Hypoxic Rat Heart Research Articles

Selection of optimal reference genes for gene expression studies in chronically hypoxic rat heart

Adaptation to chronic hypoxia renders the heart more tolerant to ischemia/reperfusion injury. To evaluate changes in gene expression after adaptation to chronic hypoxia by RT-qPCR, it is essential to select suitable reference genes. In a chronically hypoxic rat model, no specific reference genes have been identified in the myocardium. This study aimed to select the best reference genes in the left (LV) and right (RV) ventricles of chronically hypoxic and normoxic rats. Sprague-Dawley rats were adapted to continuous normobaric hypoxia (CNH; 12% O2 or 10% O2) for 3weeks. The expression levels of candidate genes were assessed by RT-qPCR. The stability of genes was evaluated by NormFinder, geNorm and BestKeeper algorithms. The best five reference genes in the LV were Top1, Nupl2, Rplp1, Ywhaz, Hprt1 for the milder CNH and Top1, Ywhaz, Sdha, Nupl2, Tomm22 for the stronger CNH. In the RV, the top five genes were Hprt1, Nupl2, Gapdh, Top1, Rplp1 for the milder CNH and Tomm22, Gapdh, Hprt1, Nupl2, Top1 for the stronger CNH. This study provides validation of reference genes in LV and RV of CNH rats and shows that suitable reference genes differ in the two ventricles and depend on experimental protocol.

Redox states of hemoglobin determine left ventricle pressure recovery and activity of mitochondrial complex IV in hypoxic rat hearts

Cardiovascular effects were reported to occur in humans and in animal models during transfusion with hemoglobin (Hb)-based oxygen therapeutics. The effects of Hb's iron redox states on cardiac parameters during hypoxia/reoxygenation are however poorly defined. We hypothesize that acute exposures to ferric Hb during hypoxia leads to cardiomyocyte injury and an impaired left ventricular response accompanied by cardiac mitochondrial bioenergetic dysfunction. Recovery of left ventricular functions in an isolated rat heart Langendorff perfusion system was observed following perfusion with ferrous but not with ferric Hb. Ferric Hb induced the development of heart lesions, and impairment of the respiratory chain complex activity. Under normoxia, a sharp decline in cardiac parameters was observed following co-perfusion of low (20 μM) and high (100 μM) ascorbic acid (Asc) with ferrous Hb. This trend continued with ferric Hb co-perfusion, but only at the higher concentration of Asc. These observations suggest that perfusion of the hypoxic heart with ferric Hb increases oxidative stress thereby resulting in cardiac dysfunction. Intervention with Asc to reduce ferric Hb may offer a strategy to control Hb toxicity; however, timing of administration, and dosage of Asc may require individual optimization to target specific redox forms of Hb.

Myocardial interstitial matrix as novel target for succinic acid treatment strategies during experimental hypobaric hypoxia

Objective — Quantitative morphological studies of myocardial cellular elements. Aim of research is to study myocardial interstitial matrix with and without succinic acid treatment in the rats exposed to intermittent hypobaric hypoxia. Material and Methods — The study was conducted on 26 adult males of Wistar rats weighing 220-310 g, divided into 3 groups. The first control group consisted of 6 intact animals. The second group included 10 rats which were exposed to hypobaric hypoxia without medication for 30 days. Third group was composed of 10 rats, which were medicated by succinic acid solution which was injected intraperitoneally once a day at the rate of 0.5 ml/100 g of animal body weight 15 minutes before hypoxic exposure for 30 days. Results — In the second group almost the entire stroma was consisting of the thickened collagen fibers and proliferating cells of the connective tissue. Their total number increased relatively to the control by 31% initiating the processes of collagenases. An increase in the number of myofibroblasts which was observed in the hypoxic rat heart in the second group followed by high MPP-9 expression. Hypoxic effects in the third group were not significantly different from control according to the observed changes in the stroma. In rats of the third group the capillary diameter exceeded the indicator of the second group, but was significantly lower than the control indicator. Accordingly, compared with the series without correction, the cross-sectional area of the capillaries was increased – 28.44±0.14 μm2 against 24.53±0.20 μm2. The total cross-sectional area of the capillaries is 0.77±0.10 x103 μm2. The relative surface area of the vascular area was lower than the control by 21.8%. Conclusion ― Fibrosis in the myocardium inevitably leads to increased myocardial stiffness, resulting in systolic and diastolic dysfunction, neurohormonal activation and, ultimately, heart failure caused by hypobaric hypoxia. Reduced oxygen delivery by microvascular damage, increased perivascular fibrosis may contribute to contractile failure. Succinic acid combined with inosine acts as a high-energy phosphate reserve, to maintain adenosine triphosphate at levels sufficient to support contractile function.

Rhodiola crenulata extract counteracts the effect of hypobaric hypoxia in rat heart via redirection of the nitric oxide and arginase 1 pathway

BackgroundRhodiola crenulata is traditionally used as a folk medicine in Tibet for preventing high-altitude illnesses, including sudden cardiac death (SCD). The cardio-protective effects of Rhodiola crenulata root extract (RCE) against hypoxia in vivo have been recently confirmed. However, the way in which RCE produces these effects remains unclear. The present study is designed to confirm the protective effects of RCE on the heart in acute hypobaric hypoxia exposure and examine the mechanisms by which this occurs.MethodsSprague–Dawley (SD) rats were pretreated with or without RCE and then exposed to a simulated altitude of 8000 m in a hypobaric hypoxia chamber for 9 h. The expression of cardiac arginase 1 (Arg-1) and endothelial nitric oxide synthase (eNOS) and the activity of associated signaling pathways was examined.ResultsHypoxia reduced cardiac eNOS phosphorylation and increased Arg-1 expression, but both responses were reversed by RCE pre-treatment. In addition, RCE decreased the hypoxia-induced oxidative stress markers of reactive oxygen species (ROS) production, malondialdehyde (MDA) level, and protein carbonyl content. Furthermore, RCE protected cardiomyocytes from hypoxia-induced cardiac apoptosis and restored the phosphorylation level of AKT and p38 MAPK as well as the superoxide dismutase 2 (SOD2) content in hypoxic animals.ConclusionThe findings provide evidence that the effects of Rhodiola crenulata against altitude illness are partially mediated by modulation of eNOS and Arg-1 pathways in the heart.

Energy metabolism and the high-altitude environment.

At high altitude the barometric pressure falls, challenging oxygen delivery to the tissues. Thus, whilst hypoxia is not the only physiological stress encountered at high altitude, low arterial P(O2) is a sustained feature, even after allowing adequate time for acclimatization. Cardiac and skeletal muscle energy metabolism is altered in subjects at, or returning from, high altitude. In the heart, energetic reserve falls, as indicated by lower phosphocreatine-to-ATP ratios. The underlying mechanism is unknown, but in the hypoxic rat heart fatty acid oxidation and respiratory capacity are decreased, whilst pyruvate oxidation is also lower after sustained hypoxic exposure. In skeletal muscle, there is not a consensus. With prolonged exposure to extreme high altitude (>5500 m) a loss of muscle mitochondrial density is seen, but this was not observed in a simulated ascent of Everest in hypobaric chambers. At more moderate high altitude, decreased respiratory capacity may occur without changes in mitochondrial volume density, and fat oxidation may be downregulated, although this is not seen in all studies. The underlying mechanisms, including the possible role of hypoxia-signalling pathways, remain to be resolved, particularly in light of confounding factors in the high-altitude environment. In high-altitude-adapted Tibetan natives, however, there is evidence of natural selection centred around the hypoxia-inducible factor pathway, and metabolic features in this population (e.g. low cardiac phosphocreatine-to-ATP ratios, increased cardiac glucose uptake and lower muscle mitochondrial densities) share similarities with those in acclimatized lowlanders, supporting a possible role for the hypoxia-inducible factor pathway in the metabolic response of cardiac and skeletal muscle energy metabolism to high altitude.

Dietary nitrate increases arginine availability and protects mitochondrial complex I and energetics in the hypoxic rat heart.

Hypoxic exposure is associated with impaired cardiac energetics in humans and altered mitochondrial function, with suppressed complex I-supported respiration, in rat heart. This response might limit reactive oxygen species generation, but at the cost of impaired electron transport chain (ETC) activity. Dietary nitrate supplementation improves mitochondrial efficiency and can promote tissue oxygenation by enhancing blood flow. We therefore hypothesised that ETC dysfunction, impaired energetics and oxidative damage in the hearts of rats exposed to chronic hypoxia could be alleviated by sustained administration of a moderate dose of dietary nitrate. Male Wistar rats (n = 40) were given water supplemented with 0.7 mmol l−1 NaCl (as control) or 0.7 mmol l−1 NaNO3, elevating plasma nitrate levels by 80%, and were exposed to 13% O2 (hypoxia) or normoxia (n = 10 per group) for 14 days. Respiration rates, ETC protein levels, mitochondrial density, ATP content and protein carbonylation were measured in cardiac muscle. Complex I respiration rates and protein levels were 33% lower in hypoxic/NaCl rats compared with normoxic/NaCl controls. Protein carbonylation was 65% higher in hearts of hypoxic rats compared with controls, indicating increased oxidative stress, whilst ATP levels were 62% lower. Respiration rates, complex I protein and activity, protein carbonylation and ATP levels were all fully protected in the hearts of nitrate-supplemented hypoxic rats. Both in normoxia and hypoxia, dietary nitrate suppressed cardiac arginase expression and activity and markedly elevated cardiac l-arginine concentrations, unmasking a novel mechanism of action by which nitrate enhances tissue NO bioavailability. Dietary nitrate therefore alleviates metabolic abnormalities in the hypoxic heart, improving myocardial energetics.

234 IMPAIRED METABOLIC AND FUNCTIONAL ADAPTATION TO HYPOXIA IN THE TYPE 2 DIABETIC HEART

AimsCardiovascular disease is the leading cause of mortality in people with type 2 diabetes. Following a myocardial infarction, the heart must adapt to the decreased oxygen availability by switching to...

Effect of Subchronic Hypobaric Hypoxia on Oxidative Stress in Rat Heart

We examined the effect of subchronic hypobaric hypoxia in rat heart. Adult male Sprague-Dawley rats were exposed at 25,000 ft for different time periods (2 and 5 days). Susceptibility of their hearts to oxidative stress as well as modulation in gene expression was evaluated. The results showed a crosstalk between reactive oxygen species (ROS) and nitric oxide (NO), initial response was accompanied by increase in ROS generation and development of oxidative stress as confirmed by increased lipid peroxidation, protein oxidation and accumulation of 2, 4-dinitrophenyl hydrazine and 4-hydroxy-2-nonenal adducts. At the same time, glutathione activity decreased; however, antioxidant enzymatic activities of superoxide dismutases, glutathione-S-transferase, and glutathione peroxidase rose in response to 5-days hypoxia. Interestingly, NO level increased till 5 days, however ROS decreased after 5 days; this observation suggests that ROS/NO balance plays an important role in cardioprotection. This observation is further supported by upregulation of antioxidant genes hemeoxygenase (HO-1) and metallothionein (MT). In addition, hypoxia also induces gradual upregulation of hypoxia-inducible transcription factor (HIF-1α), which in turn induces the expression of adaptive genes erythropoiesis, vascular endothelial growth factor, glucose transporter-1, nitric oxide synthase. Collectively, our data suggests a reciprocal regulation of ROS and NO and this effect is mediated by the increase in antioxidant proteins HO-1 and MT. Along with this HIF-1-mediated induction of various cardioprotective genes also plays an important role in acclimatization.

Cross talk betweenS-nitrosylation andS-glutathionylation in control of the Na,K-ATPase regulation in hypoxic heart

Oxygen-induced regulation of Na,K-ATPase was studied in rat myocardium. In rat heart, Na,K-ATPase responded to hypoxia with a dose-dependent inhibition in hydrolytic activity. Inhibition of Na,K-ATPase in hypoxic rat heart was associated with decrease in nitric oxide (NO) production and progressive oxidative stress. Accumulation of oxidized glutathione (GSSG) and decrease in NO availability in hypoxic rat heart were followed by a decrease in S-nitrosylation and upregulation of S-glutathionylation of the catalytic α-subunit of the Na,K-ATPase. Induction of S-glutathionylation of the α-subunit by treatment of tissue homogenate with GSSG resulted in complete inhibition of the enzyme in rat a myocardial tissue homogenate. Inhibitory effect of GSSG in rat sarcolemma could be significantly decreased upon activation of NO synthases. We have further tested whether oxidative stress and suppression of the Na,K-ATPase activity are observed in hypoxic heart of two subterranean hypoxia-tolerant blind mole species (Spalax galili and Spalax judaei). In both hypoxia-tolerant Spalax species activity of the enzyme and tissue redox state were maintained under hypoxic conditions. However, localization of cysteines within the catalytic subunit of the Na,K-ATPase was preserved and induction of S-glutathionylation by GSSG in tissue homogenate inhibited the Spalax ATPase as efficiently as in rat heart. The obtained data indicate that oxygen-induced regulation of the Na,K-ATPase in the heart is mediated by a switch between S-glutathionylation and S-nitrosylation of the regulatory thiol groups localized at the catalytic subunit of the enzyme.

N-acetylcysteine Treatment Prevents the Up-Regulation of MnSOD in Chronically Hypoxic Rat Hearts

Chronic intermittent hypoxia (CIH) is associated with increased production of reactive oxygen species that contributes to the adaptive mechanism underlying the improved myocardial ischemic tolerance. The aim was to find out whether the antioxidative enzyme manganese superoxide dismutase (MnSOD) can play a role in CIH-induced cardioprotection. Adult male Wistar rats were exposed to intermittent hypobaric hypoxia (7000 m, 8 h/day, 25 exposures) (n=14) or kept at normoxia (n=14). Half of the animals from each group received N-acetylcysteine (NAC, 100 mg/kg) daily before the hypoxic exposure. The activity and expression of MnSOD were increased by 66 % and 23 %, respectively, in the mitochondrial fraction of CIH hearts as compared with the normoxic group; these effects were suppressed by NAC treatment. The negative correlation between MnSOD activity and myocardial infarct size suggests that MnSOD can contribute to the improved ischemic tolerance of CIH hearts.

Hypoxic preconditioning facilitates acclimatization to hypobaric hypoxia in rat heart

Acute systemic hypoxia induces delayed cardioprotection against ischaemia-reperfusion injury in the heart. As cobalt chloride (CoCl₂) is known to elicit hypoxia-like responses, it was hypothesized that this chemical would mimic the preconditioning effect and facilitate acclimatization to hypobaric hypoxia in rat heart. Male Sprague-Dawley rats treated with distilled water or cobalt chloride (12.5 mg Co/kg for 7 days) were exposed to simulated altitude at 7622 m for different time periods (1, 2, 3 and 5 days). Hypoxic preconditioning with cobalt appreciably attenuated hypobaric hypoxia-induced oxidative damage as observed by a decrease in free radical (reactive oxygen species) generation, oxidation of lipids and proteins. Interestingly, the observed effect was due to increased expression of the antioxidant proteins hemeoxygenase and metallothionein, as no significant change was observed in antioxidant enzyme activity. Hypoxic preconditioning with cobalt increased hypoxia-inducible factor 1α (HIF-1α) expression as well as HIF-1 DNA binding activity, which further resulted in increased expression of HIF-1 regulated genes such as erythropoietin, vascular endothelial growth factor and glucose transporter. A significant decrease was observed in lactate dehydrogenase activity and lactate levels in the heart of preconditioned animals compared with non-preconditioned animals exposed to hypoxia. The results showed that hypoxic preconditioning with cobalt induces acclimatization by up-regulation of hemeoxygenase 1 and metallothionein 1 via HIF-1 stabilization.

Inducible Nitric Oxide Synthase-Activated Mitochondrial Apoptotic Pathway in Hypoxic and Aged Rat Hearts

Background: Hypoxia and aging determine on mammalian cells a stress response which implies modified production of oxidants, reactive oxygen species or reactive nitrogen species at the mitochondrial level, interfering with cell-signaling proteins and inducing mitochondrial damage, apoptosis occurrence and functional consequences. Objective: Here we report the effects of hypoxia on the in vivo morphological and biochemical response of young and aged Wistar rat hearts. Methods: Left ventricles were excised from each experimental point and processed. Investigations of vascular endothelial growth factor (VEGF) expression and apoptotic events, mitochondrial damage, were performed by light and electron microscopy, respectively; endothelial, inducible and neuronal NOS, PKCα, pPKCα, caspase-3 expression and Apaf-1/cytochrome c complex formation were assessed by Western blotting and co-immunoprecipitation analyses, respectively. Results: Besides morphological modifications, which confirm mitochondrial suffering upon hypoxia exposure in both young and aged hearts, the role played by PKCα in controlling nitric oxide synthase (NOS) protein level was investigated. Downstream PKCα activation, a dramatic iNOS expression increase, concomitant to enhanced apoptotic cell percentage and Apaf-1/cytochrome c co-immunoprecipitation, is evident in the hypoxic young, suggesting iNOS-mediated activation of the mitochondrial apoptotic pathway. Conclusions: Moreover, overexpression of iNOS and VEGF in the hypoxic young rat hearts suggests that an increased VEGF level may allow coordinated development of the lymphatic and blood vasculature, necessary for fluid homeostasis and to counteract oxidative stress. Thus the inhibition of such growth factor proposes new therapeutic possibilities for diseases associated to vascular function and for solid tumors which show pathological angiogenesis and lymphoangiogenesis.

Effect of Hypoxia and Aging on PKC δ‐Mediated SC‐35 Phosphorylation in Rat Myocardial Tissue

Nuclear speckles, which are sites of pre-mRNA splicing and/or assembly components, are diffusely distributed throughout the nucleoplasm. They are composed of splicing factors (SFs), including SC-35, which are nuclear proteins that remove introns (noncoding sequences in the genes) from precursor mRNA molecules, to form mature RNA, which will be transported to the cytoplasm, site of protein synthesis and activation. In light of such evidences, here we report that hypoxia modulates in vivo SC-35 SF phosphorylation via protein kinase C (PKC) delta in young rat heart. Trichrome Mallory staining and TUNEL analysis along with immunohistochemistry and Western blotting have been performed on left ventricles excised from young and old rats exposed to intermittent hypoxia. Although young hypoxic myocardial cells appear smaller than normoxic cells, connective and endothelial components increase, SC-35 phosphorylation is particularly evident in the endothelium and paralleled by an increased expression of vascular endothelial growth factor (VEGF). In addition, SC-35 and PKC delta coimmunoprecipitation occurs, suggesting that SC-35 phosphorylation could be PKC delta-mediated and that hypoxic young heart needs to counteract the damage through a process of neoangiogenesis involving such SF. Even though the levels of SC-35 and PKC delta are high, the similar response disclosed by normoxic and hypoxic old rat hearts (both showing a fibrotic organization, similar endothelial components and VEGF levels) could be due to the existence of an impaired oxygen sensing mechanism and thus to a low rate of angiogenesis.

Protein kinase C isoforms in chronically hypoxic rat heart

The aim was to determine the expression and subcellular distribution of PKC isoforms δ and ɛ in the left ventricular myocardium of normoxic and chronically hypoxic rats and to examine acute effects of PKC inhibitors. Adult male Wistar rats were either exposed for 5 weeks to intermittent hypobaric hypoxia of 7000 m (8 h/day, 5 days/week) or kept under normoxic conditions. After this period, both hypoxic and normoxic rats were divided into three subgroups: chelerythrine-treated (5 mg/kg), rottlerin-treated (0.3 mg/kg) and untreated controls.

MIFed About Metabolism

AMP (adenosine monophosphate)-activated protein kinase (AMPK), which modulates metabolic pathways in response to changes in cellular energy status, stimulates glucose uptake and glycolysis in the ischemic heart, limiting myocardial injury. Noting that macrophage migration inhibition factor (MIF, a cytokine implicated in the pathogenesis of various inflammatory conditions) stimulates glycolysis and is expressed in cardiomyocytes, Miller et al . investigated its role in cardiac AMPK activation. AMPK activation in hypoxic rat heart muscle was associated with increased MIF release, whereas antibodies directed against MIF inhibited hypoxia-stimulated activation of AMPK and glucose uptake. Ischemia triggered MIF release from isolated mouse heart, and phosphorylation and activation of AMPK in response to ischemia was decreased in mice lacking MIF ( Mif –/– mice), as was stimulation of glucose uptake during ischemia-reperfusion. Hearts from Mif –/– mice showed decreased post-ischemic function compared with those from wild-type mice and larger infarcts after coronary occlusion-reperfusion. Short interfering RNA directed against CD74 (the ligand-binding component of the MIF receptor complex) decreased hypoxia-stimulated AMPK phosphorylation in fibroblasts, and the effects of MIF or hypoxia on AMPK phosphorylation in COS-7/M6 cells depended on expression of CD74 and CD44 (the signal-transducing component). Human fibroblasts with MIF promoter polymorphism that led to decreased expression and secretion showed decreased AMPK phosphorylation during hypoxia, which was restored by exogenous MIF. Thus, the authors conclude that MIF acts as a local factor to activate AMPK during cardiac ischemia, linking inflammation with cardiac metabolism, and suggest that MIF genotyping could help predict risk of ischemic injury in individuals with coronary artery disease. E. J. Miller, J. Li, L. Leng, C. McDonald, T. Atsumi, R. Bucala, L. H. Young, Macrophage migration inhibitory factor stimulates AMP-activated protein kinase in the ischaemic heart. Nature 451 , 578-582 (2008). [PubMed]

Subcellular redistribution of protein kinase c in chronically hypoxic rat heart

Subcellular redistribution of protein kinase c in chronically hypoxic rat heart

Effect of N-acetylcysteine on expression and activity of MnSOD in chronically hypoxic and normoxic rat hearts

Effect of N-acetylcysteine on expression and activity of MnSOD in chronically hypoxic and normoxic rat hearts

Activity of MnSOD in chronically hypoxic rat heart: Effect of N-acetylcysteine

Activity of MnSOD in chronically hypoxic rat heart: Effect of N-acetylcysteine

Expression and subcellular redistribution of PKC isoforms in chronically hypoxic rat heart

Protein kinase C (PKC) is involved in the cardioprotective mechanism of chronic hypoxia (1). The aim of this study was to analyze acute effects of PKC inhibitors on the expression and subcellular localization of PKC isoforms δ and ε in normoxic and chronically hypoxic hearts. Adult male Wistar rats were divided into two groups that were either exposed for 5 weeks to intermittent hypobaric hypoxia of 7000m (8h/day, 5days/week) or kept under normoxic conditions. Both chronically hypoxic and normoxic rats were divided into three subgroups: chelerythrine-treated (5mg/kg), rottlerin-treated (0.3mg/kg) and untreated controls.

Molecular adaptation to acute, chronic and intermittent hypoxia in rat hearts: a study on HIF‐1 and apoptosis

Hypoxia increases apoptosis through signals possibly originating from Hypoxia-Inducible Factor 1 (HIF-1). To investigate this, we exposed rats to hypoxia (10% O2 or less) in either acute (1–24 h), chronic (15 days without reoxygenating the animal for feeding, chamber cleaning etc) or intermittent (as chronic, but with 1 h/day exposure to room air) fashion and determined time course of HIF-1 (immunofluorescence and Western blot) and apoptosis (TUNEL). Results: (1) HIF-1 increases fast within 1 h hypoxia and remains constant for the following 23 h; (2) reoxygenation reduces HIF-1 to baseline levels within 1 h; (3) the next exposure to hypoxia increases HIF-1 as the first exposure; (4) after 15 days of either intermittent and chronic hypoxia, HIF-1 reduces to 10% as that measured under acute hypoxia. Of interest, the number of TUNEL-positive nuclei, or apoptosis: (1) increases slower than HIF-1, as expected; (2) remains sustained even after 15 days of either chronic or intermittent hypoxia; (3) is higher in chronic than intermittent hypoxia despite same HIF-1 level. These observations are useful to assess the molecular mechanisms underlying HIF-1, apoptosis and the oxidative damage associated with intermittent hypoxia.