flow was lowered by 80% for 60 min, heart rate-pressure product and pressure-volume work fell 87% and 75%, respectively, and stabilised at these low levels, but fully recovered when flow was restored. Myocardial ATP phosphorylation potential fell by 67% during the first 10 min of ischaemia, but subsequently recovered to preischaemic levels despite continuing ischaemia, indicating down-regulation of myocardial energy demand. Lactate release increased about 10-fold during ischaemia and remained increased until reperfusion. Purine nucleoside release varied reciprocally with phosphorylation potential, peaking at 10 min of ischaemia, then gradually returning to the preischaemic level during the subsequent 50 min of ischaemia. The ecto 5'-nucleotidase inhibitor alpha,beta-methylene adenosine 5'-diphosphonate (50 microM) decreased ischaemic purine nucleoside release by 41%, but did not attenuate postischaemic contractile recovery. The unspecific adenosine receptor antagonist 8-p-sulphophenyl theophylline (8-SPT, 20 microM) doubled ischaemic lactate release and lowered coronary venous purine nucleoside release by 21%. 8-SPT increased phosphorylation potential at 10 min ischaemia relative to untreated hearts, but blunted the subsequent rebound of phosphorylation potential. 8-SPT treatment during ischaemia resulted in a significantly higher cytosolic phosphorylation potential at 30 min of reperfusion, but did not affect postischaemic contractile function. CONCLUSIONS: We conclude that activation of adenosine receptors results in recovery of cytosolic energy level of moderately ischaemic working myocardium, but this energetic recovery is not solely responsible for postischaemic contractile recovery.

Exercise training increases creatine kinase capacity in canine myocardium.

INTRODUCTION: The creatine kinase (CK) energy shuttle of cardiomyocytes channels metabolic energy from the mitochondria to sites of energy utilization at contracting myofibrils and sarcolemmal and sarcoplasmic reticular ion pumps. Although plasticity of the myocardial CK system in response to hemodynamic overload has been repeatedly demonstrated, the effects of aerobic exercise training on myocardial CK are less well understood. This investigation tested the hypothesis that aerobic exercise training increases the capacity of the CK system in canine myocardium. METHODS: Mongrel dogs were conditioned by a 9-wk treadmill running program or cage-rested for 4 wk. Total CK activity was measured colorimetrically; CK(MB) was separated from other CK isoforms and measured by electrophoresis. RESULTS: Relative to sedentary controls, training increased left ventricular total CK activity 46% (P < 0.05) but did not alter total CK activity in right ventricular myocardium. Also in left ventricular myocardium, training increased CK(MB) isoenzyme activity 4.5-fold and the CK(MB) fraction of total CK threefold from 1.1+/-0.4 to 3.4+/-0.8% (P < 0.05). In contrast to left ventricle, CK(MB) activity and its fraction of total CK activity were not altered by training in right ventricular myocardium. CONCLUSIONS: Aerobic exercise training increases total myocardial CK activity and CK(MB) content in canine left ventricular myocardium, although CK(MB) remains a minor component of the myocardial CK system. The right ventricular CK system was not affected by training.

Metabolic protection of post-ischemic phosphorylation potential and ventricular performance.

Relationships between cytosolic phosphorylation potential, low-flow ischemic purine release and post-ischemic left ventricular developed pressure were examined in perfused working guinea-pig heart. During moderate ischemic acidification, metabolic intervention by pyruvate attenuated cytosolic NADH accumulation and (ATP+ADP+AMP) degradation. In reperfusion, spontaneously developed ventricular pressure increased in parallel with the phosphorylation potential (R2 = 0.71), but forced restoration of function by inotropic measures occurred at the expense of the phosphorylation potential.

Pyruvate: metabolic protector of cardiac performance.

Pyruvate, a metabolic product of glycolysis and an oxidizable fuel in myocardium, increases cardiac mechanical performance and energy reserves, especially when supplied at supraphysiological concentrations. The inotropic effects of pyruvate are most impressive in hearts that have been reversibly injured (stunned) by ischemia/reperfusion stress. Glucose appears to be an essential co-substrate for pyruvate's salutary effects in stunned hearts, but other fuels including lactate, acetate, fatty acids, and ketone bodies produce little or no improvement in postischemic function over glucose alone. In contrast to pharmacological inotropism by catecholamines, metabolic inotropism by pyruvate increases cardiac energy reserves and bolsters the endogenous glutathione antioxidant system. Pyruvate enhancement of cardiac function may result from one or more of the following mechanisms: increased cytosolic ATP phosphorylation potential and Gibbs free energy of ATP hydrolysis, enhanced sarcoplasmic reticular calcium ion uptake and release, decreased cytosolic inorganic phosphate concentration, oxyradical scavenging via direct neutralization of peroxides and/or enhancement of the intracellular glutathione/NADPH antioxidant system, and/or closure of mitochondrial permeability transition pores. This review aims to summarize evidence for each of these mechanisms and to consider the potential utility of pyruvate as a therapeutic intervention for clinical management of cardiac insufficiency.

Coronary autoregulation and purine release in normoxic heart at various cytoplasmic phosphorylation potentials: disparate effects of adenosine.

The impacts of energy-yielding substrates on coronary flow autoregulation, cytoplasmic phosphorylation potential ([ATP]/([ADP][Pi])] and purine nucleoside production were studied in Langendorff-perfused guinea pig hearts. The perfusion medium was substrate-free or contained glucose alone or in combination with pyruvate, lactate, acetate, or octanoate as fatty acid. When coronary flow was adjusted for myocardial oxygen consumption, only pyruvate supported near-perfect intrinsic autoregulation at highly sustained [ATP]/([ADP][Pi]) and low interstitial adenosine concentrations ([Ado]). In contrast, hearts perfused with substrate-free medium were deenergized at very high [Ado], especially at supraphysiological pressures, which markedly impaired auto-regulatory vasoconstriction. Thus, efficient autoregulatory vasoconstriction was associated with high [ATP]/([ADP][Pi]) at low [Ado]. On the other hand, autoregulatory vasodilation at subphysiological pressures was associated with increased [Ado] and partially blocked by 28 microM theophylline demonstrating (partial) adenosine mediation. Massive accumulation of IMP, especially relative to free cytoplasmic AMP, occurred at normal intracellular pH during myocyte deenergization by substrate-free perfusion. This may indicate allosteric activation of native AMP deaminase in situ, perhaps because of collapse of [ATP]/([ADP][Pi]). Similarly, rates of adenosine plus inosine release and of total purines, also including urate, exhibited non-linear sigmoidal rather than linear or rectangular hyperbolic dependences on free cytoplasmic AMP concentration (not total AMP content). Since inclusion of IMP as a co-variable of free AMP appreciably improved the sigmoidal fits, IMP appeared to be a significant precursor of released inosine in guinea pig heart.

Insulin improves contractile function during moderate ischemia in canine left ventricle.

This study determined the effects of insulin on myocardial contractile function and glucose metabolism during moderate coronary hypoperfusion. Coronary perfusion pressure (CPP) was lowered from 100 to 60, 50, and 40 mmHg in the left anterior descending coronary artery of anesthetized, open-chest dogs. Regional glucose uptake (GU), lactate uptake, myocardial O2 consumption, and percent segment shortening (%SS) were measured without (n = 12) or with intravenous (4 U/min, n = 12) or intracoronary insulin (4 U/min, n = 6). Glucose metabolites were also measured in freeze-clamped biopsies of control heart (n = 6) and hearts treated with intravenous insulin (n = 6) at the completion of the protocol (40 mmHg CPP). GU increased with intravenous and intracoronary insulin (P < 0.01). In all groups, GU was unaffected by reduced CPP, although lactate uptake decreased significantly (P < 0.01). Myocardial O2 consumption fell (P < 0.05) as CPP was lowered in all groups and was not altered significantly by intravenous or intracoronary insulin treatment. Without insulin, %SS decreased 72% (P < 0.05) at 40 mmHg CPP, but in hearts treated with intravenous and intracoronary insulin, %SS was not reduced (P > 0.05). Myocardial glycogen, alanine, lactate, and pyruvate contents were not significantly different in untreated hearts and hearts treated with intravenous insulin. Thus, in moderately ischemic canine myocardium, insulin markedly improved regional contractile function and did not appreciably increase the products of anaerobic glucose metabolism.

Glucose requirement for postischemic recovery of perfused working heart.

The quantitative importance of glycolysis in cardiomyocyte reenergization and contractile recovery was examined in postischemic, preload-controlled, isolated working guinea pig hearts. A 25-min global but low-flow ischemia with concurrent norepinephrine infusion to exhaust cellular glycogen stores was followed by a 15-min reperfusion. With 5 mM pyruvate as sole reperfusion substrate, severe contractile failure developed despite normal sarcolemmal pyruvate transport rate and high intracellular pyruvate concentrations near 2 mM. Reperfusion dysfunction was characterized by a low cytosolic phosphorylation potential [( ATP]/[( ADP][Pi]) due to accumulations of inorganic phosphate (Pi) and lactate. In contrast, with 5 mM glucose plus pyruvate as substrates, but not with glucose as sole substrate, reperfusion phosphorylation potential and function recovered to near normal. During the critical ischemia-reperfusion transition at 30 s reperfusion the cytosolic creatine kinase appeared displaced from equilibrium, regardless of the substrate supply. When under these conditions glucose and pyruvate were coinfused, glycolytic flux was near maximum, the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction was enhanced, accumulation of Pi was attenuated, ATP content was slightly increased, and adenosine release was low. Thus, glucose prevented deterioration of the phosphorylation potential to levels incompatible with reperfusion recovery. Immediate energetic support due to maximum glycolytic ATP production and enhancement of the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction appeared to act in concert to prevent detrimental collapse of [ATP]/[( ADP][Pi]) during creatine kinase dysfunction in the ischemia-reperfusion transition. Dichloroacetate (2 mM) plus glucose stimulated glycolysis but failed fully to reenergize the reperfused heart; conversely, 10 mM 2-deoxyglucose plus pyruvate inhibited glycolysis and produced virtually instantaneous de-energization during reperfusion. The following conclusions were reached. (1) A functional glycolysis is required to prevent energetic and contractile collapse of the low-flow ischemic or reperfused heart (2). Glucose stabilization of energetics in pyruvate-perfused hearts is due in part to intensification of glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase activity. (3) 2-Deoxyglucose depletes the glyceraldehyde-3-phosphate pool and effects intracellular phosphate fixation in the form of 2-deoxyglucose 6-phosphate, but the cytosolic phosphorylation potential is not increased and reperfusion failure occurs instantly. (4) Consistent correlations exist between cytosolic ATP phosphorylation potential and reperfusion contractile function. The findings depict glycolysis as a highly adaptive emergency mechanism which can prevent deleterious myocyte deenergization during forced ischemia-reperfusion transitions in presence of excess oxidative substrate.

Jejunal epithelial glucose metabolism: effects of Na+ replacement.

The objective of this study was to characterize the effects of replacement of extracellular Na+ with a nontransportable cation, N-methyl-D-glucamine (NMDG+) on jejunal epithelial glucose metabolism. Jejunal epithelium isolated from male Sprague-Dawley rats was incubated in media containing 5 mM glucose, 0.5 mM glutamine, 0.5 mM beta-hydroxybutyrate, and 0.3 mM acetoacetate as the principal carbon sources. O2 consumption and total glucose utilization were reduced 30 and 50%, respectively, when Na+ was replaced with NMDG+. In both media, approximately 75% of utilized glucose carbon was converted to lactate. The rate of glucose metabolism via the hexose monophosphate shunt, as evaluated using specific 14CO2 yields from [1-14C]glucose and [6-14C]glucose, was not appreciably altered by Na+ replacement. Tricarboxylic acid (TCA) cycle flux was evaluated using 14CO2 production from [14C]glucose and [14C]pyruvate radioisotopes. Approximately 50% of TCA cycle flux was shunted into products other than CO2 in both media. The majority of the acetyl-CoA oxidized in the TCA cycle was derived from cytosolic pyruvate. It is concluded that removal of Na+ from the bathing medium substantially reduced glucose utilization via the Embden-Meyerhof pathway and TCA cycle in the jejunal epithelium.