Erythropoietin-mediated cardioprotection in hearts subjected to ischemia reperfusion.

Several studies provide evidence that erythropoietin (EPO) could play an important role in the recovery of the heart subjected to ischemia-reperfusion. In this regard, it has been suggested that EPO could be involved in protein kinase B (Akt) activation as a cell survival protein. The aim of the present study was to investigate the effects of EPO on the Akt/glycogen synthase kinase 3 beta (GSK-3ß) pathway in the presence or absence of wortmannin (W, Akt inhibitor) and its relationship with mitochondrial morphology and function preservation in ischemic-reperfused rat hearts. EPO improved the functional recovery of the heart subjected to ischemia-reperfusion, reduced the release of CK and the infarct size, and promoted preservation of the mitochondrial structure. Moreover, it reduced tissue lactate content and preserved glycogen in order to prevent ischemia. The results showed greater Akt activation, accompanied by preservation of swelling and mitochondrial calcium retention capacity, as well as an increase in ATP synthesis capacity. These results were accompanied by an inhibition of GSK-3ß, suggesting regulation of Akt on the opening of the mitochondrial permeability transition pore. All these beneficial effects exerted by acute treatment with EPO were prevented by W. The present study provided novel evidence that EPO not only enhances intrinsic activation of Akt during myocardial ischemia-reperfusion but also promotes GSK-3ß inhibition, contributing to mitochondrial structure and function preservation.

Role of autophagy in simulated ischemic-reperfused left atrial myocardium.

BACKGROUND AND AIM: Autophagy has recently emerged as a potential and promising therapeutic approach to maintain cardiac cellular homeostasis. The aim of the present study was to investigate the role of autophagy in the ischemic-reperfused atrial myocardium. METHODS: Isolated rat left atria subjected to simulated ischemia-reperfusion were used. The bathing medium contained either 10 mM d-glucose or 10 mM d-glucose and 1.2 mM palmitate. 3-methyladenine (3-MA) was used as pharmacological autophagy inhibitor. RESULTS: LC3-II/LC3-I ratio, an indicator of autophagosome formation, was significantly enhanced during reperfusion, this increase being slowed by the exposure to high palmitate concentration and prevented by 3-MA. Beclin-1 was significantly increased during reperfusion period in both metabolic conditions, and pharmacological inhibition of AMPK partially prevented LC3-II/LC3-I ratio increase. Autophagy inhibition significantly increased mitochondrial damage and impaired mitochondrial ATP synthesis rate at reperfusion. Tissue ATP content recovery and contractile reserve were also reduced during this period, these effects being more pronounced either in 3-MA treated atria and ischemic-reperfused atria incubated with palmitate. Moreover, severe tachyarrhythmias were observed in the presence of 3-MA, in both metabolic conditions. This phenomenon was partially prevented by mitochondrial inner membrane ion channels blocker, PK11195. CONCLUSION: Present study provides new insights into the role of autophagy in ischemic-reperfused atrial myocardium. The observation of greater deterioration in mitochondrial structure and function when this process was inhibited, suggests an association between autophagy and the structural and functional preservation of mitochondria. Exogenous metabolic substrates, to which the myocardium is exposed during ischemia-reperfusion, might not affect this process.

Role of AMPK in the protective effects exerted by triiodothyronine in ischemic-reperfused myocardium.

Recent studies have provided evidence that triiodothyronine (T3) might play an effective role in the recovery of ischemic myocardium, through the preservation of mitochondrial function and the improvement of energy substrate metabolism. To this respect, it has been suggested that T3 could activate AMP-activated protein kinase (AMPK), the cellular 'fuel-gauge' enzyme, although its role has yet to be elucidated. The aim of the present study was to investigate the effects produced by acute treatment with T3 (60 nM) and the pharmacological inhibition of AMPK by compound C on isolated rat left atria subjected to 75 min simulated ischemia-75 min reperfusion. Results showed that T3 increased AMPK activation during simulated ischemia-reperfusion, while compound C prevented it. At the end of simulated reperfusion, acute T3 treatment increased contractile function recovery and cellular viability conservation. Mitochondrial ultrastructure was better preserved in the presence of T3 as well as mitochondrial ATP production rate and tissue ATP content. Calcium retention capacity, a parameter widely used as an indicator of the resistance of mitochondrial permeability transition pore (MPTP) to opening, and GSK-3ß phosphorylation, a master switch enzyme that limits MPTP opening, were increased by T3 administration. All these beneficial effects exerted by T3 acute treatment were prevented when compound C was co-administrated. The present study provided original evidence that T3 enhances intrinsic activation of AMPK during myocardial ischemia-reperfusion, being this enzyme involved, at least in part, in the protective effects exerted by T3, contributing to mitochondrial structure and function preservation, post-ischemic contractile recovery and conservation of cellular viability.

Rosuvastatin protects isolated hearts against ischemia-reperfusion injury: role of Akt-GSK-3ß, metabolic environment, and mitochondrial permeability transition pore.

The cardioprotective activity of rosuvastatin (R) is yet to be known. The objective of this study was to research whether R perfusion before global ischemia can mitigate myocardial ischemia-reperfusion damage, considering the metabolic condition in which these effects occur, and to contemplate potential mitochondrial benefits. Protein kinase B (Akt)/glycogen synthase kinase-3ß (GSK-3ß) and mitochondrial permeability transition pore (MPTP) are key elements in myocardial injury produced by ischemia-reperfusion. Isolated rat hearts were subjected to 25-min ischemia and 1-h reperfusion in the presence or absence of R, with or without Wortmannin (W), a phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor. Akt and GSK-3ß were measured by Western blot analysis; lactate, glycogen, and G6PDH were determined; and Ca2+-induced MPTP opening was evaluated using a spectrophotometric method. Contractility was assessed by left ventricular developed pressure (LVDP), and rate-pressure product (RPP), peak rate of contraction and peak rate of relaxation (± dP/dt), and left ventricular end-diastolic pressure (LVEDP) were determined. Tissue samples were extracted to evaluate mitochondrial damage by electron microscopy and to assess infarct size. Statistical analysis employed ANOVA (n = 6/per group). Myocardial infarct size was significantly reduced by R, which also improved cardiac function. MPTP opening was delayed to 300 µM CaCl2, while use of W resulted in MPTP opening at 200 µM CaCl2. Electron microscopy showed better mitochondrial preservation with R, which reduced lactic acid production, increased glycogen consumption and G6PDH activity, as well as phosphorylation of Akt and GSK-3ß. R before ischemia is cardioprotective against ischemic and reperfusion damage, activating Akt and regulating GSK-3ß negatively and attenuating the MPTP opening.

Differential effects of AMP-activated protein kinase in isolated rat atria subjected to simulated ischemia-reperfusion depending on the energetic substrates available.

AMP-activated protein kinase (AMPK) is a serine-threonine kinase that functions primarily as a metabolic sensor to coordinate anabolic and catabolic processes in the cell, via phosphorylation of multiple proteins involved in metabolic pathways, aimed to re-establish energy homeostasis at a cell-autonomous level. Myocardial ischemia and reperfusion represents a metabolic stress situation for myocytes. Whether AMPK plays a critical role in the metabolic and functional responses involved in these conditions remains uncertain. In this study, in order to gain a deeper insight into the role of endogenous AMPK activation during myocardial ischemia and reperfusion, we explored the effects of the pharmacological inhibition of AMPK on contractile function rat, contractile reserve, tissue lactate production, tissue ATP content, and cellular viability. For this aim, isolated atria subjected to simulated 75 min ischemia-75 min reperfusion (Is-Rs) in the presence or absence of the pharmacological inhibitor of AMPK (compound C) were used. Since in most clinical situations of ischemia-reperfusion the heart is exposed to high levels of fatty acids, the influence of palmitate present in the incubation medium was also investigated. The present results suggest that AMPK activity significantly increases during Is, remaining activated during Rs. The results support that intrinsic activation of AMPK has functional protective effects in the reperfused atria when glucose is the only available energetic substrate whereas it is deleterious when palmitate is also available. Cellular viability was not affected by either of these conditions.

Restricted feeding improves postischemic recovery of Langendorff-perfused rat hearts.

The goal of the present study was to assess the effects of a restricted feeding schedule (RFS) on postischemic contractile recovery in relation to triacylglycerol (TAG), glycogen, and ATP content. Glucose-6-phosphate dehydrogenase (G6PDH) activity, reduced/oxidized glutathione ratio (GSH/GSSG), and thiobarbituric acid reactive substances (TBARS) levels were also determined. Isolated rat hearts entrained to daily RFS (2 h food access starting at 1200) or fed ad libitum (FED) for 3 weeks were Langendorff-perfused (25 min ischemia, 30 min reperfusion) with Krebs-Ringer bicarbonate solution (10 mmol/L glucose). RFS improved the recovery of contractility and reduced creatine kinase (CK) release upon reperfusion. Further, at the end of reperfusion, RFS hearts exhibited increased G6PDH activity and repletion of tissue glycogen, TAG, and ATP that was not observed in the FED hearts. GSH/GSSG at the end of reperfusion fell to the same value in both nutritional states, and TBARS levels were higher in the RFS hearts. In conclusion, RFS improved postischemic functional recovery, which was accompanied by a reduction in CK release and a striking energy recovery. Although enhanced G6PDH activity was displayed, RFS was unable to reduce lipid peroxidation, supporting a clear dissociation between protection against mechanical dysfunction and CK release on the one hand and oxidative damage on the other.

Involvement of mitochondrial permeability transition, glutathione status, pentose phosphate pathway and oxidative damage in the protective effect of fasting on the ischaemic-reperfused rat heart.

1. Fasting, which increases the catabolism of fatty acids, gives functional protection to the ischaemic-reperfused heart. To obtain further knowledge of this cardioprotective effect, changes in mitochondrial permeability transition (MPT) were measured by the entrapment of 2-deoxy-[(3)H]-glucose (2-DG). We also assessed whether MPT is associated with changes in glutathione status, the activity of glucose-6-phosphate-dehydrogenase (G6PDH) and tissue oxidative damage, estimated by the measurement of Thiobarbituric acid-reactive substances (TBARS). 2. Spontaneously beating hearts of fed and 24 h fasted rats were Langendorff perfused with Krebs'-Ringer bicarbonate solution (10 mmol/L glucose) and exposed to 25 min global ischaemia, followed by 30 min reperfusion. 3. Ischaemia-reperfusion resulted in a fourfold increase in mitochondrial entrapment of 2-DG in the fed group. This response was 29% lower in the fasted group, but there were no concomitant changes in total retention of 2-DG in the heart. Fasting increased the activity of G6PDH by a factor of 1.4 and caused a 2.8-fold increase in the ratio of reduced glutathione to oxidized glutathione (GSH:GSSG) at the end of the pre-ischaemic period. Ischaemia-reperfusion did not affect G6PDH activity, but reduced the GSH:GSSG ratio in both the fed and fasted groups by 50%. Therefore, the GSH:GSSG ratio remained higher in the fasted group. Fasting also decreased cellular levels of TBARS by approximately 25%. Lipolysis of endogenous triacylglycerol was increased during the pre-ischaemic period in the fasted group. 4. These data suggest that the enhancement of fatty acid catabolism that occurs in fasting activates mechanisms that tend to reduce oxidative damage and limit MPT.