Among the mechanisms of action of hyperbaric oxygenation (HBO), the chance of reducing injury by interfering with the mechanisms of redox homeostasis in the heart leads to the possibility of extending the period of viability of the myocardium at risk. This would benefit late interventions for reperfusion to the ischemic area. The objective of the present study was to investigate the changes in the redox system associated with HBO therapy maintained during the first hour after coronary occlusion in an acute myocardial infarction (MI) rat model. Surviving male rats (n=105) were randomly assigned to one of three groups: Sham (SH=26), myocardial infarction (MI=45) and infarction+hyperbaric therapy (HBO=34, 1 h at 2.5 atm). After 90 min of coronary occlusion, a sample of the heart was collected for western blot analysis of total protein levels of superoxide dismutase, catalase, peroxiredoxin and 3-nitrotyrosine. Glutathione was measured by enzyme-linked immunosorbent assay (ELISA). The detection of the superoxide radical anion was carried out by oxidation of dihydroethidium analyzed with confocal microscopy. The mortality rate of the MI group was significantly higher than that of the HBO group. No difference was noted in the myocardial infarction size. The oxidized/reduced glutathione ratio and peroxiredoxin were significantly higher in the SH and MI when compared to the HBO group. Superoxide dismutase enzymes and catalase were significantly higher in the HBO group compared to the MI and SH groups. 3-Nitrotyrosine and the superoxide radical were significantly lower in the HBO group compared to these in the MI and SH groups. These data demonstrated that hyperbaric oxygenation therapy decreased mortality by improving redox control in the hearts of rats in the acute phase of myocardial infarction.
Advances in the treatment of patients with acute myocardial infarction, through coronary reperfusion, have led to a decrease in hospital mortality rates. However, the injury generated by the reestablishment of blood flow, known as reperfusion injury (RI), still represents a great challenge (
As a way to increase myocardial tolerance to ischemia, approaches such as pre-conditioning and post-conditioning have been proposed; among these are oxidative stress: Hypoxic, ischemic and hyperoxic (
Paradoxically, the increase in O2, offered by HBO, can intensify oxidative stress due to ROS elevation (
In our previous study in a rat model, we reported that application of HBO, after induction of acute myocardial infarction (MI), promoted a decrease in the size of the infarct and an increase in rat survival rate (
In humans, Yogaratnam
The objective of the present study was to investigate the changes in the redox system associated with HBO therapy maintained during the first hour after coronary occlusion in an MI rat model. We analyzed the influence of HBO at the end of the first hour after coronary occlusion, considering that, in the rat, this period is sufficient to cause necrosis of the entire risk area (
Male Wistar rats weighing 250–330 g (11–12 weeks of age) from the Central Animal Facilities of our institution were used. The animals were housed under a 12-h light/dark cycle, at 22–23°C and 54–55% humidity. Rats were fed a pellet rodent diet (Nuvilab CR1, manufactured by Nuvital, Curitiba, Brazil),
All procedures were performed according to the principles of ethics of animal experimentation adopted by the Brazilian College of Animal Experimentation (COBEA-
The rats were anesthetized with halothane, intubated and ventilated with a ventilator for rodents (model 683; Harvard Apparatus Co., South Natik, MA, USA). The rats were randomized between the Sham (SH), myocardial infarction (MI) and hyperbaric therapy (HBO) groups. MI was produced in the animals of the MI and HBO groups by ligation of the anterior descending coronary artery, as described previously (
All experiments were initiated at the same time (13:00) in order to avoid influence of the different biological rhythms.
Immediately after recovery from anesthesia (approximately 2 min), animals from the HBO group were exposed to 100% O2 under a pressure of 2.5 atmospheres absolute (ATA) for 60 min. within a hyperbaric chamber for small rodents, as previously described (
Animals in the SH and MI groups were maintained under oxygenation at ambient pressure for the same time as the treated group. At the end of the protocol, the surviving rats (SH=26, MI=45, and HBO=34). were sacrificed under halothane anesthesia and each heart was removed and sectioned transversely at the middle of the left ventricle (LV). The basal half was used to measure the infarct size, with triphenyltetrazolium (TTZ), according to a previously described protocol (
Proteins were extracted as previously described (
Samples were homogenized in 100 mM phosphate buffer (pH 7.0; 250 µl/10 mg tissue) and centrifuged (12,000 × g for 10 min, at 4°C). To the supernatant (200 µl), 5% of sulphosalicylic acid (200 µl) was added to precipitate the proteins. Detect X® kit (Arbor Assays, Ann Arbor, MI, USA) was then utilized to quantify the total concentration of glutathione (GSH). After incubating the mixture with ThioStar® (Detect X, Arbor Assays) for 15 min, at room temperature, the fluorescence was determined (excitation 390 nm, emission 510 nm), using a spectrophotometer (U-2810; Hitachi), and the reduced GSH concentration measured.
Subsequently, a reaction mixture was added to convert all oxidized glutathione (GSSG) into free GSH, which then reacted with the excess ThioStar® to yield the signal related to the total GSH content. A standard curve was used to calculate the total and reduced glutathione concentrations. The oxidized glutathione concentration was obtained using the following calculation: GSSG=(total GSH-reduced GSH)/2. Detection limits ranged between 38 nM, for free GSH, and 42 nM, for the total GSH. Results were normalized to the muscle fragment weight for between-animal comparisons. All assays were performed in triplicate.
Myocardial homogenates were prepared under liquid N2. After centrifugation (13,400 × g for 20 min, at 4°C), 20 µl aliquots were injected into NOA (Nitric Oxide Analyzer model 280; Sievers Instruments, USA), with VCl3 and HCl (at 95°C) used as reductants, as previously described (
The data are expressed as mean ± SEM. The Student t-test was used for comparisons of infarct sizes and the Chi-square test was used to compare mortality. Two-way ANOVA was applied to parametric data using Newman-Keuls to identify statistical differences. Kruskal-Wallis was performed on non-parametric data, associated with the Dunn's test to identify statistical differences. The statistical program used was GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA). Differences with P≤0.05 were considered significant.
Immediate mortality was established when it occurred in the period between the introduction of the animal into the HBO chamber until the end of the decompression (~90 min). In the MI group, 27 animals died during this period, whereas HBO therapy was effective in improving post-infarction survival, with only 6 immediate deaths noted.
In order to investigate whether redox processes are involved in the protective effect induced by HBO therapy, we first addressed the redox status in LV homogenates, by measuring the amount of reduced and oxidized glutathione. The infarcted groups showed (
The higher oxidative environment promoted by MI may be caused by increased oxidant generation or by reduced antioxidant defense. First, we assessed the potential to generate ROS by addressing the total amount of DHE-oxidation by-products by microfluorotopography on slides of the myocardium in the SH group (
Due to the higher levels of superoxide promoted by MI, combined with the protective effect induced by HBO, the antioxidant capacity was investigated by assessing the expression of SOD, whose values showed that HBO therapy significantly increased enzyme expression in the HBO group (1.0±0.06 AU/µg) relative to the MI (0.79±0.04 AU/µg) and SH (0.69±0.08 AU/µg) groups, with no significant difference between the SH and MI groups (
Nitric oxide and its oxidation-derived by-products show a dual role during normal and pathological conditions, including MI (
In the present study, the mortality rate of the animals in the hyperbaric oxygenation (HBO) group was significantly lower than that of the animals in the myocardial infarction (MI) group, indicating a favorable action of hyperbaric oxygenation in survival. This result is in accordance with a recent review of the literature published by Bennett
Our results indicate that therapy with 2.5 ATA of HBO resulted in similar mean infarct sizes in the MI and HBO group. In a previous study by our group (
The lower level found in the GSH/GSSG ratio in the HBO group suggests an increase in glutathione (GSH) use, indicating a higher efficiency of the antioxidant mechanisms to protect against oxidative stress present in these animals. The same response was not observed in the untreated animals of the MI group. The higher glutathione disulfide (GSSG) formation indicates the increased levels of reactive oxygen species (ROS) formation induced by MI.
Importantly, the improved buffering capacity of antioxidants promoted by HBO therapy is supported by the higher amount of superoxide dismutase (SOD) and catalase (CAT), which resulted in reduced levels of oxidant generation and their post-translational modifications, such as ROS and 3-nitrotyrosine, respectively. Moreover, the lower levels of Prx-SO2/3 strengthened such a protective effect. Collectively, the pro-survival effect of HBO, may be promoted by a hormetic-like effect (
The lower levels of ROS indicated by DHE oxidation may have resulted from increased SOD and CAT expression. Although the mechanisms by which HBO therapy induces an antioxidant response remain unclear, it has been reported to involve a Nrf2-dependent effect (
The transient sulfinic acid peroxiredoxin (Prx) intermediate has been attributed to control redox signaling. Such reversibility, which is consistent with signaling events, is lost by its hyperoxidation to sulfinic and sulfonic irreversible forms (
Regarding reactive nitrogen species, we observed lower values of 3-nitrotyrosine in the animals of the HBO group in relation to the SH and MI groups, indicating lower nitration of proteins by peroxynitrite resulting from the interaction of NO with •O2− (
One limitation of the present study must be mentioned. The aim of assessing the acute effects of HBO following coronary occlusion restricted our interests solely to the immediate effects of the treatment. The effects of HBO on the evolution of MI in the long term, remains to be studied.
In conclusion, our data showed that HBO animals had greater ability to control pro-oxidants and antioxidants during myocardial ischemia. This action should be the reason for the reduced mortality rate in the treated rats.
The authors would like to thank Mr. Victor Debbas and Ms. Ana Lucia Garippo (Laboratory of Vascular Biology, Heart Institute, University of São Paulo) for technical assistance.
Financial support was provided by CAPES, FAPESP (grant no. 09/54225-8) and CNPq (grant no. 478740-5).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
MO, EA, LB and LS contributed to the design of the study, acquisition of data, and result analyses. AS performed statistical analyses and LT introduced a new research technique, analyzed the data and revised the manuscript for important intellectual content. JK, FL and PT raised grant funding, contributed to the design of the study, and revised the manuscript. All the authors read and approved the final manuscript. All authors take responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation.
All procedures were performed according to the principles of ethics of animal experimentation adopted by the Brazilian College of Animal Experimentation (COBEA-
Not applicable.
The authors declare that they have no competing interests.
hyperbaric oxygenation
reactive oxygen species
reperfusion injury
acute myocardial infarction
atmospheres absolute
glutathione
glutathione disulfide
dihydroethidium
nitric oxide
nitrate
nitrite
superoxide dismutase
catalase
peroxiredoxin
(A) Mortality rate, as expressed as percentages of infarcted animals, without therapy (MI) and infarcted animals submitted to hyperbaric oxygenation (HBO). (B) MI size as expressed as a percentage of the LV perimeter. (C) GSH values. (D) GSSG values. (E) GSH/GSSG ratio. The oxidized glutathione concentration was obtained using the following calculation: GSSG=(total GSH-reduced GSH)/2. *P<0.05 in relation to the MI group, #P<0.05 in relation to the SH group. Groups: SH, Sham; MI, myocardial infarction; HBO, hyperbaric therapy; GSH, glutathione; GSSG, glutatione disulfide; LV, left ventricle.
Microfluorotopography of dihydroethidium (DHE) oxidation products, on slides of the myocardium in the (A) SH group, (B) MI group, (C) HBO group and (D) MI group. The slides were incubated with 500 U/ml Peg-SOD. (E) The total fluorescence intensities of the studied groups. *P<0.05 relative to the MI group; #P<0.05 relative to the SH group, &P<0.05 between the SH, MI, and HBO. Groups: SH, Sham; MI, myocardial infarction; HBO, hyperbaric therapy.
Values (mean ± SEM) for (A) SOD expression, (B) CAT expression and (C) Prx-SO2/3 expression in the SH, MI, and HBO groups. (D) Immunoblots. *P<0.05 in relation to MI, #P<0.05 in relation to SH. Groups: SH, Sham; MI, myocardial infarction; HBO, hyperbaric therapy; SOD, superoxide dismutase; CAT, catalase; Prx, peroxiredoxin.
Graphs showing values (mean ± SEM) for (A) NO3−, (B) NO2− and (C) nitrated proteins. *P<0.05 in relation to MI, #P<0.05 in relation to SH. Groups: SH, Sham; MI, myocardial infarction; HBO, hyperbaric therapy.