Rafael Oliveira Fernandes

Redox-sensitive prosurvival and proapoptotic protein expression in the myocardial remodeling post-infarction in rats

In this study, we investigated the oxidative stress influence in some prosurvival and proapoptotic proteins after myocardial infarction (MI). Male Wistar rats were divided in two groups: Sham-operated (control) and MI. MI was induced by left coronary artery occlusion. 28-days after surgery, echocardiographic, morphometric, and hemodynamic parameters were evaluated. Redox status (reduced to oxidized glutathione ratio, GSH/GSSG) and hydrogen peroxide levels (H2O2) were measured in heart tissue. The p-ERK/ERK, p-Akt/Akt, p-mTOR/mTOR and p-GSK-3β/GSK-3β ratios, as well as apoptosis-inducing factor (AIF) myocardial protein expression were quantified by Western blot. MI group showed an increase in cardiac hypertrophy (23%) associated with a decrease in ejection fraction (38%) and increase in left ventricular end-diastolic pressure (82%) when compared to control, characterizing ventricular dysfunction. Redox status imbalance was seen in MI animals, as evidenced by the decrease in the GSH/GSSG ratio (30%) and increased levels of H2O2 (45%). This group also showed an increase in the ERK phosphorylation and a reduction of Akt and mTOR phosphorylation when compared to control. Moreover, we showed a reduction in the GSK-3β phosphorylation and an increase in AIF protein expression in MI group. Taken together, our results show increased H2O2 levels and cellular redox imbalance associated to a higher p-ERK and AIF immunocontent, which would contribute to a maladaptive hypertrophy phenotype.

Cardioprotective effects of thyroid hormones in a rat model of myocardial infarction are associated with oxidative stress reduction.

Reactive oxygen species (ROS) are involved with progression from infarction to heart failure. Studies show that thyroid hormones (TH) present cardioprotective effects. This study aims to evaluate whether TH effects after infarction are associated to redox balance modulation. Male Wistar rats were divided into four groups: Sham-operated (SHAM), infarcted (AMI), sham-operated+TH (SHAMT), and infarcted+TH (AMIT). During 26 days, animals received T3 (2 μg/100g/day) and T4 (8 μg/100g/day) by gavage. Echocardiographic parameters were assessed and heart tissue was collected to biochemical analysis. AMIT rats presented absence of lung congestion, less cardiac dilatation, and normalization in myocardial performance index, compared with AMI. AMI rats presented an increase in hydrogen peroxide levels and in lipid peroxidation and a decrease in GSH/GSSG. TH prevented these alterations in AMIT. In conclusion, TH seem to reduce the levels of ROS, preventing oxidative stress, and improving cardiac function in infarcted rats.

Time course of hydrogen peroxide–thioredoxin balance and its influence on the intracellular signalling in myocardial infarction

We investigated the myocardial thioredoxin‐1 and hydrogen peroxide concentrations and their association with some prosurvival and pro‐apoptotic proteins, during the transition from myocardial infarction (MI) to heart failure in rats. Male Wistar rats were divided into the following six groups: three sham‐operated groups and three MI groups, each at at 2, 7 and 28 days postsurgery. Cardiac function was analysed by echocardiography; the concentration of H2O2 and the ratio of reduced to oxidized glutathione were measured spectrophotometrically, while the myocardial immunocontent of thioredoxin‐1, angiotensin II, angiotensin II type 1 and type 2 receptors, p‐JNK/JNK, p‐ERK/ERK, p‐Akt/Akt, p‐mTOR/mTOR and p‐GSK3β/GSK3β was evaluated by Western blot. Our results show that thioredoxin‐1 appears to make an important contribution to the reduced H2O2 concentration. It was associated with lower JNK expression in the early period post‐MI (2 days). However, thioredoxin‐1 decreased, while renin–angiotensin system markers and levels of H2O2 increased, over 28 days post‐MI, in parallel with some signalling proteins involved in maladaptative cardiac remodelling and ventricular dysfunction. These findings provide insight into the time course profile of endogenous antioxidant adaptation to ischaemic injury, which may be useful for the design of therapeutical strategies targeting oxidative stress post‐MI.

Sulforaphane effects on postinfarction cardiac remodeling in rats: modulation of redox-sensitive prosurvival and proapoptotic proteins

This study investigated whether sulforaphane (SFN), a compound found in cruciferous vegetables, could attenuate the progression of post-myocardial infarction (MI) cardiac remodeling. Male Wistar rats (350 g) were allocated to four groups: SHAM (n=8), SHAM+SFN (n=7), MI (n=8) and MI+SFN (n=5). On the third day after surgery, cardiac function was assessed and SFN treatment (5 mg/kg/day) was started. At the end of 25 days of treatment, cardiac function was assessed and heart was collected to measure collagen content, oxidative stress and protein kinase. MI and MI+SFN groups presented cardiac dysfunction, without signs of congestion. Sulforaphane reduced fibrosis (2.1-fold) in infarcted rats, which was associated with a slight attenuation in the cardiac remodeling process. Both infarcted groups presented increases in the oxidative markers xanthine oxidase and 4-hydroxinonenal, as well as a parallel increase in the antioxidant enzymes glutathione peroxidase and superoxide dismutase. Moreover, sulforaphane stimulated the cytoprotective heme oxygenase-1 (HO-1) (38%). Oxidative markers correlated with ERK 1/2 activation. In the MI+SFN group, up-regulation of ERK 1/2 (34%) and Akt (35%), as well as down-regulation of p38 (52%), was observed. This change in the prosurvival kinase balance in the MI+SFN group was related to a down-regulation of apoptosis pathways (Bax/Bcl-2/caspase-3). Sulforaphane was unable to modulate autophagy. Taken together, sulforaphane increased HO-1, which may generate a redox environment in the cardiac tissue favorable to activation of prosurvival and deactivation of prodeath pathways. In conclusion, this natural compound contributes to attenuation of the fibrotic process, which may contribute to mitigation against the progression of cardiac remodeling postinfarction.

The role of AT1-receptor blockade on reactive oxygen species and cardiac autonomic drive in experimental hyperthyroidism

The objective of this study was to explore the influence of the renin-angiotensin system on cardiac prooxidants and antioxidants levels and its association to autonomic imbalance induced by hyperthyroidism. Male Wistar rats were divided into four groups: control, losartan (10mg/kg/day by gavage, 28 day), thyroxine (T4) (12 mg/L in drinking water for 28 days), and T4+losartan. Spectral analysis (autonomic balance), angiotensin II receptor (AT1R), NADPH oxidase, Nrf2 and heme-oxygenase-1 (HO-1) myocardial protein expression, and hydrogen peroxide (H2O2) concentration were quantified. Autonomic imbalance induced by hyperthyroidism (~770%) was attenuated in the T4+losartan group (~32%) (P<0.05). AT1R, NADPH oxidase, H2O2, as well as concentration, Nrf2 and HO-1 protein expression were elevated (~172%, 43%, 40%, 133%, and 154%, respectively) in T4 group (P<0.05). H2O2 and HO-1 levels were returned to control values in the T4+losartan group (P<0.05). The overall results demonstrate a positive impact of RAS blockade in the autonomic control of heart rate, which was associated with an attenuation of H2O2 levels, as well as with a reduced counter-regulatory response of HO-1 in experimental hyperthyroidism.

Redox status and pro-survival/pro-apoptotic protein expression in the early cardiac hypertrophy induced by experimental hyperthyroidism.

This study was conducted to analyse the redox status and redox‐sensitive proteins that may contribute to a non‐genomic mechanism of cardiac hypertrophy induction by hyperthyroidism. Wistar rats, treated with L‐thyroxine (T4) during 2 weeks (12 mg·l‐1 in drinking water), presented cardiac hypertrophy (68% higher than control), without signals of liver or lung congestion. Myocardial reduction of the reduced glutathione: oxidized glutathione (GSSG) ratio (45%) (redox status) and elevation in hydrogen peroxide concentration (H2O2) (28%) were observed in hyperthyroid as compared with the control. No significant difference was found in thioredoxin (Trx), Trx reductase activity and Nrf2 (a transcriptional factor) protein expression between groups. Redox‐sensitive proteins, quantified using Western blot, presented the following results: increased p‐ERK: total extracellular‐regulated kinase (ERK) (200%) and Bax : Bcl‐2 (62%) ratios and reduced total‐Akt (63%) and p‐Akt (53%) expressions in the hyperthyroid rats as compared with the control. The redox imbalance, associated with increased immunocontent of a protein related to maladaptative growth (ERK) and reduced immunocontent of protein related to cytoprotection/survival (Akt), may suggest that the molecular scenario could favour the decompensation process of cardiac hypertrophy induced by experimental hyperthyroidism. Copyright

Sulforaphane improves oxidative status without attenuating the inflammatory response or cardiac impairment induced by ischemia–reperfusion in rats

Sulforaphane, a natural isothiocyanate, demonstrates cardioprotection associated with its capacity to stimulate endogenous antioxidants and to inhibit inflammation. The aim of this study was to investigate whether sulforaphane is capable of attenuating oxidative stress and inflammatory responses through the TLR4/MyD88/NFκB pathway, and thereby could modulate post-ischemic ventricular function in isolated rat hearts submitted to ischemia and reperfusion. Male Wistar rats received sulforaphane (10 mg·kg(-1)·day(-1)) or vehicle i.p. for 3 days. Global ischemia was performed using isolated hearts, 24 h after the last injection, by interruption of the perfusion flow. The protocol included a 20 min pre-ischemic period followed by 20 min of ischemia and a 20 min reperfusion. Although no changes in mechanical function were observed, sulforaphane induced a significant increase in superoxide dismutase and heme oxygenase-1 expression (both 66%) and significantly reduced reactive oxygen species levels (7%). No differences were observed for catalase and glutathione peroxidase expression or their activities, nor for thioredoxin reductase, glutaredoxin reductase and glutathione-S-transferase. No differences were found in lipid peroxidation or TLR4, MyD88, and NF-κB expression. In conclusion, although sulforaphane was able to stimulate endogenous antioxidants modestly, this result did not impact inflammatory signaling or cardiac function of hearts submitted to ischemia and reperfusion.

Thyroid hormones effects on oxidative stress and cardiac remodeling in the right ventricle of infarcted rats

UNLABELLED Right ventricle (RV) dysfunction post-myocardial infarction (MI) was associated with a worsened prognosis. In this scenario, reactive oxygen species (ROS) are related with the progression from MI to heart failure. Previous work showed that thyroid hormones (TH) are cardioprotective after MI. AIMS This study aims to investigate the effect of T3 and T4 administration on oxidative stress and angiogenesis parameters in the RV after MI. MAIN METHODS Wistar rats were allocated into four groups: Sham-operated (SHAM), infarcted (AMI), sham-operated + TH (SHAMT), and infarcted+TH (AMIT). The treated groups received T3 (2 μg/100g/day) and T4 (8 μg/100g/day) by gavage for 26 days. After this, echocardiographic analysis was performed and the RV was collected to western blot and biochemical analysis. KEY FINDINGS Infarcted treated rats showed RV hypertrophy compared with AMI and SHAMT. Hydrogen peroxide levels were decrease and SOD activity and expression were increased in the infarcted treated rats. Besides that, the hormonal administration increased eNOS expression and prevented the reduction of VEGF levels in AMIT rats. SIGNIFICANCE In conclusion, TH seems to improve oxidative stress parameters, to promote physiological hypertrophy and to increase the expression of proteins involved with angiogenesis in the right heart.

Catalase Influence in the Regulation of Coronary Resistance by Estrogen: Joint Action of Nitric Oxide and Hydrogen Peroxide

We tested the influence of estrogen on coronary resistance regulation by modulating nitric oxide (NO) and hydrogen peroxide (H2O2) levels in female rats. For this, estrogen levels were manipulated and the hearts were immediately excised and perfused at a constant flow using a Langendorffs apparatus. Higher estrogen levels were associated with a lower coronary resistance, increased nitric oxide bioavailability, and higher levels of H2O2. When oxide nitric synthase blockade by L-NAME was performed, no significant changes were found in coronary resistance of ovariectomized rats. Additionally, we found an inverse association between NO levels and catalase activity. Taken together, our data suggest that, in the absence of estrogen influence and, therefore, reduced NO bioavailability, coronary resistance regulation seems to be more dependent on the H2O2 that is maintained at low levels by increased catalase activity.

DHEA treatment effects on redox environment in skeletal muscle of young and aged healthy rats

Background: Dehydroepiandrosterone (DHEA) is an important precursor of active ster-oid hormone, produced abundantly by the adrenal cortex with an age-dependent pattern. Objective: We investigated whether chronic DHEA administration impacts on redox status and on Akt protein activation in skeletal muscle during the aging process (3 and 24 months-old rats). Methods: Rats received one weekly dose/5 weeks of DHEA (10 mg/kg) or vehicle. Gastrocnemius muscle was removed to evaluate glutathione system, hydrogen peroxide, antioxidant enzymes, and expression of Akt kinase protein. Results: In the 3-months-old rats DHEA induced an increase in hydrogen peroxide when compared both to its control (276%) and the 24-months-old DHEA group (485%). Moreover, in the 24-months-old rats DHEA caused an increase in GSSG (41 and 28%), a decrease in reduced-GSH (55 and 51%), and a more oxidized redox status (reduction in GSH/GSSG ratio, 47 and 65%) when compared to 3-month-old DHEA and to 24-months-old control groups, respectively. Both older groups had increased G6PDH (2.7 fold) and GST (1.7 fold) activities when compared to younger groups, independently of any DHEA treatment. However, there was no modulation of Akt protein (phosphorylated/total isoform). Conclusion: The results show that chronic DHEA administration to 3 and 24-months-old rats may not present positive effects regarding the redox environment in skeletal muscle without modulation of pro-survival Akt kinase. Due to the large-scale self-administration of DHEA as an “anti-aging” die-tary supplement, it is crucial to investigate its molecular mechanisms over oxidative stress-induced related diseases.