[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.

Strategic leukocyte depletion reduces pulmonary microvascular pressure and improves pulmonary status post-cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) precipitates inflammation that causes marked pulmonary dysfunction. Leukocyte filtration has been proposed to reduce these deleterious effects. Other studies show an improvement with aprotinin. We proposed that a combination of these two therapies would synergistically improve pulmonary outcomes. Two hundred and twenty-five patients participated in a randomized prospective study comparing pulmonary microvascular function and pulmonary shunt fraction postcoronary artery bypass grafting (CABG). The study group underwent leukocyte depletion with aprotinin during the procedure. Pulmonary microvascular function was assessed by pulmonary microvascular pressure (PMVP), a measure of pulmonary capillary edema, and pulmonary function was evaluated by comparing pulmonary shunt fractions. Elevated PMVP and increased pulmonary shunting compromise pulmonary performance. The leukocyte-depleted group had significantly reduced PMVP and pulmonary shunt fraction for at least the first 24 hours postbypass. The combination of strategic leukocyte filtration and aprotinin therapy can effectively reduce postoperative decline in pulmonary function. Cardiopulmonary bypass precipitates a variety of inflammatory effects that can cause marked pulmonary dysfunction to the point of respiratory failure, necessitating prolonged mechanical ventilation. Leukocyte filtration has been investigated previously and appears to be beneficial in improving pulmonary outcome by preventing direct neutrophil-induced inflammatory injury. Recent studies of leukocyte reduction profiles suggest that leukoreduction via leukofiltration is short lived with filter saturation occurring 30-45 minutes after onset of filtration. This phenomenon may explain the limited utility observed with higher risk patients. These patients typically require longer pump runs, so leukocyte reduction capability is suboptimal at the time of pulmonary vascular reperfusion. To more effectively protect the lung from reperfusion injury, leukocyte filtration can be delayed so that reduction of activated neutrophils is maximal at the time of pulmonary vascular reperfusion. It is, thus, conceivable that a timely use of arterial line leukoreducing filters may improve, more substantially, pulmonary function postbypass. Two hundred and twenty-five isolated coronary revascularization patients participated in this prospective, randomized trial. The patients received moderately hypothermic CBP alone (control group: n = 110) or combined with leukocyte depletion, initiated 30 minutes before crossclamp release, with filters placed in the bypass circuit (study group: n = 115). All patients also received full Hammersmith aprotinin dosing during the operation. Pulmonary microvascular pressures were lower in the study group at three hours postbypass, and continued to fall until 24 hours postbypass. In contrast, the control group measured a rise in PMVP and a continued plateau throughout 24 hours postbypass (p < 0.028). The calculated pulmonary shunt fraction also was reduced significantly throughout the study interval, with the greatest reduction occurring approximately three to six hours post-CPB (p < 0.002). Shunt fractions eventually converged at 24 hours postbypass. Outcome measures included hospital charges and length of stay, which were also markedly reduced in the treatment group. Increasing PMVPs are a direct reflection of pulmonary capillary edema, which, in conjunction with increased pulmonary shunt ratio, lead to an overall worsening of pulmonary function. Intraoperative strategic leukocyte filtration combined with aprotinin treatment improves post-CPB lung performance by reducing significantly the reperfusion inflammatory response and its sequelae. These benefits are manifested by reductions in ventilator times, hospital stay and patient morbidity.

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.

Dobutamine enhances both contractile function and energy reserves in hypoperfused canine right ventricle.

Although the beta(1)-adrenergic agent dobutamine is used clinically to provide inotropic support to the failing myocardium, it could jeopardize the myocardium by depleting energy reserves. This investigation delineated the contractile and energetic effects of low versus high dobutamine doses in the hypoperfused right ventricular (RV) myocardium. The right coronary artery (RCA) of anesthetized dogs was cannulated for controlled perfusion with arterial blood, and regional RV contractile function was measured. RCA perfusion pressure was lowered from 100 mmHg baseline to 40 mmHg, and flow fell by 54%. At 15-min hypoperfusion, dobutamine was infused into the RCA at either 0.01 (low-dose dobutamine) or 0.06 microgram. kg(-1). min(-1) (high-dose dobutamine) for 15 min. Regional power (systolic segment shortening x isometric developed force x heart rate) stabilized at 63% of baseline during hypoperfusion. Low-dose dobutamine restored power to baseline but did not increase RV myocardial O(2) consumption (MVO(2)) and thus increased myocardial O(2) utilization efficiency (O(2)UE:power/MVO(2)). At 5 min, high-dose dobutamine enhancement of power was similar to that of low-dose dobutamine, but by 15 min, power and O(2)UE fell to untreated levels. Remarkably, low-dose dobutamine tripled cytosolic phosphorylation potential; in contrast, high-dose dobutamine lowered phosphorylation potential to 45% of the untreated value. Analyses of glucose uptake and glycolytic intermediates revealed sustained enhancement of glycolysis by low-dose dobutamine, but glycolysis became limited at glyceraldehyde 3-phosphate dehydrogenase during high-dose dobutamine treatment. In summary, low-dose dobutamine improved mechanical performance and efficiency of the hypoperfused RV myocardium while increasing myocardial energy reserves, but high-dose dobutamine failed to sustain improved function and depleted energy reserves. Dobutamine is capable of improving both contractile function and cellular energetics in the hypoperfused RV myocardium, but dosage should be carefully selected.

Hyperlipidemia with hypoglycemia reduces myocardial oxygen utilization efficiency but not contractile function during coronary hypoperfusion.

This study was designed to determine changes in myocardial contractile function and fuel selection during moderate coronary hypoperfusion in the presence of elevated plasma free fatty acid (FFA) at normal and reduced blood glucose concentrations. Coronary perfusion pressure (CPP) was sequentially lowered from 100 to 60, 50, and 40 mmHg in the left anterior descending coronary artery (LAD) of anesthetized, open-chest dogs. Regional glucose uptake (GU), fatty acid uptake (FAU), percentage segment shortening (%SS), and oxygen consumption (MV O(2)) were determined with normal arterial plasma FFA concentrations (Group 1) or with elevated FFA concentrations (Groups 2 and 3). In Group 3, glucose in the coronary perfusate blood was reduced from 3.53+/-0.36 to 0.15+/-0.03 m M by hemodialysis. In Group 1, FAU fell by 85% as CPP was lowered to 60 mmHg and remained depressed as CPP was reduced further; GU did not fall significantly. Hyperlipidemia in Group 2 did not alter GU at any CPP, but maintained FAU at baseline levels until CPP was lowered to 40 mmHg. At 40 mmHg CPP, myocardial function and metabolic variables were similar in Groups 1 and 2. In Group 3 at 40 mmHg, FAU increased four-fold and MV O(2)doubled v Groups 1 and 2, and GU fell to zero. Despite these metabolic changes, %SS in Group 3 was unchanged relative to Group 2. Addition of glucose to the dialysate prevented the effects of dialysis on FAU, GU, and MV O(2). Thus, preferential glucose oxidation sustains myocardial oxygen utilization efficiency [(heart rate x %SS x maximum left ventricular pressure)/MV O(2)] during hypoperfusion. Blocking preferential glucose oxidation by combined hyperlipidemia and hypoglycemia lowers oxygen utilization efficiency, but does not compromise myocardial contractile function.

Exercise training enhances glycolytic and oxidative enzymes in canine ventricular myocardium.

Aerobic exercise training evokes adaptations in the myocardial contractile machinery that enhance cardiac functional capacity; in comparison, the effects of training on the myocardium's energy generating pathways are less well characterized. This study tested the hypothesis that aerobic exercise training can increase the capacities of the major pathways of intermediary metabolism in canine myocardium. Mongrel dogs were conditioned by a 9-week treadmill running program or cage rested for 4 weeks. Exercise conditioning was evidenced by 26% and 22% decreases (P<0.05) in respective heart rates at rest and during submaximal exercise and by a 40% increase (P<0.05) in citrate synthase (CS) activity of the vastus lateralis. Glycolytic, TCA cycle, and beta-oxidative enzymes were assayed in myocardial extracts at 37 degrees C. Relative to sedentary controls, training increased glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity by 49% in left and 33% in right ventricle, and pyruvate kinase, CS, and 3-hydroxyacyl CoA dehydrogenase (HADH) activities by 74%, 91%, and 77%, respectively, in left ventricle (P<0.05). Immunoblotting further confirmed that training increased left ventricular contents of CS and GAPDH. Other measured enzymes (hexokinase, phosphofructokinase, lactate dehydrogenase, alpha-ketoglutarate dehydrogenase, malate dehydrogenase) were not altered by training in either ventricle. Kinetic analyses revealed increased maximum rates but unaltered substrate affinities of GAPDH, CS and HADH following training. Thus, aerobic exercise training augments the intermediary metabolic capacity of canine myocardium by selectively increasing the concentrations of regulatory enzymes of glycolysis and oxidative metabolism.

Myocardial oxygen consumption modulates adenosine formation by canine right ventricle in absence of hypoxia.

Myocardial adenosine formation varies with myocardial oxygen consumption (MVO(2)), but whether concurrent hypoxia is required for adenosine formation is uncertain. Changes in right coronary (RC) perfusion pressure (RCP) produce directionally similar alterations in right ventricular (RV) MVO(2)and in RC venous P O(2)(P(v)O(2)), an index of myocardial P O(2). RCP was varied in 10 anesthetized, open chest dogs to determine if, under these conditions, RV formation of adenosine would increase with MVO(2)in absence of myocardial hypoxia. Dialysis probes were implanted in the mid myocardium of RV free wall for collecting dialysate samples for HPLC analyses to estimate interstitial adenosine and other purines. Coronary venous blood was sampled from a superficial vein draining the RC artery (RCA) perfusion territory. At 115+/-3 mmHg baseline RCP, RC blood flow (RCBF)=0.51+/-0.04 ml/min/g, MVO(2)=4.6+/-0.5 ml/min/100 g, P(v)O(2)=34+/-1.5 mmHg, and dialysate adenosine=0. 27+/-0.03microM. When RCP was lowered to 61+/-1 mmHg by adjusting an occluder on the proximal RCA, RCBF decreased to 0.36+/-0.03 ml/min/g, MVO(2)fell to 3.7+/-0.4 ml/min/100 g, lactate uptake remained positive, P(v)O(2)fell to 30+/-1.7 mmHg, and dialysate adenosine decreased to 0.20+/-0.03microM. Reactive hyperemia of 1.25+/-0.13 ml/min/g was observed when the RCA constriction was released, although dialysate adenosine had fallen. When RCP was elevated to 164+/-2 mmHg by inflating a balloon catheter in the descending aorta, RCBF increased to 0.70+/-0.06 ml/min/g, MVO(2)increased to 5.8+/-1. 0 ml/min/100 g, P(v)O(2)rose to 39+/-2.3 mmHg, and dialysate adenosine increased to 0.33+/-0.04microM. These data indicate that (1) RV oxygen demand varies with RCP; (2) if RV ischemia is absent, myocardial adenosine formation is modulated by MVO(2), with no requirement for hypoxia; (3) pressure-flow autoregulation is relatively ineffective in the RC circulation, where adenosine does not mediate and may even blunt autoregulation.

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.

Antioxidant properties of pyruvate mediate its potentiation of beta-adrenergic inotropism in stunned myocardium.

UNLABELLED: This study tested the hypothesis that pyruvate's antioxidant actions, particularly its enhancement of the endogenous glutathione system, mediate its potentiation of beta-adrenergic inotropism in stunned myocardium. Isolated working guinea pig hearts, metabolizing 10 m M glucose and stunned by 45 min of low flow ischemia, were treated with 5 m M pyruvate, 5 m M N-acetylcysteine (NAC) and/or 2 n M isoproterenol beginning 15 min after reperfusion. The antioxidant NAC alone did not increase cardiac power (mJ/min/g wet: 11 +/- 1 in untreated and 15 +/- 2 in NAC treated stunned hearts), but NAC potentiated the increase in power produced by 2 n M isoproterenol (isoproterenol alone: 50+/-10; NAC plus isoproterenol: 133 +/- 24). Addition of NAC doubled cyclic AMP content but lowered cytosolic phosphorylation potential by 32% in isoproterenol-stimulated hearts. Stunning decreased the glutathione antioxidant ratio (GSH/GSSG) by 68%. The antioxidant ratio was completely restored by pyruvate alone or in combination with isoproterenol, but only partially restored by isoproterenol alone. Combining isoproterenol and NAC increased the GSH/GSSG ratio by an additional 36%. The combined treatment of pyruvate and isoproterenol increased the NADPH/NADP(+) ratio almost three-fold, and produced the greatest accumulation of glucose-6-phosphate of any treatment. CONCLUSIONS: like pyruvate, the antioxidant NAC potentiated beta-adrenergic inotropism of stunned myocardium. Unlike pyruvate, NAC did not increase cellular energy reserves, thus effectively limiting its potentiation of beta-adrenergic stimulation. Thus, pyruvate's potentiation of beta-adrenergic stimulation in stunned myocardium is most likely the result of the combined effects of its antioxidant and energetic properties.

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.

Insulin improves cardiac contractile function and oxygen utilization efficiency during moderate ischemia without compromising myocardial energetics.

Insulin improves myocardial contractile function during moderate ischemia, but the mechanism is unknown. To determine effects of insulin on myocardial oxygen utilization efficiency (O2UE) and energetics, regional left coronary perfusion pressure (CPP) was lowered sequentially from 100 to 60, 50, and 40 mmHg in 24 anesthetized, open-chest dogs. Regional power index (PI), myocardial oxygen consumption (MVO2), and O2UE index (PI/MVO2) were determined in untreated and insulin treated (4 U/min, i.v.) hearts. Biopsies were obtained from six untreated and six insulin-treated hearts at CPP=40 mmHg for determining high energy phosphates and the cytosolic phosphorylation potential. Measurements were compared with data from normal, untreated myocardium (n=11). MVO2 fell (P<0.05) in all hearts as CPP was lowered to 40 mmHg, and was unaffected by insulin treatment. PI decreased 32 and 75% in untreated hearts at CPP=50 and 40 mmHg, respectively (P<0.05). In insulin treated hearts, PI was not significantly depressed at CPP>40 mmHg, and fell only 26% at CPP=40 mmHg. O2UE increased (P<0.05) in all hearts at CPP=60 mmHg. In insulin treated hearts, O2UE was greater (P<0.05) at CPP=50 and 40 mmHg than at CPP=100 mmHg, and greater (P<0.05) than in untreated hearts at CPP=40 mmHg. Reducing CPP to 40 mmHg produced similar metabolic changes in all hearts. Compared to normal myocardium, ATP content of untreated and treated hearts was unchanged, creatine phosphate content decreased 21 and 14%, creatine content increased 24 and 30%, inorganic phosphate concentration increased 108 and 140%, and phosphorylation potential decreased 80 and 77%. We conclude that insulin markedly improves PI and O2UE without altering cytosolic energetics during moderate myocardial ischemia.

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.

Pyruvate potentiates beta-adrenergic inotropism of stunned guinea-pig myocardium.

UNLABELLED: This study tested the hypotheses that the sensitivity of stunned myocardium to beta-adrenergic stimulation is diminished, and that metabolic intervention with pyruvate can restore beta-adrenergic responsiveness to pre-ischemic levels. Isolated working guinea-pig hearts metabolizing 10 mM glucose were stunned by 45 min of low flow ischemia, and pyruvate and/or isoproterenol treatments were initiated 15 and 30 min after reperfusion, respectively. The dose: response for cardiac power from 0.1-100 nM isoproterenol was significantly shifted to the right in stunned hearts: EC50 (nm) increased from 0.3 +/- 0.06 to 5.2 +/- 1.86. Pyruvate (5 mM) largely restored isoproterenol responsiveness of stunned myocardium, lowering EC50 to 1.1 +/- 0.34 nM. Maximum power was similar in each group. Additional stunned hearts were treated with intermediate (2 nM) or high (30 nM) isoproterenol concentrations with or without pyruvate. Combining treatments produced a significant interaction at the low dose of isoproterenol, increasing cardiac power (mJ x min(-1) x g(-1)) to 149 +/- 20, twice the sum of the individual treatments (2 nM isoproterenol: 34 +/- 11; pyruvate: 33 +/- 8). Cyclic AMP content was unaltered by isoproterenol or pyruvate alone but was increased 41% by the combination. Power was maximized by 30 nM isoproterenol, which tripled cyclic AMP content; pyruvate did not augment these responses, but lessened the isoproterenol-induced decline in cytosolic phosphorylation potential. CONCLUSIONS: Beta-adrenergic inotropism is attenuated in stunned myocardium, although the maximal response is unchanged. Pyruvate potentiated the effects of sub-maximal doses of isoproterenol without depleting cellular energy reserves further, and attenuated energy depletion by high doses of isoproterenol. Pyruvate may allow restoration of contractile performance with lower, energetically less costly doses of beta-adrenergic agents.

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.

Stability of high-energy phosphates in right ventricle: myocardial energetics during right coronary hypotension.

This study was conducted to determine if mechanisms that reduce right coronary (RC) blood flow (RCBF) and right ventricular (RV) oxygen consumption (MVO2) during moderate RC hypotension preserve RV high-energy phosphates. RC arteries of anesthetized dogs were cannulated and perfused with arterial blood supplied by a pressurized extracorporeal circuit. RC perfusion pressure (RCPP) was either kept constant at 100 mmHg or reduced to 60 or 30 mmHg for 20 min followed by a freeze-clamp biopsy of RV. Left ventricular (LV) biopsy was also performed to compare energy metabolism between RV and LV.RCBF and MVO2 significantly decreased when RCPP was reduced to 60 mmHg, but RV segment shortening (%SS) was unchanged; ATP, creating phosphate (CrP) and phosphorylation state of CrP ([CrP]/[Cr][Pi]) did not differ from control values. RV %SS, CrP, and phosphorylation state fell markedly at 30 mmHg RCPP. At 100 mmHg RCPP, CrP phosphorylation state in RV was only 35% of that in LV. These results indicate that RV increases its energetic efficiency without significant changes in high-energy phosphates or CrP phosphorylation state during moderate RC hypotension. Furthermore, the RV myocardium maintains a much lower energy level than LV myocardium, commensurate with its lower energy requirements.

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.

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.

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.

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.