Altered Oxygen Utilisation in Rat Left Ventricle and Soleus after 14 Days,but Not 2 Days,of Environmental Hypoxia

The effects of environmental hypoxia on cardiac and skeletal muscle metabolism are dependent on the duration and severity of hypoxic exposure, though factors which dictate the nature of the metabolic response to hypoxia are poorly understood. We therefore set out to investigate the time-dependence of metabolic acclimatisation to hypoxia in rat cardiac and skeletal muscle. Rats were housed under normoxic conditions, or exposed to short-term (2 d) or sustained (14 d) hypoxia (10% O₂), after which samples were obtained from the left ventricle of the heart and the soleus for assessment of metabolic regulation and mitochondrial function. Mass-corrected maximal oxidative phosphorylation was 20% lower in the left ventricle following sustained but not short-term hypoxia, though no change was observed in the soleus. After sustained hypoxia, the ratio of octanoyl carnitine- to pyruvate- supported respiration was 11% and 12% lower in the left ventricle and soleus, respectively, whilst hexokinase activity increased by 33% and 2.1-fold in these tissues. mRNA levels of PPARα targets fell after sustained hypoxia in both tissues, but those of PPARα remained unchanged. Despite decreased Ucp3 expression after short-term hypoxia, UCP3 protein levels and mitochondrial coupling remained unchanged. Protein carbonylation was 40% higher after short-term but not sustained hypoxic exposure in the left ventricle, but was unchanged in the soleus at both timepoints. Our findings therefore demonstrate that 14 days, but not 2 days, of hypoxia induces a loss of oxidative capacity in the left ventricle but not the soleus, and a substrate switch away from fatty acid oxidation in both tissues.     

Antihypoxic effects of quinones connected with restoration of electro-transport and function of the respiratory chain of the isolated rat heart

The effect of hypoxia of middle severity (H50) has been investigated on the contractile activity, oxidative metabolism and bioenergetic function of the myocardium in the isolated rat heart. It has been shown that differences in the functional-metabolic parameters sensitivity in the resistant and non-resistant to hypoxia animals are defined by the primary utilization of different pathways of substrates oxidation--the succinate-oxidative pathway of oxidation or the NADH-oxidative pathway, correspondingly. It is possible to correct the early hypoxic injuries of the electron-transfer function of the myocardium respiratory chain at the NADH-CoQ region (the first enzyme complex) with the help of quinones (vitamin K3, hydroquinone). The mechanism of this correction depends on the donor-acceptor properties of these compounds and it is the same both in the myocardium of the isolated heart and in the isolated mitochondria. Vitamin K3 is recommended to be utilized on the early stages of hypoxic injuries in myocardium as an antihypoxant of the energized type.     

Modular control analysis of effects of chronic hypoxia on mouse heart

Modular control analysis (MoCA; Diolez P, Deschodt-Arsac V, Raffard G, Simon C, Santos PD, Thiaudiere E, Arsac L, Franconi JM. Am J Physiol Regul Integr Comp Physiol 293: R13-R19, 2007) was applied here on perfused hearts to describe the modifications of the regulation of heart energetics induced in mice exposed to 3-wk chronic hypoxia. MoCA combines 31P-NMR spectroscopy and modular (top down) control analysis to describe the integrative regulation of energy metabolism in the intact beating heart, on the basis of two modules [ATP/phosphocreatine (PCr) production and ATP/PCr consumption] connected by the energetic intermediates. In contrast with previous results in rat heart, in which all control of contraction was on ATP demand, mouse heart energetics presented a shared control of contraction between ATP/PCr-producing and -consuming modules. In chronic hypoxic mice, the decrease in heart contractile activity and PCr-to-ATP ratio was surprisingly associated with an important and significant higher response of ATP/PCr production (elasticity) to PCr changes compared with control hearts (-10.4 vs. -2.46). By contrast, no changes were observed in ATP/PCr consumption since comparable elasticities were observed. Since elasticities determine the regulation of energetics of heart contraction, the present results show that this new parameter may be used to uncover the origin of the observed dysfunctions under chronic hypoxia conditions. Considering the decrease in mitochondrial content reported after exposure to chronic hypoxia, it appears that the improvement of ATP/PCr production response to ATP demand may be viewed as a positive adaptative mechanism. It now appears crucial to understand the very processes responsible for ATP/PCr producer elasticity toward the energetic intermediates, as well as their regulation.     

Skeletal muscle HIF-1alpha expression is dependent on muscle fiber type

Oxygen homeostasis is an essential regulation system for cell energy production and survival. The oxygen-sensitive subunit alpha of the hypoxia inducible factor-1 (HIF-1) complex is a key protein of this system. In this work, we analyzed mouse and rat HIF-1alpha protein and mRNA expression in parallel to energetic metabolism variations within skeletal muscle. Two physiological situations were studied using HIF-1alpha-specific Western blotting and semiquantitative RT-PCR. First, we compared HIF-1alpha expression between the predominantly oxidative soleus muscle and three predominantly glycolytic muscles. Second, HIF-1alpha expression was assessed in an energy metabolism switch model that was based on muscle disuse. These two in vivo situations were compared with the in vitro HIF-1alpha induction by CoCl(2) treatment on C(2)C(12) mouse muscle cells. HIF-1alpha mRNA and protein levels were found to be constitutively higher in the more glycolytic muscles compared with the more oxidative muscles. Our results gave rise to the hypothesis that the oxygen homeostasis regulation system depends on the fiber type.     

Electron microscopy and microcalorimetry of the postnatal rat heart (Rattus norvegicus)

The interplay of ultrastructure and tissue metabolism was examined in neonatal, infant and adult rat hearts by electron microscopy and microcalorimetry. Morphometry was used to determine parameters of oxygen diffusion capacity (distance between capillaries and mitochondria, capillary surface density) and oxidative metabolic capacity (mitochondrial volume fraction). Thin slices and large samples of living tissue were examined calorimetrically to quantify aerobic metabolism and ischemia tolerance, respectively. After birth, rat hearts grow in parallel to body mass and show characteristics of cellular hypertrophy. Capillary surface density increases from neonatal to infant rats, and decreases to an intermediate value in adult rats. The distance between capillaries and mitochondria shows no significant changes throughout postnatal development. Mitochondrial volume fraction increases continuously until adulthood. The specific aerobic tissue metabolic rate is higher in the neonatal than in the infant and adult rat. However, the ischemic decline in metabolic rate is much slower in the neonatal rat, reflecting an elevated hypoxia tolerance. In conclusion, the neonatal rat heart exhibits a high metabolic rate despite a low mitochondrial volume fraction. The subsequent structural rearrangements can be interpreted as long-term adaptations to the increased postnatal workload and may contribute to the progressive loss of hypoxia tolerance.     

S-thiolation of cytoplasmic cardiac creatine kinase in heart cells treated with diamide

Two methods for quantitation of protein S-thiolation, by isoelectric focusing or by enzyme activity, were used for studying S-thiolation of cytoplasmic cardiac creatine kinase. With these methods, creatine kinase was identified as a major S-thiolated protein in both bovine and rat heart. In rat heart cell cultures, creatine kinase became 10% S-thiolated during a 10 min incubation with 0.2 mM diamide. This enzyme became S-thiolated more slowly than other heart cell proteins and it also dethiolated more slowly. Two sequential additions of diamide at a 25 min interval caused twice as much S-thiolation after the second addition as compared to the first. This increased sensitivity to the second diamide treatment may have resulted from glutathione loss during the first addition which produced a higher GSSG-to-GSH ratio after the second treatment. The GSSG-to-GSH ratio was highest prior to the maximum S-thiolation of creatine kinase, but, in general, the time course of glutathione was similar to the S-thiolation of creatine kinase. This study demonstrates that cytoplasmic creatine kinase is S-thiolated and, therefore, inhibited during a diamide-induced oxidative stress in heart cells. Implications for regulation of cardiac metabolism during oxidative stress are discussed.     

Hypoxia induces mitochondrial DNA damage and stimulates expression of a DNA repair enzyme, the Escherichia coli MutY DNA glycosylase homolog (MYH), in vivo, in the rat brain

Hypoxia-associated, acutely reduced blood oxygenation can compromise energy metabolism, alter oxidant/antioxidant balance and damage cellular components, including DNA. We show in vivo, in the rat brain that respiratory hypoxia leads to formation of the oxidative DNA lesion, 8-hydroxy-2'-deoxyguanosine (oh8dG), a biomarker for oxidative DNA damage and to increased expression of a DNA repair enzyme involved in protection of the genome from the mutagenic consequences of oh8dG. The enzyme is a homolog of the Escherichia coli MutY DNA glycosylase (MYH), which excises adenine residues misincorporated opposite the oxidized base, oh8dG. We have cloned a full-length rat MYH (rMYH) cDNA, which encodes 516 amino acids, and by in situ hybridization analysis obtained expression patterns of rMYH mRNA in hippocampal, cortical and cerebellar regions. Ensuing hypoxia, mitochondrial DNA damage was induced and rMYH expression strongly elevated. This is the first evidence for a regulated expression of a DNA repair enzyme in the context of respiratory hypoxia. Our findings support the premise that oxidative DNA damage is repaired in neurons and the possibility that the hypoxia-induced expression of a DNA repair enzyme in the brain represents an adaptive mechanism for protection of neuronal DNA from injurious consequences of disrupted energy metabolism and oxidant/antioxidant homeostasis.     

Oxygen recovery up-regulates avian UCP and ANT in newly hatched ducklings

At hatching, breaking eggshell induces a surge in oxygen availability that is likely to generate oxidative stress in newborn chicks. To investigate the involvement of potential adaptive antioxidant mechanisms, we explored some markers of oxidative stress and the regulation of muscle avian uncoupling protein (avUCP) and adenine nucleotide translocase (ANT) in ducklings in the peri-hatching period. When compared with pre-hatching levels, the amount of peroxidized lipids were increased 24 h after external pipping in gastrocnemius muscle (+37%) and heart (+39%) as well as the muscle avUCP mRNA expression (+60%) but the susceptibility of red blood cells to free radicals (a functional test of oxidative status) was not affected. In order to relate these changes to the oxidative transition of hatching, an imposed hypoxia/re-oxygenation protocol was used. Hatched chicks that had spent the last 24 h of incubation in artificial severe hypoxia showed a rise in muscle (+50%) and heart (+69%) lipid peroxidation, an increased susceptibility of red blood cells to free radicals, a marked over-expression of avUCP mRNA (+105%) and a rise in mitochondrial ANT content (+54%). These results suggest that avian UCP and ANT may contribute to prepare incubating eggs to the oxidative stress generated by the hypoxia/re-oxygenation transition naturally occurring at hatching.     

Stanniocalcin-1 is induced by hypoxia inducible factor in rat alveolar epithelial cells

Alveolar type II (ATII) cells remain differentiated and express surfactant proteins when cultured at an air–liquid (A/L) interface. When cultured under submerged conditions, ATII cells dedifferentiate and change their gene expression profile. We have previously shown that gene expression under submerged conditions is regulated by hypoxia inducible factor (HIF) signaling due to focal hypoxia resulting from ATII cell metabolism. Herein, we sought to further define gene expression changes in ATII cells cultured under submerged conditions. We performed a genome wide microarray on RNA extracted from rat ATII cells cultured under submerged conditions for 24–48 h after switching from an A/L interface. We found significant alterations in gene expression, including upregulation of the HIF target genes stanniocalcin-1 (STC1), tyrosine hydroxylase (Th), enolase (Eno) 2, and matrix metalloproteinase (MMP) 13, and we verified upregulation of these genes by RT-PCR. Because STC1, a highly evolutionarily conserved glycoprotein with anti-inflammatory, anti-apoptotic, anti-oxidant, and wound healing properties, is widely expressed in the lung, we further explored the potential functions of STC1 in the alveolar epithelium. We found that STC1 was induced by hypoxia and HIF in rat ATII cells, and this induction occurred rapidly and reversibly. We also showed that recombinant human STC1 (rhSTC1) enhanced cell motility with extended lamellipodia formation in alveolar epithelial cell (AEC) monolayers but did not inhibit the oxidative damage induced by LPS. We also confirmed that STC1 was upregulated by hypoxia and HIF in human lung epithelial cells. In this study, we have found that several HIF target genes including STC1 are upregulated in AECs by a submerged condition, that STC1 is regulated by hypoxia and HIF, that this regulation is rapidly and reversibly, and that STC1 enhances wound healing moderately in AEC monolayers. However, STC1 did not inhibit oxidative damage in rat AECs stimulated by LPS in vitro. Therefore, alterations in gene expression by ATII cells under submerged conditions including STC1 were largely induced by hypoxia and HIF, which may be relevant to our understanding of the pathogenesis of various lung diseases in which the alveolar epithelium is exposed to relative hypoxia.     

Leptin protects the cardiac myocyte cultures from hypoxic damage

Leptin, a circulating hormone mainly produced by adipose tissue, regulates fatty acid metabolism and causes multiple systemic biological actions even the regulation of cardiovascular function. It is previously known that leptin is a hypoxia-inducible hormone, that hypoxic conditions increase the expression of this peptide in various tissues such as placenta, pancreas and also in the heart. Since leptin receptors are present in the heart, we hypothesized that whether leptin was a protector response for tissues especially for the heart against the deleterious effects of hypoxia. Cultured cardiomyocytes from newborn rats were initially treated with 3000 ng/ml leptin incubation for 1, 5 and 20 h separately, then subjected to 120 min of hypoxia. Hypoxic damage of myocytes was assayed using the measurements of both lactate dehydrogenase and creatine kinase releases into the medium and performing morphological observations (ultrastructural and immunocytochemical) of plates. The obtained results from leptin treated and non-treated control groups were compared to each other, and these data have demonstrated that 5 h of leptin treatment before hypoxia provides a significant protection for cardiomyocytes against hypoxia. Neither 1- nor 20-h leptin treated groups exhibited sufficient protection against hypoxia. In conclusion, leptin protects the cardiomyocyte cultures from hypoxia, but this effect is selective and evident only in the 5-h treated myocytes.     

The AMP-Activated Protein Kinase Is Involved in the Regulation of Ketone Body Production by Astrocytes

The possible role of the AMP-activated protein kinase (AMPK), a highly conserved stress-activated kinase, in the regulation of ketone body production by astrocytes was studied. AMPK activity in rat cortical astrocytes was three times higher than in rat cortical neurons. AMPK in astrocytes was shown to be functionally active. Thus, incubation of astrocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a cell-permeable activator of AMPK, stimulated both ketogenesis from palmitate and carnitine palmitoyltransferase I. This was concomitant to a decrease of intracellular malonyl-CoA levels and an inhibition of acetyl-CoA carboxylase/fatty acid synthesis and 3-hydroxy-3-methylglutaryl-CoA reductase/cholesterol synthesis. Moreover, in microdialysis experiments AICAR was shown to stimulate brain ketogenesis markedly. The effect of chemical hypoxia on AMPK and the ketogenic pathway was studied subsequently. Incubation of astrocytes with azide led to a remarkable drop of fatty acid beta-oxidation. However, activation of AMPK during hypoxia compensated the depression of beta-oxidation, thereby sustaining ketone body production. This effect seemed to rely on the cascade hypoxia --> increase of the AMP/ATP ratio --> AMPK stimulation --> acetyl-CoA carboxylase inhibition --> decrease of malonyl-CoA concentration --> carnitine palmitoyltransferase I deinhibition --> enhanced ketogenesis. Furthermore, incubation of neurons with azide blunted lactate oxidation, but not 3-hydroxybutyrate oxidation. Results show that (a) AMPK plays an active role in the regulation of ketone body production by astrocytes, and (b) ketone bodies produced by astrocytes during hypoxia might be a substrate for neuronal oxidative metabolism.     

Changes in enzyme activities in tissues of rats exposed to hypoxia (Short Communication)

Rats were exposed to various degrees of hypoxia and enzyme activities in their tissues were determined. In general, oxidative metabolism was not increased in response to hypoxia, nor was anaerobic metabolism. Physiological and anatomical changes were concluded to be more important than changes in cellular enzyme activities in the overall adaptation to acute hypoxia.     

Activity of neutral peptide hydrolases and oxidative enzymes in the myocardium and blood during hypoxic hypoxia

The citric cycle dehydrogenases and neutral peptide hydrolases (NPH) activity was determined in the myocardium and blood serum of Wistar rats under acute and prolonged hypoxic hypoxia. It was shown that the NPH activity and the activity of the most oxidative enzymes change in the same direction during adaptation to the high-altitude conditions. The role of the NPH in the oxidative metabolism reconstruction under acute and chronic hypoxia was discussed.     

Effects of the ionophore grisorixin on myocardial function and metabolism in isolated perfused working rat heart under normoxic and hypoxic conditions

The effects of grisorixin, a monocarboxylic ionophore, were studied on isolated working rat hearts perfused with a suspension of washed pig erythrocytes (10% hematocrit). Grisorixin (2.5 microM) induced a transient stimulation of heart work, maximal at 5 min, expressed by an increase in heart rate (+21%) and aortic flow (+17%) and by an increase in coronary flow, maximal at 10 min (+47%). Concomitantly, myocardial Vo2 was slightly enhanced and the myocardial creatine phosphate level dropped (2 min). The lactate production increased by 82% (5 min) then dropped to the control value (10 min) and increased again till the 45th min (+211%), indicating a cardiac metabolic drift towards anaerobic glycolysis due to partial inhibition of the oxidative metabolism. Owing to its properties as an ionophore, grisorixin also induced a strong and rapid increase of potassium concentration in the perfusate and a decrease of sodium. Grisorixin was tested on hearts submitted to 20 min of hypoxic conditions. The hypoxia was rather mild and induced only very slight modifications of the ultrastructure. In the control series, heart rate and aortic flow decreased regularly while coronary flow and lactate production increased. Upon reoxygenation, the heart performances were rapidly restored. Grisorixin was administered according to four different protocols. When injected at the onset of hypoxia or 5 min later, it was able to maintain the aortic flow during the first minutes and induce a higher coronary dilation. These beneficial effects were short-lasting and no deleterious effects were found on the ultrastructure of hearts subjected to grisorixin whether after hypoxia or after reoxygenation.     

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.     

Some mechanisms of antihypoxic action of taurine]

It is shown that preliminary taurine treatment prevents the disturbances of energy metabolism in the brain, heart and liver tissues of Wistar rats with acute hypoxic hypoxia. Administration of taurine restored to normal the parameters of adenine pool: the concentration of ATP increased within the cytoplasm, while that of ADP and AMP diminished; mitochondrial respiration proceeded more rapidly; the concentrations of pyruvate and malate decreased; isocitrate dehydrogenase activity, P/O and NAD/NADH ratios increased. Taurine treatment resulted in a decreased level of lipid peroxides in the rat tissues with hypoxia. The role of intracellular calcium content and biomembranes structure changes as the mechanisms of taurine action on energy metabolism and lipid peroxidation is discussed.     

Hypobaric hypoxia regulates iron metabolism in rats

Intermittent hypobaric hypoxia can produce a protective effect on both the nervous system and non-nervous system tissues. Intermittent hypobaric hypoxia preconditioning has been shown to protect rats from cardiac ischemia-reperfusion injury by decreasing cardiac iron levels and reactive oxygen species (ROS) production, thereby decreasing oxidative stress to achieve protection. However, the specific mechanism underlying the protective effect of hypobaric hypoxia is unclear. To shed light on this phenomenon, we subjected Sprague-Dawley rats to hypobaric hypoxic preconditioning (8 hours/day). The treatment was continued for 4 weeks. We then measured the iron content in the heart, liver, spleen, and kidney. The iron levels in all of the assessed tissues decreased significantly after hypobaric hypoxia treatment, corroborating previous results that hypobaric hypoxia may produce its protective effect by decreasing ROS production by limiting the levels of catalytic iron in the tissue. We next assessed the expression levels of several proteins involved in iron metabolism (transferrin receptor, L-ferritin, and ferroportin1 [FPN1]). The increased transferrin receptor and decreased L-ferritin levels after hypobaric hypoxia were indicative of a low-iron phenotype, while FPN1 levels remained unchanged. We also examined hepcidin, transmembrane serine proteases 6 (TMPRSS6), erythroferrone (ERFE), and erythropoietin (EPO) levels, all of which play a role in the regulation of systemic iron metabolism. The expression of hepcidin decreased significantly after hypobaric hypoxia treatment, whereas the expression of TMPRSS6 and ERFE and EPO increased sharply. Finally, we measured serum iron and total iron binding capacity in the serum, as well as red blood cell count, mean corpuscular volume, hematocrit, red blood cell distribution width SD, and red blood cell distribution width CV. As expected, all of these values increased after the hypobaric hypoxia treatment. Taken together, our results show that hypobaric hypoxia can stimulate erythropoiesis, which systemically draws iron away from nonhematopoietic tissue through decreased hepcidin levels.