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Introduction: It has been demonstrated the dysregulation of the cardiac endocannabinoid system in cardiovascular diseases. Thus, the modulation of this system through the administration of phytocannabinoids present in medicinal cannabis oil (CO) emerges as a promising therapeutic approach. Furthermore, phytocannabinoids exhibit potent antioxidant properties, making them highly desirable in the treatment of cardiac pathologies, such as hypertension-induced cardiac hypertrophy (CH). Objective: To evaluate the effect of CO treatment on hypertrophy and mitochondrial status in spontaneously hypertensive rat (SHR) hearts. Methods: Three-month-old male SHR were randomly assigned to CO or olive oil (vehicle) oral treatment for 1 month. We evaluated cardiac mass and histology, mitochondrial dynamics, membrane potential, area and density, myocardial reactive oxygen species (ROS) production, superoxide dismutase (SOD), and citrate synthase (CS) activity and expression. Data are presented as mean ± SEM (n) and compared by t -test, or two-way ANOVA and Bonferroni post hoc test were used as appropriate. p < 0.05 was considered statistically significant. Results: CH was reduced by CO treatment, as indicated by the left ventricular weight/tibia length ratio, left ventricular mass index, myocyte cross-sectional area, and left ventricle collagen volume fraction. The ejection fraction was preserved in the CO-treated group despite the persistence of elevated systolic blood pressure and the reduction in CH. Mitochondrial membrane potential was improved and mitochondrial biogenesis, dynamics, area, and density were all increased by treatment. Moreover, the activity and expression of the CS were enhanced by treatment, whereas ROS production was decreased and the antioxidant activity of SOD increased by CO administration. Conclusion: Based on the mentioned results, we propose that 1-month oral treatment with CO is effective to reduce hypertrophy, improve the mitochondrial pool and increase the antioxidant capacity in SHR hearts.

Background: It has been previously demonstrated that the maintenance of ischemic acidic pH or the delay of intracellular pH recovery at the onset of reperfusion decreases ischemic-induced cardiomyocyte death., Objective: To examine the role played by nitric oxide synthase (NOS)/NO-dependent pathways in the effects of acidic reperfusion in a regional ischemia model., Methods: Isolated rat hearts perfused by Langendorff technique were submitted to 40 min of left coronary artery occlusion followed by 60 min of reperfusion (IC). A group of hearts received an acid solution (pH = 6.4) during the first 2 min of reperfusion (AR) in absence or in presence of l-NAME (NOS inhibitor). Infarct size (IS) and myocardial function were determined. In cardiac homogenates, the expression of P-Akt, P-endothelial and inducible isoforms of NOS (P-eNOS and iNOS) and the level of 3-nitrotyrosine were measured. In isolated cardiomyocytes, the intracellular NO production was assessed by confocal microscopy, under control and acidic conditions. Mitochondrial swelling after Ca 2+ addition and mitochondrial membrane potential (Δψ) were also determined under control and acidosis., Results: AR decreased IS, improved postischemic myocardial function recovery, increased P-Akt and P-eNOS, and decreased iNOS and 3-nitrotyrosine. NO production increased while mitochondrial swelling and Δψ decreased in acidic conditions. l-NAME prevented the beneficial effects of AR., Conclusions: Our data strongly supports that a brief acidic reperfusion protects the myocardium against the ischemia-reperfusion injury through eNOS/NO-dependent pathways., (Copyright © 2024. Published by Elsevier Inc.)

The increase of intracellular Ca 2+ concentration, produced principally by its influx through the L-type Ca 2+ channels, is one of the major contributors to the ischemia-reperfusion injury. The inhibition of those channels in different experimental models was effective to ameliorate the post-ischemic damage. However, at a clinical level, the results were contradictory. Recent results of our group obtained in an ¨ ex vivo ¨ heart model demonstrated that a chemical derived from acetazolamide, the N-methylacetazolamide (NMA) protected the heart against ischemia-reperfusion injury, diminishing the infarct size and improving the post-ischemic recovery of myocardial function and mitochondrial dynamic. A significant inhibitory action on L-type Ca 2+ channels was also detected after NMA treatment, suggesting this action as responsible for the beneficial effects on myocardium exerted by this compound. Although these results were promising, the effectiveness of NMA in the treatment of ischemic heart disease in humans as well as the advantages or disadvantages in comparison to the classic calcium antagonists needs to be investigated., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pardo, Díaz Zegarra, González Arbeláez, Aiello and Mosca.)

Our objective was to examine the effects of N-methylacetazolamide (NMA), a non‑carbonic anhydrase inhibitor, on ischemia-reperfusion injury. Isolated rat hearts were assigned to the following groups: 1) Non-ischemic control (NIC):110 min of perfusion and 2) Ischemic control (IC): 30 min of global ischemia and 60 min of reperfusion (R). Both groups were repeated in presence of NMA (5 μM), administered during the first 10 min of R. Infarct size (IS) was measured by TTC staining. Developed pressure (LVDP) and end-diastolic pressure (LVEDP) of the left ventricle were used to assess systolic and diastolic function, respectively. The content of P-Akt, P-PKCε, P-Drp1 and calcineurin Aβ were measured. In cardiomyocytes the L-type Ca 2+ current (ICaL) was recorded with the whole-cell configuration of patch-clamp technique. The addition of NMA to non-ischemic hearts decreased 15% the contractility. In ischemic hearts (IC group), NMA decreased IS (22 ± 2% vs 32 ± 2%, p < 0.05) and improved the post-ischemic recovery of myocardial function. At the end of R, LVDP was 54 ± 7% vs 18 ± 3% and LVEDP was 23 ± 8 vs. 55 ± 7 mmHg ¨p < 0.05¨. The level of P-Akt, P-PKCε and P-Drp1 increased and the expression of calcineurin Aβ decreased in NMA treated hearts. Peak ICaL density recorded at 0 mV was smaller in myocytes treated with NMA than in non-treated cells (-1.91 ± 0.15 pA/pF vs -2.32 ± 0.17 pA/pF, p < 0.05). These data suggest that NMA protects the myocardium against ischemia-reperfusion injury through an attenuation of mitochondrial fission by calcineurin/Akt/PKCε-dependent pathways associated to the decrease of ICaL current., (Copyright © 2022 Elsevier B.V. All rights reserved.)

We have previously demonstrated that inhibition of extracellularly oriented carbonic anhydrase (CA) isoforms protects the myocardium against ischemia-reperfusion injury. In this study, our aim was to assess the possible further contribution of CA intracellular isoforms examining the actions of the highly diffusible cell membrane permeant inhibitor of CA, ethoxzolamide (ETZ). Isolated rat hearts, after 20 min of stabilization, were assigned to the following groups: (1) Nonischemic control: 90 min of perfusion; (2) Ischemic control: 30 min of global ischemia and 60 min of reperfusion (R); and (3) ETZ: ETZ at a concentration of 100 μM was administered for 10 min before the onset of ischemia and then during the first 10 min of reperfusion. In additional groups, ETZ was administered in the presence of SB202190 (SB, a p38MAPK inhibitor) or chelerythrine (Chel, a protein kinase C [PKC] inhibitor). Infarct size, myocardial function, and the expression of phosphorylated forms of p38MAPK, PKCε, HSP27, and Drp1, and calcineurin Aβ content were assessed. In isolated mitochondria, the Ca 2+ response, Ca 2+ retention capacity, and membrane potential were measured. ETZ decreased infarct size by 60%, improved postischemic recovery of myocardial contractile and diastolic relaxation increased P-p38MAPK, P-PKCε, P-HSP27, and P-Drp1 expression, decreased calcineurin content, and normalized calcium and membrane potential parameters measured in isolated mitochondria. These effects were significantly attenuated when ETZ was administered in the presence of SB or Chel. These data show that ETZ protects the myocardium and mitochondria against ischemia-reperfusion injury through p38MAPK- and PKCε-dependent pathways and reinforces the role of CA as a possible target in the management of acute cardiac ischemic diseases., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Purpose: To determine the actions of isoespintanol (Isoesp) on post-ischemic myocardial and mitochondrial alterations., Methods: Hearts removed from Wistar rats were perfused by 20 min. After this period, the coronary flow was interrupted by half an hour and re-established during 1 h. In the treated group, Isoesp was administered at the beginning of reperfusion. To assess the participation of ε isoform of protein kinase C (PKCε), protein kinase B (PKB/Akt), and nitric oxide synthase (NOS), hearts were treated with Isoesp plus the respective inhibitors (chelerythrine, wortmannin, and N-nitro-L-arginine methyl ester). Cell death was determined by triphenyl tetrazolium chloride staining technique. Post-ischemic recovery of contractility, oxidative stress, and content of phosphorylated forms of PKCε, Akt, and eNOS were also examined. Mitochondrial state was assessed through the measurement of calcium-mediated response, calcium retention capacity, and mitochondrial potential., Results: Isoesp limited cell death, decreased post-ischemic dysfunction and oxidative stress, improved mitochondrial state, and increased the expression of PKCε, Akt, and eNOS phosphorylated. All these beneficial effects achieved by Isoesp were annulled by the inhibitors., Conclusion: These findings suggest that activation of Akt/eNOS and PKCε signaling pathways are involved in the development of Isoesp-induced cardiac and mitochondria tolerance to ischemia-reperfusion.

The electrogenic sodium bicarbonate co-transporter isoform 1 (NBCe1) plays an important role in ischemia-reperfusion injury. The cardioprotective action of an antibody directed to the extracellular loop 3 (a-L3) of NBCe1 was previously demonstrated by us. However, the role of a-L3 on mitochondrial post-ischemic alterations has not yet been determined. In this study, we aimed to elucidate the effects of a-L3 on post-ischemic mitochondrial state and dynamics analysing the involved mechanisms. Isolated rat hearts were assigned to the following groups: 1) Non-ischemic control (NIC): 110 min of perfusion; 2) Ischemic control (IC): 30 min of global ischemia and 60 min of reperfusion (R); 3) a-L3: a-L3 was administered during the initial 10 min of R; 4) SB + a-L3: SB202190 (p38MAPK inhibitor) plus a-L3. Infarct size (IS) was measured by TTC staining. Developed pressure (LVDP), maximal velocities of rise and decay of LVP (+dP/dt max, -dP/dt max) and end-diastolic pressure (LVEDP) of the left ventricle were used to assess systolic and diastolic function. Mitochondrial Ca 2+ response (CaR), Ca 2+ retention capacity (CRC), membrane potential (ΔΨm) and MnSOD levels were measured. The expression of P-p38MAPK, calcineurin, P-HSP27, P-Drp1, Drp1, and OPA1 were determined. a-L3 decreased IS, improved post-ischemic recovery of myocardial function, increased P-p38MAPK, P-HSP27, P-Drp1, cytosolic Drp1, and OPA1 expression and decreased calcineurin. These effects were abolished by p38MAPK inhibition with SB. These data show that NBCe1 inhibition by a-L3 limits the cell death, improves myocardial post-ischemic contractility and mitochondrial state and dynamic through calcium decrease/calcineurin inhibition-mediated p38MAPK activation and p38MAPK/HSP27-dependent pathways. Thus, we demonstrated that a-L3 is a potential therapeutic strategy in post-ischemic alterations., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Myocardial ischemia is the leading cause of death worldwide. Despite better outcomes with early coronary artery reperfusion strategies, morbidity and mortality remain significant. The principal myocardial hallmark of myocardial ischemia is cell death and the associated impairment of cardiac contractility. In this way, the use of extracts from medicinal plants versus synthetic drugs to mitigate post-ischemic damage constitutes an alternative. Despite their proven beneficial effects in cardiovascular disorders, the use of many plants is questioned. Our aim is to update the clinical and experimental studies about the actions of medicinal plants and polyphenol-enriched extracts against ischemia-reperfusion injury and the involved mechanisms. A review of the recent scientific literature (last ten years) on cardioprotective medicinal plants was developed using the following bibliographic databases: PubMed, Scopus, Web of Knowledge and Google Scholar. Herein, the clinical and experimental studies on medicinal plants and their phenolic compounds have been reviewed. The second part of this review was centered on the search for medicinal plant extracts and natural products isolated from them as potential cardioprotective agents. The botanical names of the cited plants have been authenticated by searching the Plant List and Royal Botanical Garden, Kew databases. The data collected show that treatment with natural products diminishes post-ischemic damage through an improvement of the mitochondrial functionality mainly mediated by enhanced nitric oxide bioavailability. Despite these results, further studies must be carried out to validate their use to prevent or mitigate ischemia-reperfusion injury in the clinical setting.

Background: Recent studies from our laboratory show the cardioprotective action of benzolamide (BZ, carbonic anhydrase inhibitor) against ischemia-reperfusion injury. However, the mechanisms involved have not been fully elucidated., Objective: To examine the participation of the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) in the effects of BZ in a model of regional ischemia., Methods: Isolated rat hearts perfused by Langendorff technique were submitted to 40 min of coronary artery occlusion followed by 60 min of reperfusion (IC). Other hearts received BZ during the first 10 min of reperfusion in absence or presence of L-NAME, NOS inhibitor. The infarct size (IS) and the post-ischemic recovery of myocardial function were measured. Oxidative/nitrosative damage were assessed by reduced glutathione (GSH) content, thiobarbituric acid reactive substances (TBARS) and 3-nitrotyrosine levels. The expression of phosphorylated forms of Akt, p38MAPK and eNOS, and the concentration of inducible nitric oxide synthase (iNOS) were also determined., Results: BZ significantly decreased IS (6.2 ± 0.5% vs. 34 ± 4%), improved post-ischemic contractility, preserved GSH levels and diminished TBARS and 3-nitrotyrosine. In IC hearts, P-Akt, P-p38MAPK and P-eNOS decreased and iNOS increased. After BZ addition the levels of P-kinases and P-eNOS increased and iNOS decreased. Except for P-Akt, P-p38MAPK and iNOS, the effects of BZ were abolished by L-NAME., Conclusions: Our data demonstrate that the treatment with BZ at the onset of reperfusion was effective to reduce cell death, contractile dysfunction and oxidative/nitrosative damage produced by coronary artery occlusion. These BZ-mediated beneficial actions appear mediated by eNOS/NO-dependent pathways., (Copyright © 2018 Elsevier Inc. All rights reserved.)

During ischemia, increased anaerobic glycolysis results in intracellular acidosis. Activation of alkalinizing transport mechanisms associated with carbonic anhydrases (CAs) leads to myocardial intracellular Ca 2+ increase. We characterize the effects of inhibition of CA with benzolamide (BZ) during cardiac ischemia-reperfusion (I/R). Langendorff-perfused isolated rat hearts were subjected to 30 min of global ischemia and 60 min of reperfusion. Other hearts were treated with BZ (5 μM) during the initial 10 min of reperfusion or perfused with acid solution (AR, pH 6.4) during the first 3 min of reperfusion. p38MAPK, a kinase linked to membrane transporters and involved in cardioprotection, was examined in hearts treated with BZ in presence of the p38MAPK inhibitor SB202190 (10 μM). Infarct size (IZ) and myocardial function were assessed, and phosphorylated forms of p38MAPK, Akt, and PKCε were evaluated by immunoblotting. We determined the rate of intracellular pH (pH i ) normalization after transient acid loading in the absence and presence of BZ or BZ + SB202190 in heart papillary muscles (HPMs). Mitochondrial membrane potential (ΔΨ m ), Ca 2+ retention capacity and Ca 2+ -mediated swelling after I/R were also measured. BZ, similarly to AR, reduced IZ, improved postischemic recovery of myocardial contractility, increased phosphorylation of Akt, PKCε, and p38MAPK, and normalized ΔΨ m and Ca 2+ homeostasis, effects abolished after p38MAPK inhibition. In HPMs, BZ slowed pH i recovery, an effect that was restored after p38MAPK inhibition. We conclude that prolongation of acidic conditions during reperfusion by BZ could be responsible for the cardioprotective benefits of reduced infarction and better myocontractile function, through p38MAPK-dependent pathways. NEW & NOTEWORTHY Carbonic anhydrase inhibition by benzolamide (BZ) maintains acidity, decreases infarct size, and improves postischemic myocardial dysfunction in ischemia-reperfusion (I/R) hearts. Protection afforded by BZ mimicked the beneficial effects elicited by an acidic solution (AR). Increased phosphorylation of p38MAPK occurs in I/R hearts reperfused with BZ or with AR. Mitochondria from I/R hearts possess abnormal Ca 2+ handling and a more depolarized membrane potential compared with control hearts, and these changes were restored by treatment with BZ or AR.

The response to ischemia/reperfusion and the effects of ischemic post-conditioning (IPC) are sex-dependent, but the mechanisms have not been clarified. Male (M) and female (F) rat hearts isolated and perfused using the Langendorff technique were subject to 30 min of global ischemia (GI) and 60 min reperfusion (R). In IPC hearts, three cycles of 30-sec GI/30-sec R were applied at the beginning of R. Infarct size and myocardial function were assessed. Superoxide production, antioxidant systems, and expressions of phosphorylated forms of serine/threonine kinase (Akt), glycogen synthase kinase 3β (GSK-3β), protein kinase C ε (PKCε), endothelial nitric oxide synthase (eNOS), and apoptosis were measured. In the basal state, superoxide production and apoptosis were lower, and antioxidant systems and phospho-kinase expressions were higher in F rather than in M hearts. After ischemia-reperfusion, infarct size was less in F hearts, and post-ischemic recovery of myocardial function was higher in F rather than in M hearts. Superoxide production, phospho-kinase activity, phospho-eNOS, and apoptosis increased in both sexes while antioxidants decreased in both sexes. After IPC, infarct size, superoxide production, and apoptosis decreased and phospho-eNOS increased in F and M hearts but phospho-kinase expressions and post-ischemic recovery of myocardial function improved only in M hearts. These results show that Akt/GSK-3β/PKCε/eNOS-dependent pathways-mediated superoxide production and apoptosis appear as important factors involved in the observed gender differences., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Our objective was to determine the effects of a polyphenol-enriched cocoa extract (PCE) on myocardial postischemic alterations in normotensive (Wistar rats, W) and spontaneously hypertensive rats (SHR). Isolated hearts were submitted to 110 min of perfusion or 20 min stabilization, 30 min global ischemia, and 60 min reperfusion (R). Other hearts were treated with PCE at the onset of R. Infarct size, the reduced glutathione (GSH), and the expression of phospho-Akt, P-GSK-3β, and P-eNOS were assessed. In isolated mitochondria, the Ca(2+)-mediated response of mitochondrial permeability transition pore (mPTP), membrane potential (Δψm), and superoxide production were determined. PCE decreased infarct size, partly preserved GSH, increased the P-Akt, P-GSK-3β, and P-eNOS contents, improved mPTP response to Ca(2+), decreased the superoxide production, and restored Δψm. These data show that PCE decreases the cardiac postischemic damage in W rats and SHR and suggest that Akt/GSK-3β/eNOS dependent pathways are involved.

Our aim was to assess the action of cyclosporine-A (CsA) against reperfusion injury in spontaneously hypertensive rats (SHR) compared to the effects of ischemic pre- (IP) and postconditioning (IPC), examining the role played by PKCε. Isolated hearts were submitted to the following protocols: IC: 45 min global ischemia (GI) and 1h reperfusion (R); IP: a cycle of 5 min GI and 10 min of R prior to 45 min-GI; and IPC: three cycles of 30s-GI/30s-R at the start of R. Other hearts of the IC, IP and IPC groups received CsA (mitochondrial permeability transition pore inhibitor) or chelerythrine (Che, non-selective PKC inhibitor). Infarct size (IS) was assessed. TBARS and reduced glutathione (GSH) content - as parameters of oxidative damage, the expression of P-Akt, P-GSK-3β, P-PKCε and cytochrome c (Cyc) release - as an index of mitochondrial permeability and the response of isolated mitochondria to Ca(2+) were also measured. IS similarly decreased in preconditioned, postconditioned and CsA treated heart showing the highest values in the combinations IP+CsA and IPC+CsA. TBARS decreased and GSH was partially preserved after all interventions. The content of P-Akt, P-GSK-3β and P-PKCε increased in cytosol and decreased in mitochondria after IP and IPC. In CsA treated hearts these enzymes increased in both fractions reaching the highest values. Cyc release was attenuated and the response of mitochondria to Ca(2+) was improved by the interventions. The beneficial effects of IP and IPC were annulled when PKC was inhibited with Che. A PKCε/VDAC association was also detected. These data show that, in SHR, the CsA treatment mimicked and reinforced the cardioprotective action afforded by IP and IPC in which PKCε-mediated attenuation of mitochondrial permeability appears as the main mechanism involved., (Copyright © 2016. Published by Elsevier Inc.)

In association with the published article "Mitochondrial calcium handling in normotensive and spontaneously hypertensive rats: correlation with systolic blood pressure levels" [1], this data article contains information about calcium handling of cardiac mitochondria isolated from female of both rats strains (WKY and SHR). Dataset of mitochondrial permeability transition pore (mPTP) resistance to opening Ca(2+)-mediated, Ca(2+) retention capacity (CRC), time constants and mitochondrial membrane potential (ΔΨm) are showed.

Tea made from Ilex paraguariensis (IP) dried and minced leaves is a beverage widely consumed by large populations in South America as a source of caffeine (stimulant action) and for its medicinal properties. However, there is little information about the action of IP on the myocardium in the ischemia-reperfusion condition. Therefore, the objective of this study was to examine the effects of an aqueous extract of IP on infarct size in a model of regional ischemia. Isolated rat hearts were perfused by the Langendorff technique and subjected to 40 min of coronary artery occlusion followed by 60 min of reperfusion (ischemic control hearts). Other hearts received IP 30 μg mL(-1) during the first 10 min of reperfusion in the absence or presence of l(G)-nitro-l-arginine methyl ester [l-NAME, a nitric oxide synthase (NOS) inhibitor]. The infarct size was determined by triphenyltetrazolium chloride (TTC) staining. Post-ischemic myocardial function and coronary perfusion were also assessed. Cardiac oxidative damage was evaluated by using the thiobarbituric acid reactive substance (TBARS) concentration and the reduced glutathione (GSH) content. To analyze the mechanisms involved, the expressions of phosphorylated forms of eNOS and Akt were measured. In isolated mitochondria the Ca(2+)-induced mitochondrial permeability transition pore (mPTP) opening was determined. IP significantly decreased the infarct size and improved post-ischemic myocardial function and coronary perfusion. TBARS decreased, GSH was partially preserved, the levels of P-eNOS and P-Akt increased and mPTP opening diminished after IP addition. These changes were abolished by l-NAME. Therefore, our data demonstrate that acute treatment with IP only during reperfusion was effective in reducing myocardial post-ischemic alterations. These actions would be mediated by a decrease of mitochondrial permeability through IP-activated Akt/eNOS-dependent pathways.

The aim was to study the mitochondrial Ca(2+) handling of mitochondria isolated from normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) hearts and to establish a possible correlation with systolic blood pressure (SBP). Mitochondrial swelling after Ca(2+) addition, Ca(2+)-retention capacity (CRC) by calcium green method, and membrane potential (ΔΨm) were assessed. SBP was 124±1 (WKY) and 235±6mmHg (SHR). CRC, Ca(2+) response and ΔΨm were lower in SHR than WKY mitochondria. The conclusion is: the more depolarized state of SHR than WKY mitochondria results in an abnormal Ca(2+) handling and this event is closely associated with the SBP., (Copyright © 2014 © Elsevier B.V. and Mitochondria Research Society. Published by Elsevier B.V. All rights reserved.)

Previous results show that prolonged treatment with EMD-87580 (EMD) NHE-1 blocker attenuates and reverses postinfarction remodelling. Our aim was to evaluate the effects of the treatment of EMD compared to ischemic postconditioning (IPO) in a model of regional ischemia. Isolated hearts were subjected to 40-min coronary occlusion followed by 60-min reperfusion (IC). Other hearts were treated with EMD 5μM during the first 10min of reperfusion or submitted to one cycle of 2min of reperfusion and 2min of ischemia as IPO protocol. Infarct sizes (IS), postischemic myocardial and vascular functions were assessed. The concentration of thiobarbituric reactive substances (TBARS), reduced glutathione (GSH) and expression of phosphorylated forms of ERK1/2, Akt, GSK-3β, eNOS were analyzed. MnSOD cytosolic activity - as an index of mitochondrial permeability - was also measured. EMD treatment and IPO decreased IS~50% and significantly improved the postischemic recovery of contractility and coronary perfusion. TBARS decreased and GSH increased after interventions compared to the values observed in IC hearts. MnSOD cytosolic activity increased in IC group and was significantly attenuated by EMD and abolished in IPO hearts. The content of P-ERK1/2 increased whereas P-Akt, P-GSK-3β and P-eNOS decreased in IC hearts. EMD treatment and IPO reversed these changes. The present data show that EMD treatment at the beginning of reperfusion-similarly to IPO- limited infarct size and attenuated the postischemic impairment of myocardial function through reactive oxygen species-mediated ERK1/2/Akt/GSK-3β/eNOS pathways., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Although the participation of the electrogenic sodium/bicarbonate cotransporter (NBCe1) in the recovery from an intracellular acid load is recognized, its role in ischemia-reperfusion is still unclear., Methods and Results: Our objective was to assess the role of NBCe1 in reperfusion injury. We use selective functional antibodies against extracellular loop 3 (a-L3) and loop 4 (a-L4) of NBCe1. a-L3 inhibits and a-L4 stimulates NBCe1 activity. Isolated rat hearts were submitted to 40 min of coronary occlusion and 1 h of reperfusion. a-L3, a-L4 or S0859--selective Na(+)-HCO3(-) co-transport inhibitor--were administered during the initial 10 min of reperfusion. The infarct size (IS) was measured by triphenyltetrazolium chloride staining technique. Postischemic systolic and diastolic functions were also assessed. a-L3 and S0859 treatments decreased significantly (P < .05) the IS (16 ± 3% for a-L3 vs. 32 ± 5% in hearts treated with control nonimmune serum and 19 ± 3% for S0859 vs. 39 ± 2% in untreated hearts). Myocardial function during reperfusion improved after a-L3 treatment, but it was not modified by S0859. The infusion of a-L4 did not modify neither the IS nor myocardial function., Conclusions: The NBCe1 hyperactivity during reperfusion leads to Na(+) and Ca(2+) loading, conducing to Ca(2+) overload and myocardial damage. Consistently, we have shown herein that the selective NBCe1 blockade with a-L3 exerted cardioprotection. This beneficial action strongly suggests that NBCe1 could be a potential target for the treatment of coronary disease., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: The mitochondrial permeability transition pore (mPTP) plays an important role in ischemia-reperfusion in normotensive animals. Our study aims to define their participation in the ischemic preconditioning (IP) in hypertrophied hearts and to assess the role played by NO and mitochondrial ATP-dependent K channels (mitoKATP)., Material and Methods: Isolated hearts from spontaneously hypertensive rats (SHR) and age-matched normotensive rats Wistar Kyoto (WKY) were subjected to 35-min or 50-min global ischemia (GI) followed by 2-hour reperfusion (R). IP was induced by a single cycle of 5-min GI and 10-min R (IP1) or three cycles of 2-min GI and 5-min R (IP3) applied before to prolonged ischemia. L-NAME (NOS inhibitor) or 5-HD (mitoKATP blocker) to investigate the role played by NO and mitoKATP, respectively were administered. Infarct size (IS), myocardial function, reduced glutathione (GSH) - as marker of oxidative stress and MnSOD cytosolic activity - as an index of mPTP opening were determined., Results: IP1 significantly decreased the IS in WKY hearts at both ischemia duration times. In SHR, IP1 decreased the IS observed in GI35 but it did not modify that detected at 50-min GI, which was limited by IP3. IP preserved GSH content and decreased MnSOD cytosolic activity in both rat strains. These protective effects were annulled by L-NAME and 5-HD for both ischemic periods in SHR, whereas in WKY they were only effective for 50-min GI., Conclusion: Our data demonstrate that the cardioprotection achieved by ischemic preconditioning in hearts from SHR hearts involves an attenuation of mPTP opening NO and mitoKATP-mediated., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The beneficial effects of N-(2-mercaptopropionyl)-glycine (MPG) against ischemia-reperfusion injury in normotensive animals have been previously studied. Our objective was to test the action of MPG during ischemia and reperfusion in hearts from spontaneously hypertensive rats (SHR). Isolated hearts from SHR and age-matched normotensive rats Wistar Kyoto (WKY) were subjected to 50-min global ischemia (GI) and 2-hour reperfusion (R). In other hearts MPG 2mM was administered during 10 min before GI and the first 10 min of R. Infarct size (IS) was assessed by TTC staining technique and expressed as percentage of risk area. Postischemic recovery of myocardial function was assessed. Reduced glutathione (GSH), thiobarbituric acid reactive substances (TBARS) and SOD cytosolic activity - as estimators of oxidative stress and MnSOD cytosolic activity - as an index of (mPTP) opening were determined. In isolated mitochondria H(2)O(2)-induced mPTP opening was also measured. The treatment with MPG decreased infarct size, preserved GSH levels and decreased SOD and MnSOD cytosolic activities, TBARS concentration, and H(2)O(2) induced-mPTP opening in both rat strains. Our results show that in both hypertrophied and normal hearts an attenuation of mPTP opening via reduction of oxidative stress appears to be the predominant mechanism involved in the cardioprotection against reperfusion injury MPG-mediated., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The aim of this study was to examine the effects of GSK-3 β inhibitors compared with PRE and POS in spontaneously hypertensive rats (SHR). Isolated hearts were submitted to the following protocols: IC: 45 min global ischemia (GI) and 1-hour reperfusion (R); PRE: a cycle of 5 min GI and 10 minutes of R prior to 45 min GI; POS: three cycles of 30 sec GI/30 sec R at the start of R. Other hearts received lithium chloride (LiCl) or indirubin-3'-monoxime,5-iodo-(IMI) as GSK-3 β inhibitors. All interventions reduced the infarct size observed in IC group. The expressions of P-GSK-3 β and P-Akt decreased in IC and were restored after PRE, POS, and GSK-3 β inhibitors treatments. An increase of cytosolic MnSOD activity and lipid peroxidation and a decrease of GSH content observed in IC hearts were attenuated in PRE, POS, and LiCl or IMI treatments. An increase of P-GSK-3 β /VDAC physical association and a partial recovery of mitochondrial permeability were also detected after interventions. These data show that, in SHR hearts, GSK-3 β inhibitors mimic the cardioprotection afforded by PRE and POS and suggest that a decrease in mitochondrial permeability mediated by P-GSK-3 β /VDAC interaction is a crucial event.

Growing in vitro evidence suggests NHE-1, a known target for reactive oxygen species (ROS), as a key mediator in cardiac hypertrophy (CH). Moreover, NHE-1 inhibition was shown effective in preventing CH and failure; so has been the case for AT1 receptor (AT1R) blockers. Previous experiments indicate that myocardial stretch promotes angiotensin II release and post-translational NHE-1 activation; however, in vivo data supporting this mechanism and its long-term consequences are scanty. In this work, we thought of providing in vivo evidence linking AT1R with ROS and NHE-1 activation in mediating CH. CH was induced in mice by TAC. A group of animals was treated with the AT1R blocker losartan. Cardiac contractility was assessed by echocardiography and pressure-volume loop hemodynamics. After 7 weeks, TAC increased left ventricular (LV) mass by ~45% vs. sham and deteriorated LV systolic function. CH was accompanied by activation of the redox-sensitive kinase p90(RSK) with the consequent increase in NHE-1 phosphorylation. Losartan prevented p90(RSK) and NHE-1 phosphorylation, ameliorated CH and restored cardiac function despite decreased LV wall thickness and similar LV systolic pressures and diastolic dimensions (increased LV wall stress). In conclusion, AT1R blockade prevented excessive oxidative stress, p90(RSK) and NHE-1 phosphorylation, and decreased CH independently of hemodynamic changes. In addition, cardiac performance improved despite a higher work load.

The objective of this study is to assess the participation of mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels in the cardioprotective effects of the Na(+)/H(+) exchanger (NHE-1) blocker cariporide in isolated rat hearts. Regional ischemia was induced by occlusion of left anterior descending coronary artery during 40 min followed by 2-h reperfusion (IC). Cariporide (C, 10 μΜ), or C plus 5-hydroxydecanoate (5-HD, 100 μM, a selective mitoK(ATP) channel inhibitor), or C plus chelerythrine (Chele, 1 μM, a PKC inhibitor), or an opener of mitoK(ATP) channels, diazoxide (Dz, 100 μM) was applied at the onset of reperfusion. Infarct size (IS) and myocardial function were evaluated. The calcium-induced permeability transition pore (mPTP) opening was determined by measuring the light scattering decrease (LSD, a.u.) in isolated mitochondria in the absence and presence of C, C + 5-HD and Dz. IS was 33 ± 2% of the risk area in IC and was significantly diminished by C (15 ± 2%, p < 0.05), which also improved myocardial function [LVDP = 58 ± 5% (IC) vs 80 ± 5% (C)] and blunted LSD [0.80 ± 0.04 (IC) vs 0.51 ± 0.04 (C) a.u.]. 5-HD and Chele were both able to abolish the cardioprotective effects of C on IS. Dz treatment decreased IS and LSD to a similar extent to that produced by C (15 ± 4% and 0.52 ± 0.04 a.u., respectively). The present data suggest that attenuation of mPTP opening after PKC-mediated mitoK(ATP) channel activation is a crucial step for the cardioprotective effects of cariporide.

Background/aims: Flow restoration to ischemic myocardium reduces infarct size (IS), but it also promotes reperfusion injury. A burst of reactive oxygen species (ROS) and/or NHE-1 reactivation were proposed to explain this injury. Our study was aimed to shed light on this unresolved issue., Methods: Regional infarction (40 min-ischemia/2 hs-reperfusion) was induced in isolated and perfused rat hearts. Maximal doses of N-(2-mercaptopropionyl)-glycine (MPG 2mmol/L, ROS scavenger), cariporide (10μmol/L, NHE-1 inhibitor), or sildenafil (1μmol/L, phosphodiesterase5A inhibitor) were applied at reperfusion onset. Their effects on IS, myocardial concentration of thiobarbituric acid reactive substances (TBARS), ERK1/2, p90(RSK), and NHE-1 phosphorylation were analyzed., Results: All treatments decreased IS ∼ 50% vs. control. No further protection was obtained by combining cariporide or MPG with sildenafil. Myocardial TBARS increased after infarction and were decreased by MPG or cariporide, but unaffected by sildenafil. In line with the fact that ROS induce MAPK-mediated NHE-1 activation, myocardial infarction increased ERK1/2, p90(RSK), and NHE-1 phosphorylation. MPG and cariporide cancelled these effects. Sildenafil did not reduce the phosphorylated ERK1/2-p90(RSK) levels but blunted NHE-1 phosphorylation suggesting a direct dephosphorylating action., Conclusions: 1) Reperfusion injury would result from ROS-triggered MAPK-mediated NHE-1 phosphorylation (and reactivation) during reperfusion; 2) sildenafil protects the myocardium by favouring NHE-1 dephosphorylation and bypassing ROS generation., (Copyright © 2011 S. Karger AG, Basel.)

The aim of this investigation was to study the connection between body size, fatty acid composition and sensitivity to lipid peroxidation of heart mitochondria and microsomes isolated from different size bird species: manon (Lonchura striata), quail (Coturnix coturnix var japonica), pigeon (Columba livia), duck (Cairina moschata) and goose (Anser anser), representing a 372-fold range of body mass. Fatty acids of total lipids were determined using gas chromatography and lipid peroxidation was evaluated with a chemiluminescence assay. The fatty acids present in heart organelles of the different bird species analyzed showed a small number of significant allometric trends. In mitochondria, from the individual fatty acid data, palmitoleic acid (C16:1 n7) increased allometrically (r=0.878), while stearic acid (C18:0) was negatively related to body mass (r=-0.903). Interestingly, none of the calculated fatty acid variables, the average fatty acid saturated, monounsaturated, polyunsaturated (PUFA) and the unsaturation index (UI) was established to show significant body size-related variations. In heart microsomes, the content of C18:0 was significantly smaller (r=-0.970) in the birds of greater size. A significant allometric increase in linoleic acid (C18:2 n6) (r=0.986), polyunsaturated (r=0.990) and UI (r=0.904) was observed in the larger birds. The total n6 fatty acids of heart mitochondria did not show significant differences when it was correlated to body mass of the birds. Moreover, positive allometric relationships were shown for microsomes. The total n3 fatty acids of heart mitochondria and microsomes indicated no significant correlations to body mass of birds. The C16:1 n7, C18:0 in mitochondria and C18:0, C18:2 n6, PUFA, UI and PUFA n6 in microsomes showed significant differences when they were correlated to maximum life span (MLSP) of birds. As light emission=chemiluminescence originated from heart organelles was not statistically significant, a lack of correlation between the sensitivity to lipid peroxidation and body size or maximum life span was obtained. These results indicate that the high resistance of bird hearts to the attack by free radicals is body size-independent and would be related to the preservation of cardiac function.

The possibility of a direct mitochondrial action of Na(+)/H(+) exchanger-1 (NHE-1) inhibitors decreasing reactive oxygen species (ROS) production was assessed in cat myocardium. Angiotensin II and endothelin-1 induced an NADPH oxidase (NOX)-dependent increase in anion superoxide (O(2)(-)) production detected by chemiluminescence. Three different NHE-1 inhibitors [cariporide, BIIB-723, and EMD-87580] with no ROS scavenger activity prevented this increase. The mitochondria appeared to be the source of the NOX-dependent ROS released by the "ROS-induced ROS release mechanism" that was blunted by the mitochondrial ATP-sensitive potassium channel blockers 5-hydroxydecanoate and glibenclamide, inhibition of complex I of the electron transport chain with rotenone, and inhibition of the permeability transition pore (MPTP) by cyclosporin A. Cariporide also prevented O(2)(-) production induced by the opening of mK(ATP) with diazoxide. Ca(2+)-induced swelling was evaluated in isolated mitochondria as an indicator of MPTP formation. Cariporide decreased mitochondrial swelling to the same extent as cyclosporin A and bongkrekic acid, confirming its direct mitochondrial action. Increased O(2)(-) production, as expected, stimulated ERK1/2 and p90 ribosomal S6 kinase phosphorylation. This was also prevented by cariporide, giving additional support to the existence of a direct mitochondrial action of NHE-1 inhibitors in preventing ROS release. In conclusion, we report a mitochondrial action of NHE-1 inhibitors that should lead us to revisit or reinterpret previous landmark observations about their beneficial effect in several cardiac diseases, such as ischemia-reperfusion injury and cardiac hypertrophy and failure. Further studies are needed to clarify the precise mechanism and site of action of these drugs in blunting MPTP formation and ROS release.

The objective of this investigation was to examine the relationship between body size, fatty acid composition and sensitivity to lipid peroxidation of mitochondria and microsomes isolated from the brain of different size bird species: manon, quail, pigeon, duck and goose, representing a 372-fold range of body mass. Fatty acids of total lipids were determined using gas chromatography and lipid peroxidation was evaluated using a chemiluminescence assay. The allometric study of the fatty acids present in brain mitochondria and microsomes of the different bird species showed a small number of significant allometric trends. In mitochondria the percentage of monounsaturated fatty acids, was significantly lower in the larger birds (r=-0.965; P<0.008). The significant allometric increase in 18:2 n-6; linoleic acid (r=0.986; P<0.0143), polyunsaturated (r=0.993; P<0.007) and total unsaturated (r=0.966; P<0.034) in brain microsomes but not in mitochondria may indicate a preferential incorporation of this fatty acid in the brain endoplasmic reticulum of the larger bird species. The brain of all birds studied had a high content of docosahexaenoic acid. However brain mitochondria but not microsomes isolated from all the birds analyzed showed a significant decrease of arachidonic and docosahexaenoic acids during lipid peroxidation. The allometric analyses of chemiluminescence were not statistically significant. In conclusion our results show absence of correlation between the sensitivity to lipid peroxidation of brain mitochondria and microsomes with body size and maximum life span.

The aim of this work was to assess the possible correlation between oxidative damage and the development of cardiac hypertrophy in heart tissue from young (40-d-old) and older (4-, 11- and 19-month-old) spontaneously hypertensive rats (SHR) in comparison with age-matched Wistar (W) rats. To this end, levels of thiobarbituric acid reactive substances (TBARS), nitrotyrosine contents, NAD(P)H oxidase activity, superoxide production, and the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were determined. Compared to age-matched normotensive rats, SHR showed a significant increase in systolic blood pressure from 40 d of age and left ventricular hypertrophy (LVH) was significantly evident from 4 months of age. W rats (11- and 19-month-old) also showed an increase in LVH with aging. TBARS and nitrotyrosine levels were similar in young rats from both strains and were significantly increased with age in both strains, with the values in SHR being significantly higher than those in age-matched W rats. NAD(P)H activity was similar in young SHR and W rats, whereas it was higher in aged SHR compared with age-matched W rats. Compared to W rats, superoxide production was higher in aged SHR, and was abolished by NAD(P)H inhibition with apocynin. CAT activity was increased in the hearts of 4-month-old SHR compared to age-matched W rats and was decreased in the hearts of the oldest SHR compared to the oldest W rats. SOD and GPx activities decreased in both rat strains with aging. Moreover, an increase in collagen deposition with aging was evident in both rat strains. Taken together, these data showed that aged SHR exhibited higher cardiac hypertrophy and oxidative damage compared to W rats, indicating that the two undesirable effects are associated. That is, oxidative stress appears to be a cause and/or consequence of hypertrophy development in this animal model.

To determine whether sarcolemmal and/or mitochondrial ATP-sensitive potassium (K(ATP)) channels (sarcK(ATP), mitoK(ATP)) are involved in stretch-induced protection, isolated isovolumic rat hearts were assigned to the following protocols: nonstretched hearts were subjected to 20 min of global ischemia (Is) and 30 min of reperfusion, and before Is stretched hearts received 5 min of stretch + 10 min of no intervention. Stretch was induced by a transient increase in left ventricular end-diastolic pressure (LVEDP) from 10 to 40 mmHg. Other hearts received 5-hydroxydecanoate (5-HD; 100 microM), a selective inhibitor of mitoK(ATP), or HMR-1098 (20 microM), a selective inhibitor of sarcK(ATP), before the stretch protocol. Systolic function was assessed through left ventricular developed pressure (LVDP) and maximal rise in velocity of left ventricular pressure (+dP/dt(max)) and diastolic function through maximal decrease in velocity of left ventricular pressure (-dP/dt(max)) and LVEDP. Lactate dehydrogenase (LDH) release and ATP content were also measured. Stretch resulted in a significant increase of postischemic recovery and attenuation of diastolic stiffness. At 30 min of reperfusion LVDP and +dP/dt(max) were 87 +/- 4% and 92 +/- 6% and -dP/dt(max) and LVEDP were 95 +/- 9% and 10 +/- 4 mmHg vs. 57 +/- 6%, 53 +/- 6%, 57 +/- 10%, and 28 +/- 5 mmHg, respectively, in nonstretched hearts. Stretch increased ATP content and did not produce LDH release. 5-HD did not modify and HMR-1098 prevented the protection achieved by stretch. Our results show that the beneficial effects of stretch on postischemic myocardial dysfunction, cellular damage, and energetic state involve the participation of sarcK(ATP) but not mitoK(ATP).

1. We reported recently the cardioprotection conferred by a non-alcoholic extract of Cabernet-Sauvignon red wine (RWE) against alterations derived from ischaemia and reperfusion in normotensive rats. The aim of the present study was to assess the effects of RWE on ischaemia/reperfusion injury in hearts isolated from spontaneously hypertensive rats (SHR). 2. After stabilization, rat isovolumic perfused hearts were exposed to a 20 min global ischaemic period followed by 30 min reperfusion in the absence (ischaemic control (IC) hearts) or presence of RWE infused prior to ischaemia and early in reperfusion. In other hearts, N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, was administered prior to RWE infusion (L-NAME + RWE). 3. Left ventricular developed pressure (LVDP), dP/dt(max) and left ventricular end-diastolic pressure (LVEDP) were used to assess myocardial function. 4. At the end of reperfusion, LVDP and dP/dt(max) decreased to 47 +/- 9 and 46 +/- 9% of baseline values, respectively, in IC. Treatment with the RWE significantly improved systolic postischaemic recovery (LVDP = 85 +/- 8%; dP/dt(max) = 83 +/- 5%) and attenuated the increase in LVEDP (23 +/- 6 and 53 +/- 8 mmHg in RWE and IC, respectively; P < 0.05). 5. In the L-NAME + RWE group, L-NAME completely abolished the systolic and diastolic protection induced by RWE (LVDP = 44 +/- 13%; dP/dt(max) = 43 +/- 13%; LVEDP = 60 +/- 10 mmHg). 6. These data are the first demonstration that a non-alcoholic extract of Cabernet-Sauvignon red wine protects SHR hearts from systolic and diastolic alterations induced by ischaemia and reperfusion through a nitric oxide-dependent mechanism.

The aim of this study was to examine the fatty acid composition and non-enzymatic lipid peroxidation (LP) of mitochondria and microsomes obtained from liver, heart and brain of Lonchura striata. The percentage of total unsaturated fatty acid was approximately 30-60% in the organelles from all tissues studied. Brain mitochondria and both organelles of liver exhibited the highest percentage of polyunsaturated fatty acid (PUFA) (30 and 18%, respectively). The arachidonic acid (AA) content was 7% in mitochondria of liver and brain and 3% in heart mitochondria. The percentage of docosahexanoic acid (DHA) was 8% in brain mitochondria and approximately 2-3% in heart and liver mitochondria. The peroxidizability index (PI) of brain mitochondria and both organelles from liver was higher than that of organelles from heart and brain microsomes. Liver organelles and brain mitochondria were affected by LP, as indicated by the increase in chemiluminescence and a decrease of AA and DHA. These changes were not observed during LP of brain microsomes and both organelles from heart. These results indicate: 1) PI positively correlates with PUFA percentage and LP; 2) The resistance to LP detected in heart organelles would contribute to the cardiac protection against oxidative damage.

Brief episodes of myocardial ischemia-reperfusion applied early in reperfusion may attenuate the reperfusion injury, strategy called ischemic postconditioning (IPO). Our objective was to examine the effects of IPO compared with ischemic preconditioning (IP) on postischemic myocardial dysfunction in spontaneously hypertensive rats (SHR). Isolated hearts from SHR and normotensive WKY rats were subjected to the following protocols: (1) Ischemic control (IC): global ischemia 20 min (GI20) and reperfusion 30 min (R). (2) IPO: three cycles of R30sec-IG30sec at the onset of R; (3) IP: a cycle of IG5-R10 previous to GI20, (4) IPO in the presence of chelerythrine, an inhibitor of protein kinase C (PKC). Systolic and diastolic function were assessed through developed pressure (LVDP) and end diastolic pressure (LVEDP), respectively. Lipid peroxidation was estimated by thiobarbituric reactive substance (TBARS) concentration. IPO significantly improved postischemic dysfunction. At the end of R, LVDP recovered to 87 +/- 7% in WKY and 94 +/- 7% in SHR vs. 55 +/- 11% and 58 +/- 12% in IC hearts. LVEDP reached values of 24 +/- 6 mmHg for WKY and 24 +/- 3 mmHg for SHR vs. 40 +/- 8 and 42 +/- 5 mmHg in IC hearts. Similar protection was achieved by IP. TBARS contents of SHR hearts were significantly diminished by IP and IPO. PKC inhibition aborted the protection of myocardial function and attenuated the diminution of lipid peroxidation conferred by IPO. These data show that IPO was as effective as IP in improving the postischemic dysfunction of hearts from SHR hearts, and that this cardioprotection appears to be associated with a diminution of ROS-induced damage involving the PKC activation.

Background: A burst of reactive oxygen species and activation of Na+/H+ exchanger take place at the beginning of reperfusion. The aim of this study was to assess the possible interrelation of the inhibition of Na+/H+ exchanger and reactive oxygen species about the determination of myocardial infarct size., Methods: Isolated rat hearts were submitted to 40 min of coronary occlusion and 2 h of reperfusion. Infarct size was determined through triphenyltetrazolium chloride staining technique and was expressed as a percentage of risk area. Lipid peroxidation, as a marker of oxidative stress, was estimated by the concentration of thiobarbituric reactive substances., Results: Treatment during the first 20 min of reperfusion with a selective inhibitor of Na+/H+ exchanger 1 isoform, HOE 642 (cariporide; 10 microM), significantly diminished infarct size (15.1+/-2.4% vs. 31+/-2% in untreated hearts). The administration of a "scavenger" of hydroxyl radical, N-(2-mercaptopropionyl)-glycine (2 mM), decreased infarct size in an extent similar to that of cariporide (18+/-3%). The combination cariporide+N-(2-mercaptopropionyl)-glycine did not produce additional protection (17+/-1.7%). Each intervention [HOE 642 or N-(2-mercaptopropionyl)-glycine] and its combination improved the postischemic recovery of myocardial systolic and diastolic functions in a similar extent. The content of the thiobarbituric reactive substances of untreated hearts (1012+/-144 nmol/g) decreased to 431+/-81, 390+/-82, and 433+/-41 after cariporide, N-(2-mercaptopropionyl)-glycine, and cariporide+N-(2-mercaptopropionyl)-glycine treatments, respectively., Conclusions: The present data support the conclusion that the cardioprotective effect of cariporide is associated with diminution of oxidative stress.

Birds -- particularly long-lived species -- have special adaptations for preventing tissue damage caused by reactive oxygen species. The objective of the present study was to analyse the fatty acid composition and non-enzymatic lipid peroxidation of mitochondria and microsomes obtained from liver, heart and brain of quail (Coturnix coturnix japonica), a short-lived bird. Fatty acids located in total lipids of rat liver, heart and brain mitochondria and microsomes were determined using gas chromatography and lipid peroxidation was evaluated using a chemiluminescence assay. The unsaturated fatty acid content found in mitochondria and microsomes of all tissue examined was approximately 50 and 40%, respectively with a prevalence of C18:1 n9. The C18:2 n6 content in brain mitochondria was significantly lower as compared to liver and heart mitochondria. Whereas the C20:4 n6 content in mitochondria from all tissues examined and brain microsomes was approximately 6%, liver and heart microsomes exhibited lower values. C22:6 n3 was absent in liver mitochondria, very low content in liver microsomes and heart organelles (between 0.5 and 1%) and high content in brain organelles, with mitochondria having the highest value (11%). Whereas liver and heart organelles were not affected when subjected to lipid peroxidation, brain mitochondria were highly affected, as indicated by the increase in chemiluminescence and a considerable decrease of C20:4 n6 and C22:6 n3. These results indicate that a low degree of fatty acid unsaturation in liver and heart organelles of quail, a short-lived bird, may confer advantage by decreasing their sensitivity to lipid peroxidation process.

Aim: To examine the effects of an Ilex paraguariensis (Ip) extract on postischemic alterations derived from 20 min of global ischemia and 30 min of reperfusion., Methods: Isolated rat hearts were treated 10 min before ischemia and the first 10 min of reperfusion with Ip 30 microg/ml. In other hearts, chelerythrine (1 microM), a protein kinase C blocker, or l(G)-nitro l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor, were administered prior to Ip infusion. Left ventricular developed pressure (LVDP), +dP/dt(max), -dP/dt(max), and left ventricular end diastolic pressure (LVEDP) were used to assess myocardial function. Thiobarbituric acid reactive substances (TBARS) were measured., Results: Ip treatment produced an improvement of postichemic recovery (LVDP=96+/-8%; +dP/dt(max)=95+/-10%; -dP/dt(max)=90+/-12% vs. 57+/-6%, 53+/-6% and 57+/-8%, respectively, in untreated hearts) and an attenuation of the increase of LVEDP and TBARS content. Chelerythrine did not modify and l-NAME abolished the protection induced by Ip., Conclusions: These data are the first demonstration that Ip extract attenuates the myocardial dysfunction provoked by ischemia and reperfusion and that this cardioprotection involves a diminution of oxidative damage through a nitric oxide-dependent mechanism.

We have recently demonstrated the cardioprotective effects of a non-alcoholic extract of Argentinian red wine (RWE) on ischemia-reperfusion injury. The aim of the present study was to assess the relative contribution of four phenolic fractions separated from RWE by liquid/liquid extraction with solvents of decreasing hydrophobicity, to the myocardial protection achieved by the original extract. Isovolumic perfused rat hearts treated with each fraction 10 min before ischemia and the first 10 min of reperfusion were submitted to a 20-min global ischemic period followed by 30 min of reperfusion. The treatment with the fraction rich in polymeric proanthocyanidins (fraction IV = aqueous residue) significantly improved the postischemic recovery of left ventricular developed pressure (LVDP) and +dP/dt (max) (111 +/- 5% and 117 +/- 6% vs 61 +/- 4%, 62 +/- 5% , respectively, detected in control hearts) and abolished the increase of left ventricular end diastolic pressure (LVEDP) (8 +/- 2 mmHg vs 42 +/- 4 mmHg in untreated hearts). However, the fraction rich in anthocyanins (III: butanol) elicited a cardioprotective action weaker than the original extract. On the other hand, the representative of either resveratrol or flavan-3-ols and flavonols (fractions I and II) failed to induce this type of response. LDH release and TBARS concentration were significantly lowered after treatment with fraction IV alone. These data show that the fraction rich in polymeric proanthocyanidins exerts a protective effect against myocardial alterations derived from ischemia and reperfusion comparable to the original RWE. This beneficial effect can be correlated to the ability of that fraction to attenuate the degree of lipid peroxidation.

Many reports have demonstrated that birds show a low degree of fatty acid unsaturation and lipid peroxidation compared with mammals of similar body size. The aim of the present study was to examine fatty acid profiles, non-enzymatic lipid peroxidation and vitamin E levels of mitochondria and microsomes obtained from liver, heart and brain of goose (Anser anser). The unsaturated fatty acid content found in mitochondria and microsomes of all tissues examined was approximately 60% with a prevalence of C18:1 n9 + C18:2 n6 = 50%. The 20:4 n6 + C22:6 n3 content was significantly higher in brain organelles (approx. 16%) compared with mitochondria and microsomes of liver and heart (approx. 4%). Whereas these organelles were not affected when subjected to lipid peroxidation, brain mitochondria were highly affected, as indicated by the increase in chemiluminescence and a considerable decrease of arachidonic and docosahexaenoic acids. These changes were not observed during lipid peroxidation of brain microsomes. Vitamin E content was higher in liver and heart than in brain mitochondria (1.77 +/- 0.06 and 1.93 +/- 0.13 vs. 0.91 +/- 0.09 nmol/mg protein). The main conclusion of this paper is that a lower degree of unsaturation of fatty acids in liver and heart mitochondria and a higher vitamin E level than in brain mitochondria protect those tissues against lipid peroxidation.

Background: The protection of high magnesium on infarct size remains controversial., Objective: To examine the effects of magnesium administered before ischemia or early in reperfusion on infarct size in a rat model of global ischemia, Methods: Isolated rat hearts were submitted to 40 min of normothermic global ischemia and 2 h of reperfusion. After 20 min of stabilization, four protocols were performed: ischemic control (IC) hearts; 15 mM of magnesium chloride administered 15 min before ischemia (MgI); 15 mM of magnesium chloride administered during the first 15 min of reperfusion (MgR); or 15 mM magnesium plus 5 mM calcium (Mg+Ca) before ischemia. Infarct size was measured by triphenyltetrazolium staining. Contractile function was assessed by left ventricular developed pressure and the maximal velocity of rise of left ventricular presssure., Results: The infarct size in IC hearts was 44+/-5%. In MgI and MgR hearts, the infarct diminished to 4.5+/-1.5% and 18+/-4%, respectively. In Mg+Ca hearts, the protection was also obtained (19+/-3%). Myocardial function also improved significantly by magnesium treatment. At the end of reperfusion, left ventricular developed pressure and maximal velocity of rise of left ventricular pressure values were 23+/-6% and 22+/-3% in MgI; and 10+/-3% and 9+/-2.6% in MgR versus 2+/-0.7% and 2.3+/-0.8% in IC hearts, respectively., Conclusion: The treatment with magnesium either before ischemia or early in reperfusion has an infarct size limiting effect in a model of global ischemia. This protective effect is partially due to its calcium antagonistic action.

The objective was to analyze the mechanism of the protection induced by a nonalcoholic extract of red wine (RWE) on ischemia/reperfusion injury. Isovolumic perfused rat hearts were exposed after stabilization to a 20-min global ischemic period followed by 30 min of reperfusion in absence and presence of RWE infused prior to ischemia and early in reperfusion. In other hearts, 5-hydroxydecanoate (5-HD, 100 microM), a selective mitochondrial K(ATP) blocker, chelerythrine (1 microM), a protein kinase C blocker, or >L(G)-nitro->L-arginine methyl ester (>L-NAME), a nitric oxide synthase inhibitor, was administered prior to RWE infusion. Left ventricular developed pressure (LVDP), +dP/dtmax, and left ventricular end-diastolic pressure (LVEDP) were used to assess myocardial function. The lactate dehydrogenase release during reperfusion was measured. After the ischemic period, LVDP decreased to 61 +/- 4% and +dP/dtmax to 62 +/- 5% of baseline values at the end of reperfusion. The infusion of RWE resulted in a complete recovery of systolic function (LVDP = 102 +/- 4%; +dP/dtmax = 101 +/- 4%) and in an attenuation of the increase of LVEDP (20 +/- 3 mm Hg versus 42 +/- 4 mm Hg, p < 0.05). The treatment with RWE did not produce lactate dehydrogenase release during reperfusion. 5-HD and chelerythrine completely abolished the protection induced by RWE (mechanical and enzymatic). >L-NAME partially abolished the systolic improvement induced by RWE but returned lactate dehydrogenase loss to ischemic control values. The diastolic protection afforded by RWE was not altered by >L-NAME. These data are the first demonstration that mitochondrial K channels and nitric oxide are involved in the protection against ischemia/reperfusion conferred by a nonalcoholic RWE.

Objective: To establish whether the total antioxidant capacity of nonalcoholic extracts of three Argentine red wines (RWE) is correlated with their protection against ischemia-reperfusion injury., Animals and Methods: The antioxidant properties of three RWE were determined using different free radical-generating systems. To examine the effects of these RWE during a 20 min global ischemic period followed by 30 min of reperfusion, isolated rat hearts received 50 mug/mL of RWE 1 (cabernet-sauvignon), RWE 2 (malbec) or RWE 3 (a commercial mixture of cabernet-sauvignon, malbec and merlot) 10 min before and after ischemia. Left ventricular developed pressure (LVDP), maximal velocity of rise of left ventricular pressure (+dP/dt(max)) and left ventricular end-diastolic pressure (LVEDP) were used to assess contractility and diastolic function., Results: All RWE inhibited lipid peroxidation induced by the Cl(4)C/NADPH system in a similar proportion (42+/-4%, 47+/-9% and 43+/-14% for RWE 1, RWE 2 and RWE 3, respectively). The scavenging activity of superoxide anion and 2,2-diphenyl-1-picryl-hydrazyl radical was about the same with the three RWE. In hearts without RWE treatment, LVDP and +dP/dt(max) were 61+/-4% and 62+/-5%, respectively, at the end of the reperfusion period. Infusion of RWE 1 and RWE 2 significantly improved postischemic recovery (LVDP and +dP/dt(max) were 102+/-4% and 101+/-4% for RWE 1 and 92+/-5% and 91+/-5% for RWE 2, respectively) and attenuated the increase of LVEDP. RWE 3 did not improve either systolic or diastolic dysfunction., Conclusion: These data show that although the three non-alcoholic RWE exhibit a similar total antioxidant capacity, only two of them protect the heart against myocardial stunning, suggesting that the protective effect is not primarily linked to the anti-oxidant properties of the extracts.

Our objective was to assess the participation of Na+/H+ exchanger (NHE) and Na+/Ca2+ exchanger (NCX) on systolic and diastolic alterations of myocardial stunning. Isolated perfused rat hearts were submitted to 20 min of global ischemia (Is) followed by 30 min of reperfusion (R). This protocol was repeated after treatment before ischemia and/or early in R. with HOE 642 1 microM, a specific blocker of NHE-1 and KB-R7943 1 microM the novel inhibitor of the reverse mode of NCX. In control ischemic hearts the contractility assessed through +dP/dtmax recovered approximately 60%. When the NHE blockade was performed before is or early in R the postischemic recovery reached 100%. The blockade of the reverse mode of NCX only improved significantly the recovery when administered before is and early in R (95 +/- 7%). The ischemic contracture decreased when the treatment with both blockers was performed before Is. During R the increase of end diastolic pressure (EDP) observed in control ischemic hearts (at 30 min of R, EDP value was 44 +/- 4 mmHg) diminished significantly by NHE (24 +/- 6 and 12 +/- 2 mmHg when the blocker was administered before or after Is) and NCX blockade performed before and after is (12 +/- 6 mmHg). These results indicate that the activation of the reverse mode of NCX secondary to the NHE activation during ischemia and reperfusion is the mechanism responsible for the Ca2+ overload involved in the diminution of contractility that characterizes myocardial stunning.

The protective effects of ischemic preconditioning (IP) and Na+/H+ exchanger blockade (NHEb) by two blockers [ethylisopropylamiloride (EIPA) and HOE 642] were compared in the isovolumic perfused rat heart. The impairment in systolic and diastolic function detected in control ischemic hearts (C) exposed to 20 min of ischemia and 30 min of reperfusion was diminished in similar extent by IP and by NHEb with EIPA and HOE 642. At the end of the reperfusion period +dP/dtmax values were 57+/-9% in C hearts and 94+/-6%, 82+/-6% and 104+/-6% after IP and NHEb with EIPA and HOE 642, respectively. A depletion of ATP levels detected in C hearts after reperfusion (from 20.2+/-0.8 micromol/g dry weight before ischemia to 6.9+/-0.7 micromol/g dry weight) was partially prevented by both IP and NHEb with EIPA (9.2+/-0.7 micromol/g dry weight and 11.1+/-0.5 micromol/g dry weight, respectively). The ischemic contracture (IC), assessed by the left ventricular end diastolic pressure (LVEDP), observed in C hearts (35+/-4 mmHg) was not decreased by IP (40+/-4 mmHg) but it was prevented by NHEb (18+/-4 mmHg and 10+/-3 mmHg with EIPA and HOE 642, respectively). The ATP levels at the end of the ischemic period were similar in C and IP hearts (4.1+/-0.2 micromol/g dry wt vs. 3.3+/-0.4 micromol/g dry wt) but they were significantly higher after NHEb with HOE 642 (7.0+/-1.0 micromol/g dry wt). PKC inhibition by chelerythrine abolished the protection induced by IP after reperfusion although not the improvement induced by NHEb with EIPA. According to the present results, we can conclude that despite the fact that IP and NHEb are protecting the postischemic function in a similar magnitude, both interventions are different in terms of modifying IC that develops during the ischemic period. IC was prevented by NHEb whereas it was not by IP. Furthermore, IP protection and not that obtained by NHEb is abolished by PKC.

The objective was to assess the action of two Argentine (from Mendoza) non-alcoholic red wine extracts (Cabernet-Sauvignon (CS) and a generic control (G)) on myocardial stunning that follows the reperfusion period. In the isolated isovolumically perfused rat heart the recovery of systolic and diastolic functions after 20 min of global ischemia were assessed through left ventricular developed pressure (LVDP) and end diastolic pressure (EDP), respectively. After 30 min of reperfusion LVDP recovered to 66 +/- 7% in control ischemic hearts. The administration of non-alcoholic extract of CS not of G wine significantly increased the post-ischemic recovery (101 +/- 4%, p < 0.05) of the hearts. The ischemic contracture was not modified by either of the wines. However, during reperfusion the CS, not the G wine, decreased significantly the increase in EDP observed in control ischemic hearts. These results present experimental evidence that the non-alcoholic extracts of CS and not of G Argentine red wine induces protection of postischemic myocardial function.

In a model of global ischemia in the isolated perfused rat heart, a 20 min ischemic period followed by 30 min of reperfusion induces a decrease in isovolumic developed pressure (LVDP) and +dP/dtmax to 61+/-6% and 61+/-7% of baseline, respectively. Left ventricular end-diastolic pressure (LVEDP) increases to 36+/-4 mmHg at the end of the reperfusion period. No significant necrotic area as assessed by triphenyltetrazolium chloride (TTC) was detected at the end of the reperfusion period. By an immunohistochemical method using antiactin monoclonal antibodies 10.8+/-1.9% of unstained cells were detected in the stunned hearts and 10.3+/-1.2% in control hearts. Preceding the ischemic episode with a cycle of 5 min of ischemia followed by 10 min of reperfusion (ischemic preconditioning) protected contractile function. LVDP and +dP/dtmax now stabilized at 89+/-5% and 94+/-5% of baseline respectively. LVEDP was 20+/-2 mmHg at the end of the reperfusion period. The protection of contractile dysfunction after 20 min of ischemia was achieved also by early reperfusion of low Ca2+-low pH perfusate. With this intervention LVDP stabilized at 87+/-5% of baseline. LVEDP was 12+/-2 mmHg at the end of the reperfusion period. A positive inotropic intervention induced by a modified postextrasystolic potentiation protocol at the end of the reperfusion period increases LVDP to levels higher than baseline in the stunned hearts. However, these values were less than those obtained in control hearts. Ischemic preconditioning significantly increased the maximal inotropic response. Therefore, ischemic preconditioning diminishes the contractile dysfunction of early stunning.

The present study provides evidences of left ventricular diastolic alterations following reperfusion in a model of global ischemia. Isolated perfused rabbit and rat hearts, were subjected to ischemia for 15 and 20 min respectively, followed by 30 min of reperfusion. In rabbit heart at the end of the reperfusion period, isovolumic left ventricular developed pressure (LVDP) and +dP/dtmax stabilized at 55 +/- 3% and 60 +/- 2% of preischemic values respectively and, in rat heart LVDP = 61 +/- 8% and +dP/dtmax = 57 +/- 9% of preischemic values. Stunned heart was then obtained from both species. Left ventricular end diastolic pressure (LVEDP) values stabilized at the end of reperfusion period at values higher than preischemic conditions in both species (38.9 +/- 4.4 mmHg and 30.3 +/- 3.1 mmHg in rabbit and rat respectively). The time constant of relaxation (T) increased early in reperfusion in both species, but then decreased and stabilized at the end of reperfusion period at values lower than preischemic values. The ratio between both maximal velocities (+P/-P), also showed a transitory impairment in relaxation, followed by normalization and stabilization at values lower than preischemic values. This biphasic pattern in relaxation was detected in both species. The changes in relaxation were dissociated from the diastolic compliance and could be the result of a transitory calcium overload and/or sarcoplasmic reticulum dysfunction. The faster myocardial relaxation at the end of reperfusion period is consistent with the decreased myofilament sensitivity, which characterizes the stunned myocardium.

Chronic treatment with nifedipine induces up-regulation of functional active Ca2+ channels in cardiac muscle membranes. Adult male New Zealand White rabbits (NZW) were treated with nifedipine (20 mg/day) for 25 days. In isovolumic perfused hearts at constant coronary flow and heart rate (HR) the left ventricular developed pressure (LVDP) and its first derivative (dP/dt) were monitored. Basal contractility and contractility at different end-diastolic volumes (EDV) were higher in nifedipine-treated animals, with no changes in diastolic chamber stiffness. Dose response to nifedipine in pretreated animals showed less decrease in contractility than in controls [ED50 = 1.09 +/- 0.09 x 10-7 (control) and 1.55 +/- 0.17 x 10-7 M nifedipine (treated) (p < 0.05)]. Ca2+ channel density was assessed by specific binding at the dihydropyridine receptor with [methyl-3H]PN 200-110. In cardiac membranes, maximal binding capacity (Bmax) was 269 +/- 38 (n = 7, control) and 429 +/- 46 fmol/mg protein (n = 7, treated) (p < 0.05), without significant changes in dissociation constant. In addition, we noted no changes in dihydropyridine (DHP) binding sites in aortic membranes. Our results offer a possible explanation for the lack of decrease in contractility despite the persistent hypotensive effect in hypertensive patients during chronic treatment with nifedipine.

The aim of the present study was to evaluate the effects of a single dose of nifedipine on myocardial stunning in isolated rabbit hearts. Hearts from rabbits pretreated with a single dose of 20 mg of nifedipine (NIF group) 1-4 h before isolation were compared to control hearts in their response to 15 min of global ischemia (37 degrees C) followed by 30 min of reperfusion. Both experimental groups showed similar baseline cardiac contractility. At the end of the reperfusion period in the control group, isovolumic left ventricular developed pressure (LVDP) and +dP/dtmax had stabilized at 45 +/- 2 and 48 +/- 2% of preischemic values respectively and in the NIF group LVDP stabilized at 63 +/- 6 and +dP/dtmax at 66 +/- 6% (P < 0.05 with respect to the control group). Left ventricular end diastolic pressure (LVEDP) values were significantly lower at the end of reperfusion in the NIF group compared to the controls (36.8 +/- 5.5 mmHg vs 53.4 +/- 3.9 mmHg, P < 0.05). The early impairment of the time constant of relaxation (tau) observed in control hearts was attenuated by pretreatment with nifedipine (control delta tau = 55 +/- 10 ms; NIF group delta tau = 29 +/- 5 ms, P < 0.05). Tissue ATP and creatine phosphate (CP) levels in the control group at the end of reperfusion were 6.9 +/- 0.7 and 8.7 +/- 0.7 mumol/g dry tissue, respectively; in the NIF group ATP and CP levels were significantly higher, 9.2 +/- 0.7 and 11.5 +/- 0.9 mumol/g dry tissue respectively (P < 0.05). Creatine kinase (CK) and lactate dehydrogenase (LDH) leakage during reperfusion were significantly higher in the control group (CK = 120 +/- 15 mU/ml and LDH = 60 +/- 8 mU/ml) compared to the NIF group (CK = 82 +/- 5 mU/ml and LDH = 41 +/- 2 mU/ml, P < 0.05). Our results demonstrate that pretreatment with a single oral dose (20 mg) of nifedipine attenuates systolic and diastolic functional alterations as well as the metabolic impairment associated with stunning in the isolated rabbit heart.

The effects of preconditioning, adenosine and dipyridamole in protecting the systolic and diastolic alterations of myocardial stunning in rabbit hearts were studied. Isovolumic left ventricular developed pressure (LVDP), and end diastolic pressure (LVEDP) were measured. The time constant of relaxation (T) was calculated. Isolated rabbit hearts were subject to 15 min of global ischemia (37 degrees C) followed by 30 min of reperfusion. LVDP and LVEDP stabilized to 55 +/- 5% and 320 +/- 28% of control values respectively (stunned group) T increased early in reperfusion (from 48.2 +/- 3.9 to 97.2 +/- 10 ms P < 0.05) but returned to control value late in reperfusion. When hearts were preconditioned by a single cycle of 5 min of ischemia LVDP and LVEDP stabilized at 89 +/- 3% and 162 +/- 34% of preischemic values respectively (P < 0.05 with respect to stunned group). The change in T was attenuated (62 +/- 6 ms at 5 min of reperfusion, P < 0.05 with respect to stunned group). Hearts treated either with adenosine (800 micrograms/min) or the nucleoside transport blocker dipyridamole (4 micrograms/min) previously to the ischemia, recovered their LVDP to 86 +/- 1% and 82 +/- 3% of preischemic values, respectively (P < 0.05 with respect to stunned group). Adenosine and dipyridamole also attenuated the increase in LVEDP (195 +/- 12% and 197 +/- 10% respectively, P < 0.05 with respect to stunned group).(ABSTRACT TRUNCATED AT 250 WORDS)

In rabbits chronically pretreated with nifedipine (20 mg/day for 25 days), we demonstrate upregulation of Ca2+ channels in mammalian heart (maximal binding capacity = 222 +/- 14 and 421 +/- 55 fmol/mg protein for control and treated, respectively; P < 0.05) without significant changes in dissociation constant. No changes in calcium sensitivity or maximal force were detected by pretreatment with nifedipine in chemically skinned fibers. Cardiac contractility at different extracellular Ca2+ concentrations was similar in control and pretreated animals. This upregulation of Ca2+ channels in mammalian heart offers an explanation for the lack of significant negative inotropic effect after chronic administration of nifedipine.