Signal transduction in myocardial ischaemia and reperfusion

Recent studies in the non-ischaemic myocardium indicated that drugs stimulating cAMP formation inhibit ₁-mediated inositol phosphate generation, while ₁-adrenergic stimulation lowered tissue CAMP levels, implicating cross-talk between ₁,- and -adrenergic signalling pathways in normal physiological conditions. Massive amounts of endogenous catecholamines, predominantly noradrenaline, are released during myocardial ischaemia and reperfusion, causing stimulation of both ₁- and -adrenergic receptors which, in turn, may contribute to intracellular Ca²⁺ overload and subsequent cell damage. Since no information is available regarding cross-talk in pathophysiological conditions, the aim of this study was to evaluate the interactions between ₁- and -adrenergic signalling pathways during different periods of ischaemia and reperfusion.Isolated rat hearts were perfused retrogradely for 30 min before being subjected to (i) 5–25 min global ischaemia and (ii) 1–5 min of reperfusion after 20 min global ischaemia. Drugs (prazosin, 10^–7 M; propranolol, 10^–6 M; phenylephrine 3 × 10^–5 M; isoproterenol 10^–9 M) were added 10 min before the onset of ischaemia and were present during reperfusion.Increasing periods of ischaemia caused an immediate rise and progressive lowering in tissue cAMP and Ins(1,4,5)P₃ levels respectively. In contrast, reperfusion caused an elevation in Ins(1,4,5)P₃ levels and reduced cAMP. Prazosin elevated cAMP levels during both ischaemia and reperfusion, while propranolol had no effects on tissue Ins(1,4,5)P_3–. The activity of the ₁-adrenergic signal transduction pathway appears to have an inhibitory effect on the activity of the -adrenergic system during ischaemia and reperfusion.     

Inhibition of adenosine kinase attenuates myocardial ischaemia/reperfusion injury

Increased adenosine helps limit infarct size in ischaemia/reperfusion-injured hearts. In cardiomyocytes, 90% of adenosine is catalysed by adenosine kinase (ADK) and ADK inhibition leads to higher concentrations of both intracellular adenosine and extracellular adenosine. However, the role of ADK inhibition in myocardial ischaemia/reperfusion (I/R) injury remains less obvious. We explored the role of ADK inhibition in myocardial I/R injury using mouse left anterior ligation model. To inhibit ADK, the inhibitor ABT-702 was intraperitoneally injected or AAV9 (adeno-associated virus)—ADK—shRNA was introduced via tail vein injection. H9c2 cells were exposed to hypoxia/reoxygenation (H/R) to elucidate the underlying mechanisms. ADK was transiently increased after myocardial I/R injury. Pharmacological or genetic ADK inhibition reduced infarct size, improved cardiac function and prevented cell apoptosis and necroptosis in I/R-injured mouse hearts. In vitro, ADK inhibition also prevented cell apoptosis and cell necroptosis in H/R-treated H9c2 cells. Cleaved caspase-9, cleaved caspase-8, cleaved caspase-3, MLKL and the phosphorylation of MLKL and CaMKII were decreased by ADK inhibition in reperfusion-injured cardiomyocytes. X-linked inhibitor of apoptosis protein (XIAP), which is phosphorylated and stabilized via the adenosine receptors A2B and A1/Akt pathways, should play a central role in the effects of ADK inhibition on cell apoptosis and necroptosis. These data suggest that ADK plays an important role in myocardial I/R injury by regulating cell apoptosis and necroptosis.     

Mitochondrial and mitochondrial-independent pathways of myocardial cell death during ischaemia and reperfusion injury

Acute myocardial infarction causes lethal injury to cardiomyocytes during both ischaemia and reperfusion (IR). It is important to define the precise mechanisms by which they die in order to develop strategies to protect the heart from IR injury. Necrosis is known to play a major role in myocardial IR injury. There is also evidence for significant myocardial death by other pathways such as apoptosis, although this has been challenged. Mitochondria play a central role in both of these pathways of cell death, as either a causal mechanism is the case of mitochondrial permeability transition leading to necrosis, or as part of the signalling pathway in mitochondrial cytochrome c release and apoptosis. Autophagy may impact this process by removing dysfunctional proteins or even entire mitochondria through a process called mitophagy. More recently, roles for other programmed mechanisms of cell death such as necroptosis and pyroptosis have been described, and inhibitors of these pathways have been shown to be cardioprotective. In this review, we discuss both mitochondrial and mitochondrial-independent pathways of the major modes of cell death, their role in IR injury and their potential to be targeted as part of a cardioprotective strategy. This article is part of a special Issue entitled ‘Mitochondria as targets of acute cardioprotection’ and emerged as part of the discussions of the European Union (EU)-CARDIOPROTECTION Cooperation in Science and Technology (COST) Action, CA16225.     

Protective effects of a plant histaminase in myocardial ischaemia and reperfusion injury in vivo

Grass pea seedling histaminase (a copper-diamine oxidase) was found to exert a significant cardioprotection against post-ischaemic reperfusion damage. Electrocardiogram (ECG) recordings from the rats subjected in vivo to ischaemia and reperfusion showed ventricular tachycardia (VT) and ventricular fibrillations (VF) occurring in 9 out of 12 untreated rats whereas no ventricular arrhythmias were found under histaminase (80U/kg body weight) treatment (n=16 rats). Computer-assisted morphometry of the ischaemic reperfused hearts stained with nitroblue tetrazolium showed the extension of damaged myocardium (area at risk and infarct size) significantly reduced in rats treated with histaminase, in comparison with the non-treated rats, whereas no protection was found with the semicarbazide inactivated histaminase. Biochemical markers of ischaemia-reperfusion myocardial tissue damage: malonyldialdehyde (MDA), tissue calcium concentration, myeloperoxidase (MPO), and apoptosis indicator caspase-3 were significantly elevated in untreated post-ischaemic reperfused rats, but significantly reduced under histaminase protection. In conclusion, plant histaminase appears to protect hearts from ischaemia-reperfusion injury by more than one mechanism, essentially involving histamine oxidation, and possibly as reactive oxygen species scavenger, presenting good perspectives for a novel therapeutic approach in treatment of ischaemic heart pathology.     

Interleukin-7 aggravates myocardial ischaemia/reperfusion injury by regulating macrophage infiltration and polarization

Interleukin (IL)-7 is known to enhance the macrophages cytotoxic activity and that macrophages play a pivotal role in the development and progression of myocardial ischaemia/reperfusion (I/R) injury. However, the effects of IL-7 on macrophages infiltration and polarization in myocardial I/R injury are currently unclear. This study aimed to evaluate the effects of the IL-7 expression on myocardial I/R injury and their relationship with macrophages. The data showed that IL-7 expression in mouse heart tissue increases following I/R injury and that IL-7 knockout or anti-IL-7 antibody treatment significantly improve I/R injury, including reduction in myocardial infarction area, a serum troponin T level decreases and an improvement in cardiac function. On the other hand, recombinant IL-7 (rIL-7) supplementation induces opposite effects and the anti-IL-7 antibody significantly reduces the cardiomyocyte apoptosis and macrophage infiltration. rIL-7 cannot directly cause apoptosis, but it can induce cardiomyocyte apoptosis through macrophages, in addition to increase the macrophages migration in vitro. Anti-IL-7 antibody affects the cytokine production in T helper (Th) 1 and Th2 cells and also promotes the macrophages differentiation to M2 macrophages. However, anti-IL-7 antibody does not reduce the M1 macrophage number, and it only increases the ratio of M2/M1 macrophages in mice heart tissues after I/R injury. Taking together, these data reveal that IL-7 plays an intensifying role in myocardial I/R injury by promoting cardiomyocyte apoptosis through the regulation of macrophage infiltration and polarization.     

Enzyme and immunohistochemical assessment of myocardial damage after ischaemia and reperfusion in a closed-chest pig model

The usefulness of different enzyme and immunohistochemical stains to distinguish reversible and irreversible myocardial cell injury after experimental coronary artery occlusion of varying duration and reperfusion with or without superoxide dismutase as adjunct was investigated. Biopsies or parts of the infarcted and non-infarcted area were rapidly frozen and sectioned in series for enzyme and immunohistochemical evaluation. Sections were stained for the demonstration of phosphorylase, myofibrillar ATPase and mitochondrial oxidative enzymes and also with periodic acid-Schiff, alizarin red S and routine histological stains. Other sections in series were stained with antibodies against fibronectin and the intermediate filament proteins desmin and vimentin. In 49 biopsies a blind quantitative estimation of the area stained for fibronectin, phosphorylase and alizarin red S was performed and evaluated statistically. Phosphorylase, periodic acid-Schiff, fibronectin and alizarin red S allowed delineation of affected myocardium after 30 min of ischaemia followed by reperfusion whereas with the other stains, affected myocardium was readily detectable only after 60 or 90 min of ischaemia followed by reperfusion as well as after 24 h of ischaemia without reperfusion. The immunostaining for fibronectin was very distinct and inversely related to the phosphorylase activity. We show that fibronectin is an excellent marker for damaged cells and that these positively stained myocytes are necrotic as confirmed ultrastructurally. Using alizarin red S as a marker of calcium accumulation in myocytes, a marked discrepancy was observed between the area of fibronectin-containing myocytes and that of myocytes stained by alizarin red S. Calcium accumulation in mitochondria is thus not a prerequisite for myocyte necrosis but does occur only in some of the irreversibly damaged cells. Of special interest is the finding that there was a significant reduction of intracellular calcium in pigs where superoxide dismutase had been used as an adjunct at reperfusion, thus supporting the theory that free radicals do play a role during reperfusion of ischaemic myocardium.     

Thyroid hormone and myocardial ischaemia

Thyroid hormone has various effects on the cardiovascular system and its effects on cardiac contractility, heart rhythm and vascular function has long been recognized. However, new evidence is emerged on the importance of thyroid hormone in the response of the myocardium to ischaemic stress and cardiac remodelling following myocardial infarction. Based on this new information, this review highlights the role of thyroid hormone in myocardial ischaemia and cardiac remodelling, the possible underlying mechanisms and the potential therapeutic implications. Thyroid hormone or analogs may prove new therapeutic agents for treating ischaemic heart disease.     

Enzyme and immunohistochemical assessment of myocardial damage after ischaemia and reperfusion in a closed-chest pig model.

The usefulness of different enzyme and immunohistochemical stains to distinguish reversible and irreversible myocardial cell injury after experimental coronary artery occlusion of varying duration and reperfusion with or without superoxide dismutase as adjunct was investigated. Biopsies or parts of the infarcted and non-infarcted area were rapidly frozen and sectioned in series for enzyme and immunohistochemical evaluation. Sections were stained for the demonstration of phosphorylase, myofibrillar ATPase and mitochondrial oxidative enzymes and also with periodic acid-Schiff, alizarin red S and routine histological stains. Other sections in series were stained with antibodies against fibronectin and the intermediate filament proteins desmin and vimentin. In 49 biopsies a blind quantitative estimation of the area stained for fibronectin, phosphorylase and alizarin red S was performed and evaluated statistically. Phosphorylase, periodic acid-Schiff, fibronectin and alizarin red S allowed delineation of affected myocardium after 30 min of ischaemia followed by reperfusion whereas with the other stains, affected myocardium was readily detectable only after 60 or 90 min of ischaemia followed by reperfusion as well as after 24 h of ischaemia without reperfusion. The immunostaining for fibronectin was very distinct and inversely related to the phosphorylase activity. We show that fibronectin is an excellent marker for damaged cells and that these positively stained myocytes are necrotic as confirmed ultrastructurally. Using alizarin red S as a marker of calcium accumulation in myocytes, a marked discrepancy was observed between the area of fibronectin-containing myocytes and that of myocytes stained by alizarin red S. Calcium accumulation in mitochondria is thus not a prerequisite for myocyte necrosis but does occur only in some of the irreversibly damaged cells. Of special interest is the finding that there was a significant reduction of intracellular calcium in pigs where superoxide dismutase had been used as an adjunct at reperfusion, thus supporting the theory that free radicals do play a role during reperfusion of ischaemic myocardium.     

Epidermal growth factor protects against myocardial ischaemia reperfusion injury through activating Nrf2 signalling pathway

Alleviating the oxidant stress associated with myocardial ischaemia reperfusion has been demonstrated as a potential therapeutic approach to limit ischaemia reperfusion (I/R)-induced cardiac damage. It is reported that EGFR/erbB2 signalling is an important cardiac survival pathway in cardiac function and activation of EGFR has a cardiovascular effect in global ischaemia. Epidermal growth factor (EGF), a typical EGFR ligand, was considered to have a significant role in activating EGFR. However, no evidence has been published whether exogenous EGF has protective effects on myocardial ischaemia reperfusion. This study aims to investigate the effects of EGF in I/R-induced heart injury and to demonstrate its mechanisms. H9c2 cells challenged with H₂O₂ were used for in vitro biological activity and mechanistic studies. The malondialdehyde (MDA) and Superoxide Dismutase (SOD) levels in H9c2 cells were determined, and the cell viability was assessed by MTT assay. Myocardial I/R mouse administrated with or without EGF were used for in vivo studies. Pretreatment of H9c2 cells with EGF activated Nrf2 signalling pathway, attenuated H₂O₂-increased MDA and H₂O₂-reduced SOD level, followed by the inhibition of H₂O₂-induced cell death. In in vivo animal models of myocardial I/R, administration of EGF reduced infarct size and myocardial apoptosis. These data support that EGF decreases oxidative stress and attenuates myocardial ischaemia reperfusion injury via activating Nrf2.     

Maternal diabetes up‐regulates NOX2 and enhances myocardial ischaemia/reperfusion injury in adult offspring

Offspring of diabetic mothers are at risk of cardiovascular diseases in adulthood. However, the underlying molecular mechanisms are not clear. We hypothesize that prenatal exposure to maternal diabetes up‐regulates myocardial NOX2 expression and enhances ischaemia/reperfusion (I/R) injury in the adult offspring. Maternal diabetes was induced in C57BL/6 mice by streptozotocin. Glucose‐tolerant adult offspring of diabetic mothers and normal controls were subjected to myocardial I/R injury. Vascular endothelial growth factor (VEGF) expression, ROS generation, myocardial apoptosis and infarct size were assessed. The VEGF‐Akt (protein kinase B)‐mammalian target of rapamycin (mTOR)‐NOX2 signalling pathway was also studied in cultured cardiomyocytes in response to high glucose level. In the hearts of adult offspring from diabetic mothers, increases were observed in VEGF expression, NOX2 protein levels and both Akt and mTOR phosphorylation levels as compared to the offspring of control mothers. After I/R, ROS generation, myocardial apoptosis and infarct size were all significantly higher in the offspring of diabetic mothers relative to offspring of control mothers, and these differences were diminished by in vivo treatment with the NADPH oxidase inhibitor apocynin. In cultured cardiomyocytes, high glucose increased mTOR phosphorylation, which was inhibited by the PI3 kinase inhibitor LY294002. Notably, high glucose‐induced NOX2 protein expression and ROS production were inhibited by rapamycin. In conclusion, maternal diabetes promotes VEGF‐Akt‐mTOR‐NOX2 signalling and enhances myocardial I/R injury in the adult offspring. Increased ROS production from NOX2 is a possible molecular mechanism responsible for developmental origins of cardiovascular disease in offspring of diabetic mothers.     

Arrhythmias associated with myocardial ischaemia and infarction

The intention of this review has been to summarise the current state of knowledge regarding the arrhythmias induced by myocardial ischaemia and infarction. Both clinical and experimental aspects were considered. There has been some progress toward understanding the electrophysiological mechanisms responsible for the genesis of such arrhythmias but understanding is far from complete. We are still unable to trace the sequence of events which begin with the electrophysiological changes induced in cells by ischaemia and progress through macromechanisms such as re-entry, automaticity, etc., to the final arrhythmia. Exactly how the changes in individual cells translate into the macromechanisms is not known. Similarly, which macromechanism actually operate, and to what extent, is not known.We have very little information regarding the biochemical events responsible for the changes in intracellular potential seen with ischaemia. Similarly, we do not know whether arrhythmogenic mediators are involved in such a process. We have a fairly complete catalogue of the changes in biochemistry induced by ischaemia, but at the moment it is difficult to find causal relationships between such changes and ischaemia-induced disturbances in electrophysiology.Finally, we are in possession of a catalogue of drugs which may reduce the arrhythmias induced by ischaemia and infarction (both clinically and experimentally), but have no clear direction as how to develop the ideal antiarrhythmic (antifibrillatory) drugs.     

Oxidative stress in myocardial ischaemia reperfusion injury: a renewed focus on a long-standing area of heart research

Advances in the treatment of coronary artery disease have seen a significant drop in mortality and morbidity particularly amongst patients with acute myocardial infarction (MI). In particular, percutaneous trans-luminal balloon angioplasty (PTCA) with stenting to re-open atherosclerotic coronary arteries has yielded marked improvement in clinical outcome for patients with acute MI. Furthermore, with the advent of drug-eluting stents occurrence rates for coronary artery restenosis, one common clinical problem associated with angioplasty and stent deployment, have declined markedly. However, coronary restenosis in diabetic patients remains an on-going problem. The success of drug-eluting stents has seen a renewed focus on myocardial ischaemia reperfusion (IR) injury as this represents one area of research where many questions remain unanswered. In particular, the relationship between myocardial IR injury and decreased myocardial micro-vasculature re-flow post PTCA (that ultimately leads to poor clinical outcome and myocardial damage/dysfunction) is one area of research with the potential to decrease current complication rates further in patients suffering myocardial IR injury sustained during MI. This review discusses the role for oxidative stress, oxidant source(s) and both gene regulation and stem-cell therapy as potential strategic targets in the ischaemic myocardium, with the ultimate aim of providing significant cardioprotection in the setting of acute MI.     

Cardioprotective effects of melatonin against myocardial ischaemia/reperfusion injury: Activation of AMPK/Nrf2 pathway

Although reperfusion is the most effective therapy for patients with acute myocardial infarction, reperfusion injury limits the therapeutic effects of early reperfusion. Oxidative stress plays a crucial role in myocardial ischaemia/reperfusion (I/R) injury. Melatonin, a circulating hormone, is well-known as an antioxidant in cardiovascular diseases. In this short communication, we show that melatonin significantly improves post-ischaemic cardiac function, reduces infarct size and decreases oxidative stress. Furthermore, melatonin markedly increases AMPK activation and Nrf2 nuclear translocation. Nevertheless, these melatonin-induced changes are abrogated by compound C. In addition, ML-385, an Nrf2 inhibitor, also withdraws the antioxidative effects of melatonin but has little effect on AMPK activation. In conclusion, our results demonstrate that melatonin alleviates myocardial I/R injury by inhibiting oxidative stress via the AMPK/Nrf2 signalling pathway.     

The non-anticoagulant heparin-like K5 polysaccharide derivative K5-N,OSepi attenuates myocardial ischaemia/reperfusion injury

Heparin and low molecular weight heparins have been demonstrated to reduce myocardial ischaemia/reperfusion (I/R) injury, although their use is hampered by the risk of haemorrhagic and thrombotic complications. Chemical and enzymatic modifications of K5 polysaccharide have shown the possibility of producing heparin-like compounds with low anticoagulant activity and strong anti-inflammatory effects. Using a rat model of regional myocardial I/R, we investigated the effects of an epimerized N-,O-sulphated K5 polysaccharide derivative, K5-N,OSepi, on infarct size and histological signs of myocardial injury caused by 30 min. ligature of the left anterior descending coronary artery followed by 1 or 24 h reperfusion. K5-N,OSepi (0.1-1 mg/kg given i.v. 15 min. before reperfusion) significantly reduced the extent of myocardial damage in a dose-dependent manner. Furthermore, we investigated the potential mechanism(s) of the cardioprotective effect(s) afforded by K5-N,OSepi. In left ventricular samples, I/R induced mast cell degranulation and a robust increase in lipid peroxidation, free radical-induced DNA damage and calcium overload. Markers of neutrophil infiltration and activation were also induced by I/R in rat hearts, specifically myeloperoxidase activity, intercellular-adhesion-molecule-1 expression, prostaglandin-E(2) and tumour-necrosis-factor-α production. The robust increase in oxidative stress and inflammatory markers was blunted by K5-N,OSepi, in a dose-dependent manner, with maximum at 1 mg/kg. Furthermore, K5-N,OSepi administration attenuated the increase in caspase 3 activity, Bid and Bax activation and ameliorated the decrease in expression of Bcl-2 within the ischaemic myocardium. In conclusion, we demonstrate that the cardioprotective effect of the non-anticoagulant K5 derivative K5-N,OSepi is secondary to a combination of anti-apoptotic and anti-inflammatory effects.     

Experimental studies in transient myocardial ischaemia

The biology of the myocardium was studied under experimental conditions similar to angina pectoris. In some dogs the myocardium was adapted to ischaemia by progressive coronary occlusion of 1-5 min followed by restoration of circulation during 5 min. In other dogs adaption was followed by 20 to 35 min ischaemia. The animals were sacrificed immediately or after 2-10 days. Transient ischaemia produced less severe alterations then abrupt coronary obstruction. Adaptation followed by 20 and 35 min ischaemia induced foci that undergo cytolysis and scarring of maximum intensity on the 8th day. Activity of enzymes in the mitochondrial suspension, especially of cytochromoxidase, decreases and lysosomal hydrolases increase with focal necroses.