Effect of drugs on the resistance of the myocardium to hypoxia

A method for the investigation of drug effects in the myocardium resistance to hypoxia has been suggested. It is based on the determination of drug effects on the performance of the isolated spontaneously contracting atrium (ISCA) of rats under hypoxic conditions. Hypoxia was induced by oxygen displacement from the nutritional solution by nitrogen. ISCA resistance to hypoxia was assessed by the mechanogram of the heart preparation (the duration and volume of ISCA performance being up to 50% of the initial amplitude). Using the inhibitor analysis, it has been demonstrated that the given model of myocardial hypoxia adequately reflects the role of energy cellular metabolism in the regulation of ISCA resistance to hypoxia and can be used in the search for myocardial antihypoxic agents.     

The Early Activation of PI3K Strongly Enhances the Resistance of Cortical Neurons to Hypoxic Injury via the Activation of Downstream Targets of the PI3K Pathway and the Normalization of the Levels of PARP Activity, ATP, and NAD+

Phosphatidylinositol 3-kinase (PI3K) plays several important roles in neuronal survival. Activation of the pathway is essential for the neuroprotective mechanisms of materials that shield neuronal cells from many stressful conditions. However, there have been no reports to date about the effect of the direct activation of the pathway in hypoxic injury of neuronal cells. We investigated whether the direct activation of the PI3K pathway inhibits neuronal cell death induced by hypoxia. Primary cultured cortical neurons (PCCNs) were exposed to hypoxic conditions (less than 1 mol% O₂) and/or treated with PI3K activator. Hypoxia reduced the viability of PCCNs in a time-dependent manner, but treatment with PI3K significantly restored viability in a concentration-dependent manner. Among the signaling proteins involved in the PI3K pathway, those associated with survival, including Akt and glycogen synthase kinase-3β, were decreased shortly after exposure to hypoxia and those associated with cell death, including BAX, apoptosis-induced factor, cytochrome c, caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP), were increased. However, treatment with PI3K activator normalized the expression levels of those signaling proteins. PARP activity and levels of ATP and NAD⁺ altered by hypoxia were also normalized with direct PI3K activation. All these findings suggest that direct and early activation is important for protecting neuronal cells from hypoxic injury.     

Overexpressing cellular repressor of E1A-stimulated genes protects mesenchymal stem cells against hypoxia- and serum deprivation-induced apoptosis by activation of PI3K/Akt

Bone marrow-derived mesenchymal stem cells (MSCs) have great potential for repair after myocardial infarction. However, poor viability of transplanted MSCs in the ischemic heart has limited their therapeutic potential. Cellular repressor of E1A-stimulated genes (CREG) has been identified as a potent inhibitor of apoptosis. The aim of this study was to investigate the anti-apoptotic effects of CREG on MSCs under hypoxic and serum deprivation (SD) conditions. We also investigated the potential mechanism(s) that may mediate the actions of CREG. All experiments were performed on rat bone marrow MSCs. Apoptosis was induced by exposure of cells to hypoxia/SD in a sealed GENbox hypoxic chamber. Effects of CREG were investigated in the absence or presence of inhibitors that target phosphoinositide 3-kinase (PI3K). We found that the overexpression of CREG markedly protected MSCs from hypoxia/SD-induced apoptosis through inhibition of the mitochondrial apoptotic pathway, leading to attenuation of caspase-3. Moreover, CREG enhanced Akt phosphorylation and decreased the expression of p53 in MSCs under hypoxic/SD conditions. The PI3K/Akt inhibitor LY294002 significantly increased the amount of p53 protein and attenuated the anti-apoptotic effects of CREG on MSCs. This study indicates that CREG is a novel and potent survival factor for MSCs, therefore, it may be a useful therapeutic adjunct for transplanting MSCs into damaged heart after myocardial infarction.     

Effect of preliminary local myocardial hypoxia on the development of immune heart damage

The changes in hemo- and cardiodynamics, myocardial contractility and its structural and histochemical alterations were studied in two series of experiments (immune and combined hypoxic and immune action on the myocardium) on 27 mongrel dogs anesthesized with chloralose and urethane. It was established that preliminary short-term hypoxia of the myocardium essentially increases the alterative effect of immune factors, causing a rise in the involved area as well as in the degree of metabolic shifts in this area and in the intensity of the disorders of heart function and hemodynamics.     

Hypoxic preconditioning is a phenomenon increasing tolerance of cardiomyocytes to hypoxia-reoxygenation

The work covers the problem of hypoxic preconditioning (HP) carried out in isolated cardiomyocytes. Papers on delayed HP in vivo are comparatively few, and only some single works are devoted to early preconditioning in vivo. It has been established that the HP limits necrosis and apoptosis of cardiomyocytes and improves contractility of the isolated heart after ischemia (hypoxia) and reperfusion (reoxygenation). It was found that adenosine was a trigger of iP in vitro. It was proved that NO* was a trigger of HP both in vitro and in vivo. It was shown that reactive oxygen species also were triggers of hypoxic preconditioning. It was shown that ERK1/2 and p38 kinase played important role in delayed HP in vitro.     

Hypoxia induces the activation of the phosphatidylinositol 3-kinase/Akt cell survival pathway in PC12 cells: protective role in apoptosis

Hypoxia is a common environmental stress that influences signaling pathways and cell function. Several cell types, including neuroendocrine chromaffin cells, have evolved to sense oxygen levels and initiate specific adaptive responses to hypoxia. Here we report that under hypoxic conditions, rat pheochromocytoma PC12 cells are resistant to apoptosis induced by serum withdrawal and chemotherapy treatment. This effect is also observed after treatment with deferoxamine, a compound that mimics many of the effects of hypoxia. The hypoxia-dependent protection from apoptosis correlates with activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is detected after 3-4 h of hypoxic or deferoxamine treatment and is sustained while hypoxic conditions are maintained. Hypoxia-induced Akt activation can be prevented by treatment with cycloheximide or actinomycin D, suggesting that de novo protein synthesis is required. Finally, inhibition of PI3K impairs both the protection against apoptosis and the activation of Akt in response to hypoxia, suggesting a functional link between these two phenomena. Thus, reduced oxygen tension regulates apoptosis in PC12 cells through activation of the PI3K/Akt survival pathway.     

Intermittent high altitude hypoxia - induced structural changes in the pulmonary myocardium in young mice

Seven-day-old mice, strain H, were exposed to intermittent high altitude hypoxia (IHA) in a barochamber (7,000 m, 4 h/day, 5 days a week); a total number of exposures was 10. It has been shown that the layer of cardiac musculature in the adventitia of the pulmonary veins, the so-called pulmonary myocardium, reacts to IHA hypoxia by enlargement even sooner than the right ventricular myocardium. The average thickness of the layer of pulmonary myocardium was significantly greater in animals exposed to IHA hypoxia as compared with the controls. Furthermore, IHA hypoxia induces the extension of the pulmonary myocardium to the periphery of the pulmonary venous bed. Ultrastructural investigation of the pulmonary myocardium in hypoxic animals revealed the presence of unoriented myofilaments in the peripheral myofibril-free sarcoplasma, increase in the number of ribosomes and appearance of profiles of granular endoplasmic reticulum. Our data provide quantitative support for the hypothesis that it is not only the contraction of pulmonary arteries, but also venoconstriction which contribute to the hypoxic pressure response in mice.     

Hypoxic preconditioning improves survival of cardiac progenitor cells: role of stromal cell derived factor-1α-CXCR4 axis

Background

Cardiac progenitor cells (CPCs) have been shown to be suitable in stem cell therapy for resurrecting damaged myocardium, but poor retention of transplanted cells in the ischemic myocardium causes ineffective cell therapy. Hypoxic preconditioning of cells can increase the expression of CXCR4 and pro-survival genes to promote better cell survival; however, it is unknown whether hypoxia preconditioning will influence the survival and retention of CPCs via the SDF-1α/CXCR4 axis.

Methods and Results

CPCs were isolated from adult mouse hearts and purified by magnetic activated cell sorting using c-kit magnetic beads. These cells were cultured at various times in either normoxic or hypoxic conditions, and cell survival was analyzed using flow cytometry and the expression of hypoxia-inducible factor-1α (HIF-1α), CXCR4, phosphorylated Akt and Bcl-2 were measured by Western blot. Results showed that the expression of pro-survival genes significantly increased after hypoxia treatment, especially in cells cultured in hypoxic conditions for six hours. Upon completion of hypoxia preconditioning from c-kit+ CPCs for six hours, the anti-apoptosis, migration and cardiac repair potential were evaluated. Results showed a significant enhancement in anti-apoptosis and migration in vitro, and better survival and cardiac function after being transplanted into acute myocardial infarction (MI) mice in vivo. The beneficial effects induced by hypoxia preconditioning of c-kit+ CPCs could largely be blocked by the addition of CXCR4 selective antagonist AMD3100.

Conclusions

Hypoxic preconditioning may improve the survival and retention of c-kit+ CPCs in the ischemic heart tissue through activating the SDF-1α/CXCR4 axis and the downstream anti-apoptosis pathway. Strategies targeting this aspect may enhance the effectiveness of cell-based cardiac regenerative therapy.     

Radiolabeled hypoxic cell sensitizers: tracers for assessment of ischemia

Hypoxic, non-functional, but viable, tissue may exist in heart and brain following an arterial occlusion. Identification of such tissue in vivo is crucial to the development of effective treatment strategies. It has been suggested that certain compounds capable of sensitizing hypoxic tumor cells to killing by x-rays (i.e., misonidazole) might serve as in vivo markers of hypoxic tissue in ischemic myocardium or brain if properly radiolabeled. To this end we have radiolabeled two fluorinated analogs of nitroimidazole based hypoxic cell sensitizers with the 110 minute half-lived positron-emitting fluorine-18. The ability of these tracers to quantitate the presence of hypoxic tissue has been studied in a gerbil stroke model. The in vivo uptake of one of these tracers [F-18]-fluoronormethyoxymisonidazole is dependent on the extent of tissue hypoxia, and thus, appears to have potential as a diagnostic indicator of non-functional but viable tissue when the tracer is used in conjunction with positron emission tomography.     

Time-Dependent Effect of Hypoxia on Tumor Progression and Liver Progenitor Cell Markers in Primary Liver Tumors

Background & Aims

Expression of liver progenitor cell (LPC) characteristics has been proposed as a negative prognostic marker in primary liver tumors. Hypoxia has been linked to activation of the Notch pathway which is responsible for activation and proliferation of LPCs and hypoxia-induced LPC activation has been shown in hepatocellular carcinoma. Our aim was to elucidate the time-dependent effects of hypoxia on the LPC niche in hepatocellular carcinoma which could aid in determining a safe time frame for use of hypoxia inducing therapies.

Methods

We used dimethyloxaloylglycine to mimic a hypoxic reaction in mice by stabilizing hypoxia-inducible factor 1 alpha at three distinct time points in diethylnitrosamine induced hepatocarcinogenesis. LPC, metastasis and Notch pathway markers were determined by quantitative PCR and (immune)histochemistry (heamatoxillin-eosin, reticulin, Sirius red and cytokeratin 19 staining).

Results

Activating the hypoxia inducible pathway early in hepatocarcinogenesis resulted in an increased incidence of both cholangioma and hepatocellular lesions, associated with high expression of LPC, metastatic and Notch pathway markers. Adversely, activating the hypoxic response during tumor development resulted in decreased incidence of hepatocellular lesions and increased cholangioma incidence, with an unaltered gene expression profile of LPC-, Notch pathway- and metastatic markers. A hypoxic insult at advanced stages of hepatocarcinogenesis severely increased the expression of LPC characteristics, however without increased expression of actors of the Notch pathway and metastatic markers and minor changes in incidence of hepatocellular and cholangioma lesions.

Conclusion

Our results indicate that increased hypoxia at the onset of tumor development has detrimental effects on tumor progression; patients with HCC developed in a background of fibrosis/cirrhosis might therefore represent a more difficult treatment group. In contrast, hypoxia during tumor development appears to favor tumor outcome, highlighting the importance of early detection. Finally, hypoxia in advanced stages resulted in increased expression of LPC characteristics indicating poor outcome.     

Relationship of the activity of cardiac cholinergic mechanisms to the state of the sympathetic innervation and its noradrenaline content in hypoxia]

Preliminary sympathectomia depletes acetylcholine (ACh) in the heart of rabbits under hypoxia. In these conditions the inhibitory action of ACh on the rat isolated heart is reduced under the noradrenaline content fall, while under increase it is potentiated. Under hypoxia noradrenaline increases concentration of potassium in the myocardium, thus stimulating ACh formation and activity. It is suggested that under deep hypoxia suppression of the sympathetic mechanisms causes functional isolation of the heart from nervous effects.     

Xanthine Oxidase is Not Responsible for Reoxygenation Injury in Isolated-Perfused Rat Heart

The massive leakage of intracellular enzymes which occurs during reoxygenation of heart tissue after hypoxic or ischemic episodes has been suggested to result from the formation of oxygen radicals. One purported source of such radicals is the xanthine oxidase-mediated metabolism of hypoxanthine and xanthine. Xanthine oxidase (O form) has been suggested to be formed in vivo by limited proteolysis of xanthine dehydrogenase (D form) during the hypoxic period (Granger el ai. Gastroenterology 81, 22 (1981)). We measured the activities of xanthine oxidase in both fresh and isolated-perfused (Langendorff) rat heart tissue. Approximately 32% of the total xanthine oxidase was in the O form in fresh and isolated-perfused rat heart. This value was unchanged following 60min of hypoxia and 30 minutes of reoxygenation. The infusion of 250/JM allopurinol throughout the perfusion completely inhibited xanthine oxidase activity but had no effect on the massive release of lactate dehydrogenase (LDH) into the coronary effluent upon reoxygenation of heart tissue subjected to 30 or 60min of hypoxia. Protection from 30min of hypoxia was also not obtained when rats were pretreated for 48 h with allopurinol at a dose of 30mg/kg/day and perfused with allopurinol containing medium. Superoxide dismutase (50 units/ml), catalase (200 units/ml), or the antioxidant cyanidanol (100μM) also had no effect on LDH release upon reoxygenation after 60 min of hypoxia. Xanthine oxidase activity was detected in a preparation enriched in cardiac endothelial cells while no allupurinol-inhibitable activity could be measured in purified isolated cardiomyocytes. It is concluded that xanthine dehydrogenase is not converted to xanthine oxidase in hypoxic tissue of the isolated perfused rat heart, and that the release of intracellular enzymes upon reoxygenation in this experimental model is mediated by factors other than reactive oxygen generated by xanthine oxidase.     

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.     

Protective effect of gap junction uncouplers given during hypoxia against reoxygenation injury in isolated rat hearts

It has been shown that cell-to-cell chemical coupling may persist during severe myocardial hypoxia or ischemia. We aimed to analyze the effects of different, chemically unrelated gap junction uncouplers on the progression of ischemic injury in hypoxic myocardium. First, we analyzed the effects of heptanol, 18alpha-glycyrrhetinic acid, and palmitoleic acid on intracellular Ca2+ concentration during simulated hypoxia (2 mM NaCN) in isolated cardiomyocytes. Next, we analyzed their effects on developed and diastolic tension and electrical impedance in 47 isolated rat hearts submitted to 40 min of hypoxia and reoxygenation. All treatments were applied only during the hypoxic period. Cell injury was determined by lactate dehydrogenase (LDH) release. Heptanol, but not 18alpha-glycyrrhetinic acid nor palmitoleic acid, attenuated the increase in cytosolic Ca2+ concentration induced by simulated ischemia in cardiomyocytes and delayed rigor development (rigor onset at 7.31 +/- 0.71 min in controls vs. 14.76 +/- 1.44 in heptanol-treated hearts, P < 0.001) and the onset of the marked changes in electrical impedance (tissue resistivity: 4.02 +/- 0.29 vs. 7.75 +/- 1.84 min, P = 0.016) in hypoxic rat hearts. LDH release from hypoxic hearts was minimal and was not significantly modified by drugs. However, all gap junction uncouplers, given during hypoxia, attenuated LDH release during subsequent reoxygenation. Dose-response analysis showed that increasing heptanol concentration beyond the level associated with maximal effects on cell coupling resulted in further protection against hypoxic injury. In conclusion, gap junction uncoupling during hypoxia has a protective effect on cell death occurring upon subsequent reoxygenation, and heptanol has, in addition, a marked protective effect independent of its uncoupling actions.     

Increased cross-bridge cycling rate in stunned myocardium

It is well known that the implantation of bone marrow mononuclear cells (BM-MNCs) into ischemic hearts can induce angiogenesis and improve cardiac function after myocardial infarction, but the precise mechanisms of these actions are unclear. We hypothesize that the cytokines produced by BM-MNCs play a key role in this cell-based therapy. BM-MNCs from rats were cultured under normoxic or hypoxic (1% O2) conditions for 24 h, and then supernatants were collected for study. ELISA and Western blotting analysis showed that various cytokines, including VEGF, IL-1 beta, PDGF, and IGF-1, were produced from BM-MNCs, some of which were enhanced significantly under hypoxia stimulation. When compared with a control blank medium, the supernatants of BM-MNCs cultured under normoxic or hypoxic conditions inhibited apoptosis significantly and preserved the contractile capacity of isolated adult rat cardiomyocytes in vitro (P < 0.05). Using a rat model of acute myocardial infarction, we injected the supernatants of BM-MNCs or control medium intramyocardially on day 0 and then intraperitoneally on days 2, 4, and 6 after infarction. When compared with the control medium, the supernatants of BM-MNCs cultured under both normoxic or hypoxic conditions increased the microvessel density and decreased the fibrotic area in the infarcted myocardium significantly, contributing to remarkable improvement in cardiac function. Various cytokines were produced by BM-MNCs, and these cytokines contributed to functional improvement of the infarcted heart by directly preserving the contractile capacity of the myocardium, inhibiting apoptosis of cardiomyocytes, and inducing therapeutic angiogenesis of the infarcted heart.     

Adenosine effect on lactate and pyruvate production in the hypoxic guineapig heart

• 1.1. It has been shown that adenosine stimulates glycolysis in some cells and this ability of adenosine was tested in the hypoxic guineapig heart.
• 2.2. Adenosine (10 μM) activated lactate production in the isolated perfused guineapig heart under conditions of normoxia but did not under hypoxia.
• 3.3. Despite this, the nucleoside favorably influenced the energy metabolism of the hypoxic heart as revealed by the better posthypoxic functional recovery (98%) compared to the control without adenosine (78%).
• 4.4. Our findings suggest a role for the glycolytic pathway in this effect of the nucleoside as long as other cardiac energy-yielding pathways are strictly aerobic.

     

Cysteinyl leukotriene signaling aggravates myocardial hypoxia in experimental atherosclerotic heart disease

Background

Cysteinyl-leukotrienes (cys-LT) are powerful spasmogenic and immune modulating lipid mediators involved in inflammatory diseases, in particular asthma. Here, we investigated whether cys-LT signaling, in the context of atherosclerotic heart disease, compromises the myocardial microcirculation and its response to hypoxic stress. To this end, we examined Apoe^−/− mice fed a hypercholesterolemic diet and analysed the expression of key enzymes of the cys-LT pathway and their receptors (CysLT1/CysLT2) in normal and hypoxic myocardium as well as the potential contribution of cys-LT signaling to the acute myocardial response to hypoxia.

Methods and principal findings

Myocardial biopsies from Apoe^−/− mice demonstrated signs of chronic inflammation with fibrosis, increased apoptosis and expression of IL-6, as compared to biopsies from C57BL/6J control mice. In addition, we found increased leukotriene C₄ synthase (LTC₄S) and CysLT1 expression in the myocardium of Apoe^−/− mice. Acute bouts of hypoxia further induced LTC₄S expression, increased LTC₄S enzyme activity and CysLT1 expression, and were associated with increased extension of hypoxic areas within the myocardium. Inhibition of cys-LT signaling by treatment with montelukast, a selective CysLT1 receptor antagonist, during acute bouts of hypoxic stress reduced myocardial hypoxic areas in Apoe^−/− mice to levels equal to those observed under normoxic conditions. In human heart biopsies from 14 patients with chronic coronary artery disease mRNA expression levels of LTC₄S and CysLT1 were increased in chronic ischemic compared to non-ischemic myocardium, constituting a molecular basis for increased cys-LT signaling.

Conclusion

Our results suggest that CysLT1 antagonists may have protective effects on the hypoxic heart, and improve the oxygen supply to areas of myocardial ischemia, for instance during episodes of sleep apnea.     

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.