Cardioprotection of benzolamide in a regional ischemia model: Role of eNOS/NO.

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 ±â€¯0.5% vs. 34 ±â€¯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.

Protective effects of N-(2-mercaptopropionyl)-glycine against ischemia-reperfusion injury in hypertrophied hearts.

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.

Is N-methylacetazolamide a possible new therapy against ischemia-reperfusion injury?

The increase of intracellular Ca2+ concentration, produced principally by its influx through the L-type Ca2+ 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 Ca2+ 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.

Cardioprotective effects of N-methylacetazolamide mediated by inhibition of L-type Ca2+ channel current.

Our objective was to examine the effects of N-methylacetazolamide (NMA), a non­carbonic 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 Aß were measured. In cardiomyocytes the L-type Ca2+ 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 Aß 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.

In vivo key role of reactive oxygen species and NHE-1 activation in determining excessive cardiac hypertrophy.

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.

Myocardial reperfusion injury: reactive oxygen species vs. NHE-1 reactivation.

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.

Myocardial and mitochondrial effects of the anhydrase carbonic inhibitor ethoxzolamide in ischemia-reperfusion.

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 Aß content were assessed. In isolated mitochondria, the Ca2+ response, Ca2+ 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.

Isoespintanol, a monoterpene isolated from oxandra cf xylopioides, ameliorates the myocardial ischemia-reperfusion injury by AKT/PKCε/eNOS-dependent pathways.

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.

Calcineurin/P38MAPK/HSP27-dependent pathways are involved in the attenuation of postischemic mitochondrial injury afforded by sodium bicarbonate co-transporter (NBCe1) inhibition.

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 Ca2+ response (CaR), Ca2+ 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.

Na+/H+ exchanger-1 inhibitors decrease myocardial superoxide production via direct mitochondrial action.

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.

Is cardiac hypertrophy in spontaneously hypertensive rats the cause or the consequence of oxidative stress?

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.

Survival kinase-dependent pathways contribute to gender difference in the response to myocardial ischemia-reperfusion and ischemic post-conditioning.

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 3ß (GSK-3ß), 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-3ß/PKCε/eNOS-dependent pathways-mediated superoxide production and apoptosis appear as important factors involved in the observed gender differences.

Cardioprotection and natural polyphenols: an update of clinical and experimental studies.

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.

Benzolamide perpetuates acidic conditions during reperfusion and reduces myocardial ischemia-reperfusion injury.

During ischemia, increased anaerobic glycolysis results in intracellular acidosis. Activation of alkalinizing transport mechanisms associated with carbonic anhydrases (CAs) leads to myocardial intracellular Ca2+ 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 (pHi) normalization after transient acid loading in the absence and presence of BZ or BZ + SB202190 in heart papillary muscles (HPMs). Mitochondrial membrane potential (ΔΨm), Ca2+ retention capacity and Ca2+-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 ΔΨm and Ca2+ homeostasis, effects abolished after p38MAPK inhibition. In HPMs, BZ slowed pHi 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 Ca2+ handling and a more depolarized membrane potential compared with control hearts, and these changes were restored by treatment with BZ or AR.

Na+/H+ exchanger inhibition at the onset of reperfusion decreases myocardial infarct size: role of reactive oxygen species.

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.

Data supporting the cardiac mitochondria calcium handling in female normotensive and spontaneously hypertensive rats.

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.

Ex Vivo Treatment with a Polyphenol-Enriched Cocoa Extract Ameliorates Myocardial Infarct and Postischemic Mitochondrial Injury in Normotensive and Hypertensive Rats.

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-3ß, 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-3ß, 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-3ß/eNOS dependent pathways are involved.

Effect of an Ilex paraguariensis (yerba mate) extract on infarct size in isolated rat hearts: the mechanisms involved.

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.

Cardioprotective effects of Ilex paraguariensis extract: evidence for a nitric oxide-dependent mechanism.

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.

Effects of different fractions of a red wine non-alcoholic extract on ischemia-reperfusion injury.

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.