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.

Effects of 3-methyladenine on isolated left atria subjected to simulated ischaemia-reperfusion.

Although autophagy is a prominent feature of myocardial ischaemia and reperfusion, its functional significance is unclear and controversial. In order to gain a deeper insight into the role of autophagy in myocardial ischaemia-reperfusion, we explored the effects of the pharmacological inhibitor of autophagy 3-methyladenine (3-MA). Isolated rat atria subjected to simulated 75-min ischaemia/75-min reperfusion (Is-Rs) in the presence or absence of 3-MA were used. The LC3-II/LC3-I ratio, an indicator of autophagosome formation, did not increase after ischaemia either in the presence or absence of 3-MA, but there was significant enhancement during reperfusion, which was prevented by the presence of 3-MA. The autophagy inhibitor also increased p62 protein, one of the specific substrates degraded through the autophagy-lysosomal pathway. Electron micrographs showed double membrane autophagosome-like structures during reperfusion, which were absent in atria subjected to Is-Rs in the presence of 3-MA. These findings suggest that this agent inhibited the autophagic flux under the present experimental conditions. Inhibition of autophagy during Is-Rs was accompanied by a high incidence of tachyarrhythmias during reperfusion, and a decrease in the maximal inotropic response to ß-adrenergic and to calcium stimulation at the end of Is-Rs. Deterioration of mitochondrial morphology and function, without affecting cell viability, was observed in atria subjected to Is-Rs in the presence of 3-MA. The present results suggest an association between the inhibition of autophagy and functional alterations of the cells that have undergone sublethal stress, and have been able to recover in this experimental model of ischaemia-reperfusion.

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.