Myocardial oxygenation and adenosine release in isolated guinea pig hearts during changes in contractility

Previous work from this laboratory using near-infrared optical spectroscopy of myoglobin has shown that approximately 20% of the myocardium is hypoxic in buffer-perfused hearts that are perfused with fully oxygenated buffer at 37 degrees C. The present study was undertaken to determine cardiac myoglobin saturation in buffer-perfused hearts when cardiac contractility was increased with epinephrine and decreased during cardiac arrest with KCl. Infusion of epinephrine to achieve a doubling of contractility, as measured by left ventricular maximum pressure change over time (dP/dt), resulted in a decrease in mean myoglobin saturation from 79% at baseline to 65% and a decrease in coronary venous oxygen tension from 155 mmHg at baseline to 85 mmHg. Cardiac arrest with KCl increased mean myoglobin saturation to 100% and coronary venous oxygen tension to 390 mmHg. A previously developed computer model of oxygen transport in the myocardium was used to calculate the probability distribution of intracellular oxygen tension and the hypoxic fraction of the myocardium with an oxygen tension below 0.5 mmHg. The hypoxic fraction of the myocardium was approximately 15% at baseline, increased to approximately 30% during epinephrine infusion, and fell to approximately 0% during cardiac arrest. The coronary venous adenosine concentration changed in parallel with the hypoxic fraction of the myocardium during epinephrine and KCl. It is concluded that catecholamine stimulation of buffer-perfused hearts increases hypoxia in the myocardium and that the increase in venous adenosine concentration is a reflection of the larger hypoxic fraction of myocardium that is releasing adenosine.     

Cardioprotection in chronically hypoxic rabbits persists on exposure to normoxia: role of NOS and KATP channels

Hypoxia from birth increases resistance to myocardial ischemia in infant rabbits. We hypothesized that increased cardioprotection in hearts chronically hypoxic from birth persists following development in a normoxic environment and involves increased activation of nitric oxide synthase (NOS) and ATP-dependent K (K(ATP)) channels. Resistance to myocardial ischemia was determined in rabbits raised from birth to 10 days of age in a normoxic (Fi(O(2)) = 0.21) or hypoxic (Fi(O(2)) = 0.12) environment and subsequently exposed to normoxia for up to 60 days of age. Isolated hearts (n = 8/group) were subjected to 30 min of global ischemia followed by 35 min of reperfusion. At 10 days of age, resistance to myocardial ischemia (percent recovery postischemic recovery left ventricular developed pressure) was higher in chronically hypoxic hearts (68 +/- 4%) than normoxic controls (43 +/- 4%). At 10 days of age, N(G)-nitro-L-arginine methyl ester (200 microM) and glibenclamide (3 microM) abolished the cardioprotective effects of chronic hypoxia (45 +/- 4% and 46 +/- 5%, respectively) but had no effect on normoxic hearts. At 30 days of age resistance to ischemia in normoxic hearts declined (36 +/- 5%). However, in hearts subjected to chronic hypoxia from birth to 10 days and then exposed to normoxia until 30 days of age, resistance to ischemia persisted (63 +/- 4%). L-NAME or glibenclamide abolished cardioprotection in previously hypoxic hearts (37 +/- 4% and 39 +/- 5%, respectively) but had no effect on normoxic hearts. Increased cardioprotection was lost by 60 days. We conclude that cardioprotection conferred by adaptation to hypoxia from birth persists on subsequent exposure to normoxia and is associated with enhanced NOS activity and activation of K(ATP) channels.     

Cellular redistribution of inducible Hsp70 protein in the human and rabbit heart in response to the stress of chronic hypoxia: role of protein kinases,

Many infants who undergo cardiac surgery have a congenital cyanotic defect where the heart is chronically perfused with hypoxemic blood. Infant hearts adapt to chronic hypoxemia by activation of intracellular protein kinase signal transduction pathways. However, the involvement of heat shock protein 70 in adaptation to chronic hypoxemia and its role in protein kinase signaling pathways is unknown. We determined expression of message and subcellular protein distribution for inducible (Hsp70i) and constitutive heat shock protein 70 (Hsc70) in chronically hypoxic and normoxic infant human and rabbit hearts and their relationship to protein kinases. In chronically hypoxic human and rabbit hearts message levels for Hsp70i were elevated 4- to 5-fold compared with normoxic hearts, Hsp70i protein was redistributed from the particulate to the cytosolic fraction. In normoxic infants Hsp70i protein was distributed almost equally between the cytosolic and particulate fractions. Hsc70 message and subcellular distribution of Hsc70 protein were unaffected by chronic hypoxia. We then determined if protein kinases influence Hsp70i protein subcellular distribution. In rabbit hearts SB203580 and chelerythrine reduced Hsp70i message levels, whereas SB203580, chelerythrine, and curcumin reversed the subcellular redistribution of Hsp70i protein caused by chronic hypoxia, with no effect in normoxic hearts, indicating regulation of Hsp70i message and subcellular distribution of Hsp70i protein in chronically hypoxic rabbit hearts is influenced by protein kinase C and mitogen-activated protein kinases, specifically p38 MAPK and JNK. We conclude the Hsp70 signal transduction pathway plays an important role in adaptation of infant human and rabbit hearts to chronic hypoxemia.     

Inosine, not adenosine, initiates endothelial glycocalyx degradation in cardiac ischemia and hypoxia

Ischemia/reperfusion and hypoxia/reoxygenation of the heart both induce shedding of the coronary endothelial glycocalyx. The processes leading from an oxygen deficit to shedding are unknown. An involvement of resident perivascular cardiac mast cells has been proposed. We hypothesized that either adenosine or inosine or both, generated by nucleotide catabolism, attain the concentrations in the interstitial space sufficient to stimulate A3 receptors of mast cells during both myocardial ischemia/reperfusion and hypoxia/reoxygenation. Isolated hearts of guinea pigs were subjected to either normoxic perfusion (hemoglobin-free Krebs-Henseleit buffer equilibrated with 95% oxygen), 20 minutes hypoxic perfusion (buffer equilibrated with 21% oxygen) followed by 20 minutes reoxygenation, or 20 minutes stopped-flow ischemia followed by 20 minutes normoxic reperfusion (n = 7 each). Coronary venous effluent was collected separately from so-called transudate, a mixture of interstitial fluid and lymphatic fluid appearing on the epicardial surface. Adenosine and inosine were determined in both fluid compartments using high-performance liquid chromatography. Damage to the glycocalyx was evident after ischemia/reperfusion and hypoxia/reoxygenation. Adenosine concentrations rose to a level of 1 μM in coronary effluent during hypoxic perfusion, but remained one order of magnitude lower in the interstitial fluid. There was only a small rise in the level during postischemic perfusion. In contrast, inosine peaked at over 10 μM in interstitial fluid during hypoxia and also during reperfusion, while effluent levels remained relatively unchanged at lower levels. We conclude that only inosine attains levels in the interstitial fluid of hypoxic and postischemic hearts that are sufficient to explain the activation of mast cells via stimulation of A3-type receptors.     

Antiadrenergic effects of adenosine A(1) receptor-mediated protein phosphatase 2a activation in the heart

The ability of adenosine A(1) receptors to activate type 2a protein phosphatase (PP2a) and account for antiadrenergic effects was investigated in rat myocardial preparations. We observed that the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) significantly reduces the isoproterenol-induced increase in left ventricular developed pressure of isolated heats, and this effect is blocked by pretreatment of hearts with the PP2a inhibitor cantharidin. CPA alone or given in conjunction with isoproterenol stimulation decreases phosphorylation of phospholamban and troponin I in ventricular myocytes. These dephosphorylations are blocked by an adenosine A(1) receptor antagonist and by PP2a inhibition with okadaic acid. Adenosine A(1) receptor activation was also shown to increase carboxymethylation of the PP2a catalytic subunit (PP2a-C) and cause translocation of PP2a-C to the particulate fraction in ventricular myocytes. These results support the hypothesis that adenosine A(1) receptor activation leads to methylation of PP2a-C and subsequent translocation of the PP2a holoenzyme. Increases in localized PP2a activity lead to dephosphorylation of key cardiac proteins responsible for the positive inotropic effects of beta-adrenergic stimulation.     

Cardiac anaphylaxis in isolated guinea pig hearts perfused at constant flow or constant pressure

Acute responses to antigen-antibody interactions (anaphylactic reactions) in isolated guinea pig hearts are reported to include decreases in coronary flow, increases in heart rate, prolongation of impulse propagation, development of arrhythmias, and transient increases followed by substantial decreases in ventricular contractile force. It is not clear from these studies, however, whether all of the changes are direct effects of the mediators released by the antigen-antibody reaction or whether some of them are indirect results of the severe reduction in flow evoked by coronary vasoconstriction. Therefore, the present study was designed to assess cardiac anaphylactic events in isolated hearts of guinea pigs passively sensitized with IgG antibody to ovalbumin under conditions in which coronary perfusion pressure was maintained constant and to compare the responses to those of hearts in which coronary flow was maintained at a constant rate. Our data indicate that when coronary flow decreased during anaphylaxis (constant pressure perfusion), hearts responded to antigen challenge with greater prolongation of the PR interval, duration of arrhythmias, suppression of left ventricular systolic pressure, and release of histamine and adenosine plus inosine into the venous effluent than when coronary flow was maintained during anaphylaxis (constant flow perfusion). The data suggest that maintenance of coronary flow during cardiac anaphylaxis may attenuate the severity of the functional derangement.     

Effect of prenatal hypoxia on heat stress-mediated cardioprotection in adult rat heart

Fetal programming has profound effects on cardiovascular function in later adult life. We tested the hypothesis that chronic hypoxic exposure during fetal development downregulates endogenous cardioprotective mechanisms in adult rats. Time-dated pregnant rats were divided between normoxic and hypoxic (10.5% O2 from days 15 to 21 of gestation) groups. The male progeny were studied at 2 mo of age. Rats were subjected to heat stress (42 degrees C for 15 min). After 24 h, hearts were excised and subjected to 30 min of global ischemia and 1 h of reperfusion. Prenatal hypoxia did not change adult rat body weight and heart weight, but significantly increased the cross-sectional area of a left ventricular (LV) myocyte. Heat stress significantly improved postischemic recovery of LV function in normoxic control rats, but not in prenatally hypoxic rats. The infarct size in the LV resulting from ischemia-reperfusion was reduced by the heat stress pretreatment in control rats, but not in prenatally hypoxic rats. In accordance, heat stress significantly increased LV myocardial content of heat shock protein 70 only in normoxic control rats. In addition, there was a significant decrease in the LV myocardial content of the PKC-epsilon isoform in prenatally hypoxic rats compared with control rats. We conclude that prenatal hypoxia causes in utero programming of hsp70 gene in the LV, leading to an inhibition of its response to heat stress and a loss of cardioprotection in later adult life.     

Cardiac performance of an isolated eel heart: effects of hypoxia and responses to coronary artery perfusion.

The pericardial sac containing the heart was removed from large (2.7-6.3 kg) long-finned eels (Anguilla dieffenbachii). Coronary arteries were cannulated in preparation for perfusion with eel Ringer or red cell suspensions. The hearts were maintained by Ringer perfusion while the performance of the heart was assessed. Responses of the hearts to increases in filling pressure and output pressure were recorded. Maximum cardiac output was 22.3 +/- 1.4 ml/min/kg body mass (mean +/- 1 SEM; N = 9). The highest cardiac power output was measured at maximum cardiac output and was 3.39 +/- 0.32 mW/g ventricle mass (mean +/- 1 SEM; N = 9). Eel hearts could sustain output pressures near 80 cm H2O, but cardiac output was reduced and cardiac power output was 1.89 +/- 0.24 mW/g ventricular mass (mean +/- 1 SEM; N = 9). Maximum cardiac output decreased by 14% when hearts pumped hypoxic Ringer with a PO2 of 11.5 torr. At high input pressures concomitant with high output pressures (80 cm H2O), cardiac power output decreased by 38% upon exposure to hypoxic Ringer. Coronary perfusion of hypoxic hearts with red cell suspensions (mean hematocrit 10.4%) at a rate of 2% of control cardiac output (0.20 ml/min/kg body mass) had no effect on maximum cardiac output. However, coronary perfusion enabled hypoxic hearts to maintain cardiac output when output pressure was raised to 80 cm H2O. Under conditions of high input pressure and high output pressure, power output increased by 20% compared to hypoxic hearts without coronary perfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potential role for pyruvate kinase M2 in the regulation of murine cardiac glycolytic flux during in vivo chronic hypoxia

Carbohydrate metabolism in heart failure shares similarities to that following hypoxic exposure, and is thought to maintain energy homoeostasis in the face of reduced O₂ availability. As part of these in vivo adaptations during sustained hypoxia, the heart up-regulates and maintains a high glycolytic flux, but the underlying mechanism is still elusive. We followed the cardiac glycolytic responses to a chronic hypoxic (CH) intervention using [5-³H]-glucose labelling in combination with detailed and extensive enzymatic and metabolomic approaches to provide evidence of the underlying mechanism that allows heart survivability. Following 3 weeks of in vivo hypoxia (11% oxygen), murine hearts were isolated and perfused in a retrograde mode with function measured via an intraventricular balloon and glycolytic flux quantified using [5-³H]-glucose labelling. At the end of perfusion, hearts were flash-frozen and central carbon intermediates determined via liquid chromatography tandem mass spectrometry (LC-MS/MS). The maximal activity of glycolytic enzymes considered rate-limiting was assessed enzymatically, and protein abundance was determined using Western blotting. Relative to normoxic hearts, CH increased ex vivo cardiac glycolytic flux 1.7-fold with no effect on cardiac function. CH up-regulated cardiac pyruvate kinase (PK) flux 3.1-fold and cardiac pyruvate kinase muscle isoenzyme M2 (PKM2) protein content 1.4-fold compared with normoxic hearts. CH also augmented cardiac pentose phosphate pathway (PPP) flux, reflected by higher ribose-5-phosphate (R5P) content. These findings support an increase in the covalent (protein expression) and allosteric (flux) control of PKM2 as being central to the sustained up-regulation of the glycolytic flux in the chronically hypoxic heart.     

Myocardial infarct size-limiting effect of chronic hypoxia persists for five weeks of normoxic recovery

We examined cardioprotective effect of chronic hypoxia and the time course of its recovery under normoxic conditions. Adult male Wistar rats were exposed to intermittent hypobaric hypoxia (7000 m, 8 h/day, 35 exposures) and susceptibility of their hearts to ischemia-induced ventricular arrhythmias and myocardial infarction was evaluated in anesthetized open-chest animals subjected to 30-min coronary artery occlusion and 4-h reperfusion on the day after the last hypoxic exposure and at 7, 35 and 90 days of normoxic recovery. The infarct size was reduced from 69.2+/-1.7 % of the area at risk in normoxic controls to 48.0+/-2.2 % in the chronically hypoxic group and to 61.6+/-2.3 % in the group recovered for 7 days. This residual protection persisted for at least 35 days of normoxic recovery but it was absent after 90 days. In contrast to the infarct size-limitation, the antiarrhythmic protection disappeared already during the first week; the incidence of ventricular fibrillation was even significantly increased 7 and 90 days after the last hypoxic exposure. In conclusion, the duration of cardioprotection induced by chronic hypoxia differs markedly, depending on the end point of ischemia/reperfusion injury examined. Whereas the increased tolerance to lethal myocardial injury persists for at least 5 weeks after the termination of hypoxia, the antiarrhythmic protection rapidly vanishes, being replaced with transient proarrhythmic effect.     

Mitochondrial BKCa channels contribute to protection of cardiomyocytes isolated from chronically hypoxic rats

Chronic hypoxia protects the heart against injury caused by acute oxygen deprivation, but its salutary mechanism is poorly understood. The aim was to find out whether cardiomyocytes isolated from chronically hypoxic hearts retain the improved resistance to injury and whether the mitochondrial large-conductance Ca2+-activated K+ (BKCa) channels contribute to the protective effect. Adult male rats were adapted to continuous normobaric hypoxia (inspired O2 fraction 0.10) for 3 wk or kept at room air (normoxic controls). Myocytes, isolated separately from the left ventricle (LVM), septum (SEPM), and right ventricle, were exposed to 25-min metabolic inhibition with sodium cyanide, followed by 30-min reenergization (MI/R). Some LVM were treated with either 30 μM NS-1619 (BKCa opener), or 2 μM paxilline (BKCa blocker), starting 25 min before metabolic inhibition. Cell injury was detected by Trypan blue exclusion and lactate dehydrogenase (LDH) release. Chronic hypoxia doubled the number of rod-shaped LVM and SEPM surviving the MI/R insult and reduced LDH release. While NS-1619 protected cells from normoxic rats, it had no additive salutary effect in the hypoxic group. Paxilline attenuated the improved resistance of cells from hypoxic animals without affecting normoxic controls; it also abolished the protective effect of NS-1619 on LDH release in the normoxic group. While chronic hypoxia did not affect protein abundance of the BKCa channel regulatory β1-subunit, it markedly decreased its glycosylation level. It is concluded that ventricular myocytes isolated from chronically hypoxic rats retain the improved resistance against injury caused by MI/R. Activation of the mitochondrial BKCa channel likely contributes to this protective effect.     

MCC-134, a blocker of mitochondrial and opener of sarcolemmal ATP-sensitive K+ channels, abrogates cardioprotective effects of chronic hypoxia

We examined the effect of MCC-134, a novel inhibitor of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels and activator of sarcolemmal ATP-sensitive K(+) (sarcK(ATP)) channels, on cardioprotection conferred by adaptation to chronic hypoxia. Adult male Wistar rats were exposed to intermittent hypobaric hypoxia (7000 m, 8 h/day, 5-6 weeks) and susceptibility of their hearts to ventricular arrhythmias and myocardial infarction was evaluated in anesthetized open-chest animals subjected to 20-min coronary artery occlusion and 3-h reperfusion on the day after the last hypoxic exposure. MCC-134 was administered intravenously 10 min before ischemia and 5 min before reperfusion in a total dose of 0.3 mg/kg or 3 mg/kg divided into two equal boluses. The infarct size (tetrazolium staining) was reduced from 59.2+/-4.4 % of the area at risk in normoxic controls to 43.2+/-3.3 % in the chronically hypoxic group. Chronic hypoxia decreased the reperfusion arrhythmia score from 2.4+/-0.5 in normoxic animals to 0.7+/-0.5. Both doses of MCC-134 completely abolished the antiarrhythmic protection (score 2.4+/-0.7 and 2.5+/-0.5, respectively) but only the high dose blocked the infarct size-limiting effect of chronic hypoxia (54.2+/-3.7 %). MCC-134 had no effect in the normoxic group. These results support the view that the opening of mitoKATP channels but not sarcKATP channels plays a crucial role in the mechanism by which chronic hypoxia improves cardiac tolerance to ischemia/reperfusion injury.     

Intermittent high altitude hypoxia protects the heart against lethal Ca2+ overload injury

Adaptation to intermittent high altitude (IHA) hypoxia can protect the heart against ischemia-reperfusion injury. In view of the fact that both Ca2+ paradox and ischemia-reperfusion injury are associated with the intracellular Ca2+ overload, we tested the hypothesis that IHA hypoxia may protect hearts against Ca2+ paradox-induced lethal injury if its cardioprotection bases on preventing the development of intracellular Ca2+ overload. Langendorff-perfused hearts from normoxic and IHA hypoxic rats were subjected to Ca2+ paradox (5 min of Ca2+ depletion followed by 30 min of Ca2+ repletion) and the functional, biochemical and pathological changes were investigated. The Ca2+ paradox incapacitated the contractility of the normoxic hearts, whereas the IHA hypoxic hearts significantly preserved contractile activity. Furthermore, the normoxic hearts subjected to Ca2+ paradox exhibited a marked reduction in coronary flow, increase in lactate dehydrogenase release, and severe myocyte damage. In contrast, these changes were significantly prevented in IHA hypoxic hearts. We, then, tested and confirmed our hypothesis that the protective mechanisms are mediated by mitochondria ATP-sensitive potassium channels (mitoKATP) and Ca2+/calmodulin-dependent protein kinase II (CaMKII), as the protective effect of IHA hypoxia was abolished by 5-hydroxydecanoate, a selective mitoKATP blocker, and significantly attenuated by KN-93, a CaMKII inhibitor. In conclusion, our studies offer for the first time that IHA hypoxia confers cardioprotection against the lethal injury of Ca2+ paradox and give biochemical evidence for the protective mechanism of IHA hypoxia. We propose that researches in this area may lead a preventive regimen against myocardial injury associated with Ca2+ overload.     

Intermittent hypoxic training protects canine myocardium from infarction

This investigation examined cardiac protective effects of normobaric intermittent hypoxia training. Six dogs underwent intermittent hypoxic training for 20 consecutive days in a normobaric chamber ventilated intermittently with N2 to reduce fraction of inspired oxygen (FiO2) to 9.5%-10%. Hypoxic periods, initially 5 mins and increasing to 10 mins, were followed by 4-min normoxic periods. This hypoxia-normoxia protocol was repeated, initially 5 times and increasing to 8 times. The dogs showed no discomfort during intermittent hypoxic training. After 20 days of hypoxic training, the resistance of ventricular myocardium to infarction was assessed in an acute experiment. The left anterior descending (LAD) coronary artery was occluded for 60 mins and then reperfused for 5 hrs. At 30 mins of LAD occlusion, radioactive microspheres were injected through a left atrial catheter to assess coronary collateral blood flow into the ischemic region. After 5 hrs reperfusion, the heart was dyed to delineate the area at risk (AAR) of infarction and stained with triphenyl tetrazolium chloride to identify infarcted myocardium. During LAD occlusion and reperfusion, systemic hemodynamics and global left ventricular function were stable. Infarction was not detected in 4 hearts and was 1.6% of AAR in the other 2 hearts. In contrast, 6 dogs sham-trained in a chamber ventilated with compressed air and 5 untrained dogs subjected to the same LAD occlusion/reperfusion protocol had infarcts of 36.8% +/- 5.8% and 35.2% +/- 9.5% of the AAR, respectively. The reduction in infarct size of four of the six hypoxia-trained dogs could not be explained by enhanced collateral blood flow to the AAR. Hypoxia-trained dogs had no ventricular tachycardia or ventricular fibrillation. Three sham-trained dogs had ventricular tachycardia and two had ventricular fibrillation. Three untrained dogs had ventricular fibrillation. In conclusion, intermittent hypoxic training protects canine myocardium from infarction and life-threatening arrhythmias during coronary artery occlusion and reperfusion. The mechanism responsible for this potent cardioprotection merits further study.     

Hypoxia and AMP independently regulate AMP-activated protein kinase activity in heart

The hypothesis was tested that hypoxia increases AMP-activated protein kinase (AMPK) activity independently of AMP concentration ([AMP]) in heart. In isolated perfused rat hearts, cytosolic [AMP] was changed from 0.2 to 16 microM using metabolic inhibitors during both normal oxygenation (95% O2-5% CO2, normoxia) and limited oxygenation (95% N2-5% CO2, hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol.min(-1).mg protein(-1) in normoxic hearts and from 5 to 55 pmol.min(-1).mg protein(-1) in hypoxic hearts. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the measurements from individual hearts to a hyperbolic equation. The [AMP] resulting in half-maximal total AMPK activity (A0.5) was 3 +/- 1 microM for hypoxic hearts and 28 +/- 13 microM for normoxic hearts. The A0.5 for alpha2-isoform AMPK activity was 2 +/- 1 microM for hypoxic hearts and 13 +/- 8 microM for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the Thr172 residue of the AMPK alpha-subunit. In potassium-arrested hearts perfused with variable O2 content, alpha-subunit Thr172 phosphorylation increased at O2 < or = 21% even though [AMP] was <0.3 microM. Thus hypoxia or O2 < or = 21% increased AMPK phosphorylation and activity independently of cytosolic [AMP]. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr172 by an upstream kinase or reduction in the A0.5 for [AMP].     

Production of reactive oxygen by mitochondria from normoxic and hypoxic rat heart tissue.

Reactive oxygen species (ROS), which may be involved in ischemic or reperfusion heart injury, can be produced by mitochondria. Previous work indicated that coupled mitochondria from ischemic heart tissue incubated in calcium-free medium produced less ROS than normal. The effects of calcium, which may be elevated in hypoxic or ischemic tissue, were not examined. The relative production of ROS by mitochondria from normoxic or hypoxic rat heart tissue was estimated by measuring the oxidation of dichlorofluorescin to the fluorescent compound, dichlorofluorescein. ROS were detectable during succinate-stimulated State 4 respiration. In the absence of calcium, mitochondria from hypoxic (60 min) heart tissue produced less ROS than mitochondria from normoxic heart tissue. In the presence of 0.1, 1 or 10 microM calcium, ROS produced by hypoxic mitochondria were increased to normoxic levels. While function was depressed in mitochondria from hypoxic tissue, the presence of 0.1 and 1 microM calcium had no further effect. Respiration was uncoupled in the presence of 10 microM calcium in mitochondria from both normoxic and hypoxic heart tissue. ROS production was increased in mitochondria from hypoxic tissue with both increasing concentrations of calcium and increasing duration of exposure. ROS production in mitochondria from normoxic heart tissue was only stimulated after 200 or more seconds of exposure to 1 or 10 microM calcium. Production of ROS in mitochondria from hypoxic tissue in the presence of 1 microM calcium was inhibited by rotenone (80%), ruthenium red (69%), and a combination of these agents (96%). In contrast, ruthenium red had no effect on ROS production by mitochondria from normoxic heart tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

PKCε promotes cardiac mitochondrial and metabolic adaptation to chronic hypobaric hypoxia by GSK3β inhibition

PKCε is central to cardioprotection. Sub-proteome analysis demonstrated co-localization of activated cardiac PKCε (aPKCε) with metabolic, mitochondrial, and cardioprotective modulators like hypoxia-inducible factor 1α (HIF-1α). aPKCε relocates to the mitochondrion, inactivating glycogen synthase kinase 3β (GSK3β) to modulate glycogen metabolism, hypertrophy and HIF-1α. However, there is no established mechanistic link between PKCε, p-GSK3β and HIF1-α. Here we hypothesized that cardiac-restricted aPKCε improves mitochondrial response to hypobaric hypoxia by altered substrate fuel selection via a GSK3β/HIF-1α-dependent mechanism. aPKCε and wild-type (WT) mice were exposed to 14 days of hypobaric hypoxia (45 kPa, 11% O(2)) and cardiac metabolism, functional parameters, p-GSK3β/HIF-1α expression, mitochondrial function and ultrastructure analyzed versus normoxic controls. Mitochondrial ADP-dependent respiration, ATP production and membrane potential were attenuated in hypoxic WT but maintained in hypoxic aPKCε mitochondria (P < 0.005, n = 8). Electron microscopy revealed a hypoxia-associated increase in mitochondrial number with ultrastructural disarray in WT versus aPKCε hearts. Concordantly, left ventricular work was diminished in hypoxic WT but not aPKCε mice (glucose only perfusions). However, addition of palmitate abrogated this (P < 0.05 vs. WT). aPKCε hearts displayed increased glucose utilization at baseline and with hypoxia. In parallel, p-GSK3β and HIF1-α peptide levels were increased in hypoxic aPKCε hearts versus WT. Our study demonstrates that modest, sustained PKCε activation blunts cardiac pathophysiologic responses usually observed in response to chronic hypoxia. Moreover, we propose that preferential glucose utilization by PKCε hearts is orchestrated by a p-GSK3β/HIF-1α-mediated mechanism, playing a crucial role to sustain contractile function in response to chronic hypobaric hypoxia.     

Myocardial adenosine cycling rates during normoxia and under conditions of stimulated purine release.

Formation and rephosphorylation of adenosine (adenosine cycling) was studied in isolated rat hearts during normoxia and under conditions of stimulated purine formation. Hearts were infused with an inhibitor of adenosine kinase (5-iodotubercidin, 2 microM). In addition, perfusions were carried out with or without acetate, which is converted into acetyl-CoA, with simultaneous breakdown of ATP to AMP and purines. We found a linear, concentration-dependent, increase in normoxic purine release by acetate (5-20 mM). Differences in total purine release with or without iodotubercidin were taken as a measure of adenosine cycling. In normoxic hearts, iodotubercidin caused a minor increase in purine release (2.7 nmol/min per g wet wt.). Acetate (12.5 mM) increased purine release by 4.9 nmol/min per g, and its combination with inhibitor gave a large increase, by 14.2 nmol/min per g. This indicates a strongly increased adenosine cycling rate during acetate infusion. However, no significant differences in purine release were observed when iodotubercidin was infused during hypoxia, anoxia or ischaemia. The hypothesis that adenosine cycling is near-maximal during normoxia was not confirmed. Increased myocardial adenosine formation appears to be regulated by the availability of AMP and not by inhibition of adenosine kinase. This enzyme mainly functions to salvage adenosine in order to prevent excessive loss of adenine nucleotides.     

Effects of high glucose on the hypoxic isolated guinea pig heart: interactions with ATP-dependent K+ channels?

The effect of perfusion with elevated glucose concentrations on hypoxic myocardium was investigated in isolated Langendorff guinea pig hearts. For that purpose, mechanical (heart rate, systolic peak pressure and coronary flow) and electrophysiological (monophasic action potential duration=MAP, ectopic beats) data were evaluated. At the end of the experiments the hearts were examined histologically after trypan blue vital staining for quantification of irreversible myocardial damage. In the absence of insulin moderate glucose elevation (from 5 to 15 mM) exerted beneficial effects on hypoxic hearts: the depressed contraction was improved, the action potential shortening partly reversed and the percentage of irreversibly damaged myocytes diminished. Glucose did not have any effect on heart rate and arrhythmias under hypoxia or reperfusion. A contribution of cardiac ATP-dependent K+ channels to the effects of glucose could be excluded by further experiments. Thus, blocking these channels with high glibenclamide concentrations did not affect the action of glucose on MAP and contraction. To some degree the glucose effect on MAP, but not on systolic pressure, was also observable under normoxic conditions.     

The transmethylation pathway as a source for adenosine in the isolated guinea-pig heart.

In order to quantify adenosine production from the transmethylation pathway [S-adenosylmethionine (AdoMet)----S-adenosylhomocysteine (AdoHcy) in equilibrium adenosine + L-homocysteine] in the isolated guinea-pig heart under basal conditions (normoxic perfusion with 95% O2) and during elevated adenosine production (hypoxic perfusion with 30% O2), two methods were used. (1) Hearts were perfused with normoxic medium containing [2,5,8-3H]adenosine (5 microM) and L-homocysteine thiolactone (0.1 mM), which brings about net AdoHcy synthesis via reversal of the AdoHcy hydrolase reaction and labels the intracellular pool of AdoHcy. From the decrease in AdoHcy pool size and specific radioactivity of AdoHcy in the post-labelling period, the rate of transmethylation, which is equivalent to the rate of adenosine production, was calculated to be 0.98 nmol/min per g. Adenosine release from the hearts was 40-50 pmol/min per g. (2) Hearts were perfused with hypoxic medium containing [35S]homocysteine (50 microM). Owing to the hypoxia-induced increase in adenosine production, this procedure also results in expansion and labelling of the AdoHcy pool. From the dilution of the specific radioactivity of AdoHcy relative to that of [35S]homocysteine, the rate of AdoHcy synthesis from AdoMet (transmethylation) was calculated to be 1.12 nmol/min per g. It is concluded that in the oxygenated heart the transmethylation pathway is quantitatively an important intracellular source of adenosine, which exceeds the rate of adenosine wash-out by the coronary system by about 15-fold. Most of the adenosine formed by this pathway is re-incorporated into the ATP pool, most likely by adenosine kinase. The transmethylation pathway is essentially O2-independent, and the known hypoxia-induced production of adenosine must be derived from an increase in 5'-AMP hydrolysis.