Training has been shown to induce cardioprotection. The mechanisms involved remain still poorly understood. Aims of the study were to examine the relevance of training intensity on myocardial protection against ischemia/reperfusion (I/R) injury, and to which extent the beneficial effects persist after training cessation in rats. Sprague-Dawley rats trained at either low (60% \( {\dot{{V}}\text{O}}_{2\max } \)) or high (80% \( {\dot{{V}}\text{O}}_{2\max } \)) intensity for 10 weeks. An additional group of highly trained rats was detrained for 4 weeks. Untrained rats served as controls. At the end of treatment, rats of all groups were split into two subgroups. In the former, rats underwent left anterior descending artery (LAD) ligature for 30 min, followed by 90-min reperfusion, with subsequent measurement of the infarct size. In the latter, biopsies were taken to measure heat-shock proteins (HSP) 70/72, vascular endothelial growth factor (VEGF) protein levels, and superoxide dismutase (SOD) activity. Training reduced infarct size proportionally to training intensity. With detraining, infarct size increased compared to highly trained rats, maintaining some cardioprotection with respect to controls. Cardioprotection was proportional to training intensity and related to HSP70/72 upregulation and Mn-SOD activity. The relationship with Mn-SOD was lost with detraining. VEGF protein expression was not affected by either training or detraining. Stress proteins and antioxidant defenses might be involved in the beneficial effects of long-term training as a function of training intensity, while HSP70 may be one of the factors accounting for the partial persistence of myocardial protection against I/R injury in detrained rats.

OBJECTIVE. Solid tumors contain underperfused regions where hypoxia-inducible factor-1 alpha (HIF-1 alpha) over-expression induces hypoxia adaptation and cell proliferation. We test the hypothesis that systemic hypoxia promotes prostate cancer growth in vivo and examine HIF-1 alpha centrality in this effect.METHODS. Male athymic mice were xenografted with 3 x 10(6) LNCaP cells per each flank and exposed for 28 days to either chronic hypoxia (CH, 10% O-2) or CH with reoxygenation (CHReox, 3 times/week for 1 hr), with normoxia as control (n = 17, 9, and 20, respectively). At the end of the observation, mice were euthanized and tumors harvested for analyses.RESULTS. The successful xenografts grew faster in CH and CHReox than in normoxia (first-order rate constants 0.15 +/- 0.01, 0.18 +/- 0.03, and 0.09 +/- 0.01 day(-1), P < 0.05, n = 18, 15, and 25, respectively). Furthermore, the tumor masses at the end were 4.09 +/- 0.58, 3.42 +/- 0.55, and 1.86 +/- 0.25 mg/g bw (P < 0.05), respectively. HIF-1 alpha, assayed by Western blot and immuno-fluorescence, was slightly increased in CH with respect to normoxia, but markedly over-expressed (5-10 times) in CHReox (P < 0.001). The tumor hemoglobin content, higher in CH and CHReox than in normoxia, reflected the higher blood hemoglobin concentration, not neovascularization, as supported by similar expression levels of vascular endothelial growth factor (VEGF) in the three groups. By contrast, protein kinase B (Akt) was more phosphorylated in both hypoxic groups than in normoxia (P < 0.01).CONCLUSION. In vivo systemic hypoxia promotes prostate cancer growth regardless of HIF-1 alpha expression level and neovascularization, suggesting an important role for hypoxia-dependent pathways that do not involve HIF-1 alpha, as the phosphatidyl inositol-3-phosphate signaling cascade. Prostate 70: 1243-1254, 2010. (C) 2010 Wiley-Liss, Inc.

Exposure to hypoxia triggers a variety of adverse effects in the brain that arise from metabolic stress and induce neuron apoptosis. Overexpression of the hypoxia-inducible factor-1α (HIF-1α) is believed to be a major candidate in orchestrating the cell’s defense against stress. To test the impact of HIF-1α on apoptosis during chronic hypoxia in vivo, we examined the protective effect of modulating the nitric oxide (NO)/cGMP pathway by sildenafil, a selective inhibitor of phosphodiesterase-5 (PDE-5). Male ICR/CD-1 mice were divided into 3 groups ( n = 6/group): normoxic (21% O2), hypoxic (9.5% O2), and hypoxic with sildenafil (1.4-mg/kg intraperitoneal injections daily). At the end of the 8-day treatment period, the mice were euthanized and cerebral cortex biopsies were harvested for analyses. We found that sildenafil: (1) did not significantly alter the hypoxia-induced weight loss and hemoglobin increase, but did augment plasma nitrates+nitrites and the tissue content of cGMP and phosphorylated (P) NO synthase III; (2) reversed the hypoxia-induced neuron apoptosis (terminal deoxynucleotidyl transferase positivity and double-staining immunofluorescence, P = 0.009), presumably through increased bcl-2/Bax ( P = 0.0005); and (3) did not affect HIF-1α, but rather blunted the hypoxia-induced increase in P-ERK1/2 ( P = 0.0002) and P-p38 ( P = 0.004). We conclude that upregulating the NO/cGMP pathway by PDE-5 inhibition during hypoxia reduces neuron apoptosis, regardless of HIF-1α, through an interaction involving ERK1/2 and p38.

To improve current knowledge of the molecular mechanisms underlying exercise-induced cardioprotection in a rat model of mild exercise training, Sprague–Dawley rats were trained to run on a treadmill up to 55% of their maximal oxygen uptake for 1 h/day, 3 days/week, 14 weeks, with age-matched sedentary controls (n = 20/group). Rats were sacrificed 48 h after the last training session. Despite lack of cardiac hypertrophy, training decreased blood hemoglobin (7.94 ± 0.21 mM vs. 8.78 ± 0.23 mM, mean ± SE, P = 0.01) and increased both plasma malondialdehyde (0.139 ± 0.005 mM vs. 0.085 ± 0.009 mM, P = 0.05) and the activity of Mn-superoxide dismutase (11.6 ± 0.6 vs. 16.5 ± 1.6 mU/μg, P = 0.01), whereas total superoxide dismutase activity was unaffected. When subjected to 30-min ischemia followed by 90-min reperfusion, hearts from trained rats (n = 5) displayed reduced infarct size as compared to controls (37.26 ± 0.92% vs. 49.09 ± 2.11% of risk area, P = 0.04). The biochemical analyses in the myocardium, which included gene expression profiles, real-time PCR, Western blot and determination of enzymatic activity, showed training-induced upregulation of the following mRNAs and/or proteins: growth-arrest and DNA-damage induced 153 (GADD153/CHOP), heme-oxygenase-1 (HO-1), cyclooxygenase-2 (Cox-2), heat-shock protein 70/72 (HSP70/72), whereas heat-shock protein 60 (HSP60) and glucose-regulated protein 75 (GRP75) were decreased. As a whole, these data indicate that mild exercise training activates a second window of myocardial protection against ischemia/reperfusion by upregulating a number of protective genes, thereby warranting further investigation in man.

To study the in vivo dynamics of hypoxia-inducible factor 1alpha (HIF-1alpha), master regulator of O(2)-dependent gene expression, and mitogen-activated protein kinases (MAPKs) in the hypoxic myocardium, Sprague-Dawley rats (n = 4 to 6 per group) were exposed to 1-hr hypoxia (10% O(2)), 23-hr hypoxia, and 23-hr hypoxia, followed by reoxygenation. HIF-1alpha increased 15-fold after 1-hr hypoxia, remained constant for 23 hrs, and returned to baseline on reoxygenation. Extracellular signal-regulated kinases (ERK1/2) were unchanged throughout. Phosphorylated p38 increased 4-fold after 1-hr hypoxia and returned to baseline within 23-hr hypoxia. The activity of stress-activated protein kinases/c-Jun NH(2)-terminal kinases (JNKs), measured as phosphorylated c-Jun, increased 3-fold after 1-hr hypoxia and remained sustained afterward. Furthermore, HIF-1alpha was halved in rats that were administered with the p38 inhibitor SB202190 and made hypoxic for 1 hr. In conclusion, although very sensitive to the reoxygenation, HIF-1alpha is overexpressed in vivo in the hypoxic myocardium, and its acute induction by hypoxia is correlated with that of p38.

We studied the in vivo persistence of hypoxia-inducible factor-1alpha (HIF-1alpha), main transducer of hypoxia, the differential response in organs exposed to the same degree of hypoxemia and the relationship with apoptosis. We measured HIF-1alpha (immunohistochemistry peroxidase and Western blot) and apoptosis (TUNEL) in heart, liver, kidney, gastrocnemius, and brain of rats exposed to chronic normobaric hypoxia (10% O2) or normoxia (21% O2) for 2 weeks. Despite same arterial O2 pressure and increased hemoglobin concentration (219 +/- 5 vs. 124 +/- 4 g/L), the organs responded differently. While marked in brain, muscle, and kidney cortex, HIF-1alpha was undetectable in heart and liver. In kidney medulla, HIF-1alpha was high in both normoxia and hypoxia. By contrast, apoptosis was marked in heart, slight in kidney medulla, and undetectable in other organs. We conclude that the HIF-1alpha response to chronic hypoxia can be a sustained phenomenon, but not in all organs, and that apoptosis responds differently from HIF-1alpha.

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.

Although exposure to broad band (0.2–20 MHz) electromagnetic fields (EMF) is part of the treatment of several diseases, little is known as to their effects on myocardial protein expression and resistance to ischemia-reperfusion (I/R). We exposed Sprague-Dawley rats to either high (H, 10 min/day at 200 V/m, 36.1 μT) or low (L, 2 min/day at 30 V/m, 11.4 μT) intensity broad band EMF for 15 days. At the end of the treatment, myocardial HSP70 was 32 ± 8% (mean ± SEM) higher in L (P = 0.01) than in control (C), whereas in H it remained the same as in C. Electron microscopy revealed sporadic ruptures of mitochondrial cristae in H hearts, with no differences in other parameters. Malondialdehyde was increased in treated hearts (P < 0.05), but especially in H (P = 0.008). To assess the protective role of HSP70 during I/R, hearts were Langendorff-perfused with Krebs-Henseleit. After I/R, C hearts displayed depressed rate · pressure (−13 ± 7%) and increased end-diastolic (+9.2 ± 2.8 mmHg) and perfusion pressures (+30 ± 10 mmHg). In H and L, rate · pressure recovery was similar to C (−2 ± 21% and −12 ± 16%, respectively, P = NS). In contrast, both end-diastolic and perfusion pressures were higher in L than in H (30.8 ± 5.4 vs 18.2 ± 3.5, P = 0.01, and 54 ± 8 vs 21 ± 8 mmHg, P = 0.01, respectively) indicating diastolic derangement in L. In conclusion, the effects of broad band EMF on HSP70 appear to be biphasic, and HSP70 overexpression might not be directly related to improved protection against I/R.

Cobalamin-deficient (Cbl-D) central neuropathy is a pure myelinolytic disease, in which gliosis is also observed. Iron is abundant in the mammalian central nervous system, where it is required for various essential functions including myelinogenesis. It is predominantly located in the white matter and oligodendrocytes, which also actively synthesize the major iron proteins (e.g., ferritin, transferrin). We investigated the expression of the main proteins of iron metabolism in the spinal cord (SC) of totally gastrectomized Cbl-D rats 2 months after surgery (i.e., when the Cbl-D status has become severe). There were no significant changes in iron content, the activity of iron regulatory proteins, or the expression of transferrin or its receptor in the SC. We observed a significant decrease in the levels of both H and L ferritin subunits, with a more marked reduction in the latter. Postoperative cobalamin replacement therapy normalized only the H-ferritin subunits, and only in the SC. Our results therefore suggest that permanent cobalamin deficiency affects iron metabolism in the rat SC preferentially from a functional point of view, because H-ferritin is known to be involved in the uptake and release of iron. (C) 2002 Wiley-Liss, Inc.

Posttranscriptional regulation of iron homeostasis involves, among other factors, a reversible conversion of the Fe-S enzyme cytoplasmic aconitase to a mRNA-binding iron regulatory protein (IRP-1) that lacks an Fe-S cluster. Previous studies have shown that aconitase/IRP-1 may be a target of *NO or peroxynitrite (ONOO(-)), formed after reaction of *NO with superoxide anion (O(2)(*-)); however, the mechanisms and consequences of such interactions have remained uncertain. In this study, recombinant aconitase/IRP-1 was exposed to SIN-1, whose thermal decomposition releases *NO and O(2)(*-). Results showed that SIN-1 was able to induce concomitant inactivation of aconitase and activation of IRP-1, attributable to cluster disassembly induced by ONOO(-). SIN-1 was used also in lysates of J774A.1 mouse macrophages grown under control conditions, or subjected to iron loading or starvation by treatment with hemin or desferrioxamine, respectively. Three lines of evidence confirmed that ONOO(-) activated IRP-1 by removing iron from the Fe-S cluster of cytoplasmic aconitase. First, IRP-1 activation was accompanied by iron release and loss of aconitase activity. Second, aconitase activity was recovered by reassembling Fe-S clusters with cysteine and ferrous ammonium sulfate. Third, iron release and IRP-1 activation were observed in lysates from control or iron-loaded macrophages, containing increasing levels of Fe-S clusters, but not in lysates from iron-starved macrophages, in which aconitase had already undergone cluster disassembly and switched to IRP-1. *NO was less efficient than ONOO(-) in attacking the Fe-S cluster of cytoplasmic aconitase; in fact, SIN-1-dependent iron release and IRP-1 activation were diminished by superoxide dismutase, which scavenged O(2)(*-) before it reacted with *NO to form ONOO(-). Under comparable conditions, however, both *NO and ONOO(-) inactivated an IRP-2 unable to assemble an Fe-S cluster. These results indicate that *NO and ONOO(-) may activate IRP-1 by attacking the Fe-S cluster of cytoplasmic aconitase, while also inactivating the cluster-deficient IRP-2. Such divergent actions offer clues to explain links between iron homeostasis and reactive nitrogen species in macrophages involved in inflammation or other pathophysiologic conditions.

Changes in iron homeostasis have been implicated in cardiotoxicity induced by the anticancer anthracycline doxorubicin (DOX). Certain products of DOX metabolism, like the secondary alcohol doxorubicinol (DOXol) or reactive oxygen species (ROS), may contribute to cardiotoxicity by inactivating iron regulatory proteins (IRP) that modulate the fate of mRNAs for transferrin receptor and ferritin. It is important to know whether DOXol and ROS act by independent or combined mechanisms. Therefore, we monitored IRP activities in H9c2 rat embryo cardiomyocytes exposed to DOX or to analogues which were selected to achieve a higher formation of secondary alcohol metabolite (daunorubicin), a concomitant increase of alcohol metabolite and decrease of ROS (5-iminodaunorubicin), or a defective conversion to alcohol metabolite (mitoxantrone). On the basis of such multiple comparisons, we characterized that DOXol was able to remove iron from the catalytic Fe-S cluster of cytoplasmic aconitase, making this enzyme switch to the cluster-free IRP-1. ROS were not involved in this step, but they converted the IRP-1 produced by DOXol into a null protein which did not bind to mRNA, nor was it able to switch back to aconitase. DOX was also shown to inactivate IRP-2, which does not assemble or disassemble a Fe-S cluster. Comparisons between DOX and the analogues revealed that IRP-2 was inactivated only by ROS. Thus, DOX can inactivate both IRP through a sequential action of DOXol and ROS on IRP-1 or an independent action of ROS on IRP-2. This information serves guidelines for designing anthracyclines that spare iron homeostasis and induce less severe cardiotoxicity.