Zinc and myocardial ischemia/reperfusion injury

As an important trace element, zinc is required for the normal cellular structure and function, and impairment of zinc homeostasis is associated with a variety of health problems including cardiovascular disease. Zinc homeostasis is regulated through zinc transporters, zinc binding molecules, and zinc sensors. Zinc also plays a critical role in cellular signaling. Studies have documented that zinc homeostasis is impaired by ischemia/reperfusion in the heart and zinc dyshomeostasis may play a role in the pathogenesis of myocardial ischemia/reperfusion injury. Both exogenous and endogenously released zinc may play an important role in cardioprotection against ischemia/reperfusion injury. The goal of this review is to summarize the current understanding of the roles of zinc homeostasis and zinc signaling in myocardial ischemia/reperfusion injury.     

SIRT1 activation by curcumin pretreatment attenuates mitochondrial oxidative damage induced by myocardial ischemia reperfusion injury

Ischemia reperfusion (IR) injury (IRI) is harmful to the cardiovascular system and causes mitochondrial oxidative stress. Silent information regulator 1 (SIRT1), a type of histone deacetylase, contributes to IRI. Curcumin (Cur) is a strong natural antioxidant and is the active component in Curcuma longa; Cur has protective effects against IRI and may regulate the activity of SIRT1. This study was designed to investigate the protective effect of Cur pretreatment on myocardial IRI and to elucidate this potential mechanism. Isolated and in vivo rat hearts and cultured neonatal rat cardiomyocytes were subjected to IR. Prior to this procedure, the hearts or cardiomyocytes were exposed to Cur in the absence or presence of the SIRT1 inhibitor sirtinol or SIRT1 siRNA. Cur conferred a cardioprotective effect, as shown by improved postischemic cardiac function, decreased myocardial infarct size, decreased myocardial apoptotic index, and several biochemical parameters, including the up-regulation of the antiapoptotic protein Bcl2 and the down-regulation of the proapoptotic protein Bax. Sirtinol and SIRT1 siRNA each blocked the Cur-mediated cardioprotection by inhibiting SIRT1 signaling. Cur also resulted in a well-preserved mitochondrial redox potential, significantly elevated mitochondrial superoxide dismutase activity, and decreased formation of mitochondrial hydrogen peroxide and malondialdehyde. These observations indicated that the IR-induced mitochondrial oxidative damage was remarkably attenuated. However, this Cur-elevated mitochondrial function was reversed by sirtinol or SIRT1 siRNA treatment. In summary, our results demonstrate that Cur pretreatment attenuates IRI by reducing IR-induced mitochondrial oxidative damage through the activation of SIRT1 signaling.     

激动乙醛脱氢酶2对抗糖尿病大鼠心肌缺血/再灌注损伤的作用

目的:探讨激动乙醛脱氢酶2(ALDH2)在糖尿病大鼠心肌损伤中的作用。方法:腹腔注射55 mg/kg链脲佐菌素复制糖尿病大鼠模型,分为糖尿病组和乙醇+糖尿病组(n=8)。8周后行离体心肌缺血/再灌注(I/R),测定心室动力学指标和复灌期间冠脉流出液中乳酸脱氢酶(LDH)含量。测定空腹血糖、糖化血红蛋白(HbA1c)水平。RT-PCR和Western blot测定左心室前壁心尖组织线粒体ALDH2 mRNA和蛋白表达。结果:与正常大鼠心肌I/R相比,糖尿病大鼠左室发展压、左心室最大上升和下降速率、左室做功进一步下降,左室舒张末压抬高,复灌期冠脉流出液中LDH释放增多,心室ALDH2 mRNA和蛋白表达降低;与糖尿病大鼠心肌I/R相比,ALDH2激动剂乙醇明显促进左室发展压、左心室最大上升和下降速率、左室做功的恢复,降低左室舒张末压,同时降低HbA1c水平和LDH的释放,ALDH2 mRNA和蛋白表达增高。结论:糖尿病大鼠心肌缺血/再灌注时,心肌ALDH2表达降低;增强ALDH2在糖尿病大鼠心肌中的表达可发挥保护作用。     

C5aR-mediated myocardial ischemia/reperfusion injury

The complement system activation can mediate myocardial ischemia and reperfusion (I/R). Inhibition of C5a activity reveals attenuation of I/R-induced myocardial infarct size. However, the contribution of C5a receptor (C5aR) to I/R injury remains to be unknown. Here, we reported that both mRNA and protein for the C5aR were constitutively expressed on cardiomyocytes and upregulated as a function of time after I/R-induced myocardial cell injury in mice. Blockade of C5aR markedly decreased microvascular permeability in ischemic myocardial area and leukocyte adherence to coronary artery endothelium. Importantly, the blocking of C5aR with an anti-C5aR antibody was associated with inhibition in activation of protein kinase C delta (PKC-delta) and induction of PKC-mediated mitogen-activated protein kinase phosphatases-1 (MKP-1) leading to the increased activity of p42/p44 mitogen-activated protein (MAP) kinase cascade. These data provide evidence that C5aR-mediated myocardial cell injury is an important pathogenic factor, and that C5aR blockade may be useful therapeutic targets for the prevention of myocardial I/R injury.     

Cardioprotective effects of resveratrol following myocardial ischemia and reperfusion

Resveratrol (RSV), a plant origin polyphenol, has shown beneficial cardiovascular effects. In this study, isolated hearts from male Wistar rats were studied using the Langendorff technique. Following 30 min stabilization, the hearts underwent 30 min global ischemia and 120 min reperfusion. The perfusion solution in the test group contained RSV (10 μM). Hemodynamics of the hearts, the markers of myocardial damage including creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), and troponin I were studied during the study. Furthermore, the infarct size and the markers of oxidative stress including catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GPX) were assayed in the homogenates of the hearts. The release of nitrite from the hearts and the occurrence of ventricular arrhythmias were also monitored throughout the experiment. Resveratrol caused a significant improvement in the restoration of the mechanical performance of the hearts following myocardial ischemia and reperfusion (MIR). Besides, the infarct size, CK-MB, LDH, and troponin I declined in the test group. Besides, the cardiac release of nitrite increased, and the redox status of the heart was improved as indicated by the levels of CAT, SOD, GPX, and MDA. Finally, the treatment caused significant decreases in the occurrences of single and salvo arrhythmias, ventricular tachycardia, and ventricular fibrillation. The current study suggests strong cardioprotective and antiarrhythmic effects for RSV following MIR.

     

Beclin-1 overexpression regulates NLRP3 activation by promoting TNFAIP3 in microvascular injury following myocardial reperfusion

Innate immune response contributes significantly to ischemia reperfusion (I/R) injury. Targeting innate immunity seems to be a promising method for protecting the microvascular injury in ST-elevation myocardial infarction (STEMI) patients following myocardial I/R injury (MI/R). NLRP3 inflammasome is a central part of the innate immune system involved in the pathophysiological process of MI/R. However, the mechanisms regulating NLRP3 activation are yet to be clarified. Recently, autophagy has been related to the regulation of NLRP3 activation. Thus, how Beclin-1/Becn1 overexpression influences NLRP3 activation in microvascular endothelial cells (CMECs) after MI/R is yet to be investigated. The present study showed that Becn1 overexpression exhibits a significant increase in NLRP3 and IL-1β in CMEC responses to MI/R. Interestingly, Becn1 overexpression promoted TNFAIP3 expression, which restricted NLRP3 activation in vitro and in vivo. The current study also showed that inflammatory cells (CD68) and B (CDB220) lymphocytes were decreased in transgenic mice with overexpression of Beclin-1 (BECN1-Tg) in the spleen and heart. These findings highlighted Becn1 as a prospective target for treating NLRP3 mediated microvascular injury following MI/R.     

AGGF1 protects from myocardial ischemia/reperfusion injury by regulating myocardial apoptosis and angiogenesis

Angiogenic factor with G patch and FHA domains 1 (AGGF1) is a newly identified proangiogenic protein, which plays an important role in vascular disease and angiogenesis. However, its role in myocardial ischemia/reperfusion (I/R) injury remains unknown. This study investigated whether AGGF1 is involved in the pathogenesis of mouse myocardial I/R injury and the underlying mechanisms. Wild-type (WT) C57BL/6 J mice were treated at 30 min prior to I/R injury with anti-AGGF1 neutralizing antibody (3 mg/kg) or recombinant human AGGF1 (rhAGGF1, 0.25 mg/kg). After I/R injury, the infarct size, the number of TUNEL-positive cardiomyocytes, Bax/Bcl2 ratio, inflammatory cytokine expression and angiogenesis were markedly increased as compared with sham control. Treatment of WT mice with anti-AGGF1 neutralizing antibody resulted in exaggeration of myocardial I/R injury but reducing angiogenesis. In contrast, administration of rhAGGF1 markedly reversed these effects. Furthermore, anti-AGGF1- or rhAGGF1-mediated effects on I/R-induced cardiac apoptosis, inflammation and angiogenesis were dose dependent. In addition, the protective effects of AGGF1 on cardiomyocyte apoptosis and inflammation were confirmed in cultured cardiomyocytes after I/R. Finally, these effects were associated with activation of ERK1/2, Stat3 and HIF-1α/VEGF pathways and inhibition of activation of NF-κB, p53 and JNK1/2 pathways. In conclusion, we report the first in vivo and in vitro evidence that AGGF1 reduces myocardial apoptosis and inflammation and enhances angiogenesis, leading to decreased infarct size after I/R injury. These results may provide a novel therapeutic approach for ischemic heart diseases.     

Endoxin-mediated myocardial ischemia reperfusion injury in rats in vitro

Myocardial ischemia reperfusion results in an increase in intracellular sodium concentration, which secondarily increases intracellular calcium via Na(+)-Ca2+ exchange, resulting in cellular injury. Endoxin is an endogenous medium of digitalis receptor and can remarkably inhibit Na+/K(+)-ATPase activity. Although the level of plasma endoxin is significantly higher during myocardial ischemia, its practical significance is unclear. This research is to investigate whether endoxin is one of important factors involved in myocardial ischemia reperfusion injury. Ischemia reperfusion injury was induced by 30 min of global ischemia and 30 min of reperfusion in isolated rat hearts. Heart rate (HR), left ventricular developed pressure (LVDP), and its first derivative (+/-dp/dtmax) were recorded. The endoxin contents, intramitochondrial Ca2+ contents, and the Na+/K(+)-ATPase activity in myocardial tissues were measured. Myocardial damages were evaluated by electron microscopy. The endoxin and intramitochondrial Ca2+ contents in myocardial tissues were remarkably higher, myocardial membrane ATPase activity was remarkably lower, the cardiac function was significantly deteriorated, and myocardial morphological damages were severe in myocardial ischemia reperfusion group vs. control. Anti-digoxin antiserum (10, 30 mg/kg) caused a significant improvement in cardiac function (LVDP and +/-dp/dtmax), Na+/K(+)-ATPase activity, and myocardial morphology, and caused a reduction of endoxin and intramitochondrial Ca2+ contents in myocardial tissues. In the present study, the endoxin antagonist, anti-digoxin antiserum, protected the myocardium against the damages induced by ischemia reperfusion in isolated rat hearts. The results suggest that endoxin might be one of main factors mediating myocardial ischemia reperfusion injury.     

Biphasic changes in relaxation following reperfusion after myocardial ischemia

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/dt_max stabilized at 55 ± 3% and 60 ± 2% of preischemic values respectively and, in rat heart LVDP = 61 ± 8% and +dP/dt_max = 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.     

Hypothyroidism provides resistance to reperfusion injury following myocardium ischemia

A growing body of evidence has demonstrated that reperfusion injury may be mediated, in part, by mitochondrial Ca2+ overload that promotes non-selective permeability of the inner membrane. In this regard it is known that mitochondria from hypothyroid rats are resistant to membrane damage as induced by Ca2+. The purpose of this study was to evaluate the sensitivity of hearts from hypothyroid rats, to the damage by reperfusion, after an ischemic period of 5 min. The results were compared with those from control and hyperthyroid rats. Hypothyroidism was established by surgical removal of the thyroid gland; in turn hyperthyroidism was induced after a daily injection of 2 mg/kg of 3,5,3'-triiodothyronine for 4 days. ECG tracings from hypothyroid rats showed a total absence of post-reperfusion arrhythmias conversely to what was observed in control and hyperthyroid rats. The release of creatine kinase and aspartate amino transferase to the plasma in hypothyroid rats was found to be lower than that found in hyperthyroid and euthyroid rats. The histological studies showed that myocardial fibers from hypothyroid rats were in good condition and retained their striae and a remarkable near absence of edema was clearly observed.     

膜磷脂代谢与心肌缺血再灌注损伤

膜磷脂是维持细胞结构与功能的重要成份。心肌缺血—再灌注后导致膜磷脂降解,其含量明显减少,这是再灌注损伤的重要发病环节。用药物阻止膜磷脂降解可预防缺血心肌的再灌注损伤。     

Effect of duration of ischemia on myocardial proteome in ischemia/reperfusion injury

Ischemia/reperfusion (I/R) injury is a serious problem resulting from clinical setting of coronary revascularization. Despite extensive studies on I/R injury, the molecular bases of cardiac dysfunction caused by I/R are still unknown, but are likely to result from alterations in protein expression. Isolated rat hearts were subjected to 15-30 min of no-flow ischemia without (Ischemia protocol) or with 30 min of reperfusion (I/R protocol). 2-DE analysis of heart proteins from both experimental protocols showed wide-ranging changes in protein levels. In the Ischemia protocol, 39 protein spots were changed in ischemic groups and those changes correlated with duration of ischemia. Ninety percent of the affected proteins were increased. In contrast to increased protein levels, the total messenger RNA (mRNA) level decreased approximately two fold. Compared to the Ischemia protocol, changes in protein levels in the I/R protocol did not correlate with the duration of ischemia and the degree of recovery of mechanical function. The decrease of affected protein from I/R protocol was associated with the increase in total protein level in reperfusate. Our studies show that the protein increase is correlated with the mechanical function of the I/R hearts and the increase is not likely associated with an increase in protein synthesis.     

Inhibiting multiple forms of cell death optimizes ganglion cells survival after retinal ischemia reperfusion injury

Progressive retinal ganglion cells (RGCs) death that triggered by retinal ischemia reperfusion (IR), leads to irreversible visual impairment and blindness, but our knowledge of post-IR neuronal death and related mechanisms is limited. In this study, we first demonstrated that apart from necroptosis, which occurs before apoptosis, ferroptosis, which is characterized by iron deposition and lipid peroxidation, is involved in the whole course of retinal IR in mice. Correspondingly, all three types of RGCs death were found in retina samples from human glaucoma donors. Further, inhibitors of apoptosis, necroptosis, and ferroptosis (z-VAD-FMK, Necrostatin-1, and Ferrostatin-1, respectively) all exhibited marked RGC protection against IR both in mice and primary cultured RGCs, with Ferrostatin-1 conferring the best therapeutic effect, suggesting ferroptosis plays a more prominent role in the process of RGC death. We also found that activated microglia, Müller cells, immune responses, and intracellular reactive oxygen species accumulation following IR were significantly mitigated after each inhibitor treatment, albeit to varying degrees. Moreover, Ferrostatin-1 in combination with z-VAD-FMK and Necrostatin-1 prevented IR-induced RGC death better than any inhibitor alone. These findings stand to advance our knowledge of the post-IR RGC death cascade and guide future therapy for RGC protection.Subject terms: Cell death, Neurodegeneration, Cell death in the nervous system

     

Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury

Human ESC-derived mesenchymal stem cell (MSC)-conditioned medium (CM) was previously shown to mediate cardioprotection during myocardial ischemia/reperfusion injury through large complexes of 50–100 nm. Here we show that these MSCs secreted 50- to 100-nm particles. These particles could be visualized by electron microscopy and were shown to be phospholipid vesicles consisting of cholesterol, sphingomyelin, and phosphatidylcholine. They contained coimmunoprecipitating exosome-associated proteins, e.g., CD81, CD9, and Alix. These particles were purified as a homogeneous population of particles with a hydrodynamic radius of 55–65 nm by size-exclusion fractionation on a HPLC. Together these observations indicated that these particles are exosomes. These purified exosomes reduced infarct size in a mouse model of myocardial ischemia/reperfusion injury. Therefore, MSC mediated its cardioprotective paracrine effect by secreting exosomes. This novel role of exosomes highlights a new perspective into intercellular mediation of tissue injury and repair, and engenders novel approaches to the development of biologics for tissue repair.     

Oleuropein prevents oxidative myocardial injury induced by ischemia and reperfusion

The potential protective effects of oleuropein, a dietary antioxidant of olive oil, has been investigated in the isolated rat heart. The organs were subjected to 30 minutes of no-flow global ischemia and then reperfused. At different time intervals, the coronary effluent was collected and assayed for creatine kinase activity as well as for reduced and oxidized glutathione. In addition, the extent of lipid peroxidation was evaluated by measuring thiobarbituric acid reactive substance concentration in cardiac muscle. Pretreatment with 20 microg/g oleuropein before ischemia resulted in a significant decrease in creatine kinase and reduced glutathione release in the perfusate. The protective effect of oleuropein against the post-ischemic oxidative burst was investigated by measuring the release, in the coronary effluent, of oxidized glutathione, a sensitive marker of heart's exposure to oxidative stress. Reflow in ischemic hearts was accompanied by a prompt release of oxidized glutathione; in ischemic hearts pretreated with oleuropein, this release was significantly reduced. Membrane lipid peroxidation was also prevented by oleuropein. The reported data provide the first experimental evidence of a direct cardioprotective effect of oleuropein in the acute events that follow coronary occlusion, likely because of its antioxidant properties. This finding strengthens the hypothesis that the nutritional benefit of olive oil in the prevention of coronary heart disease can be also related to the high content of oleuropein and its derivatives. Moreover, our data, together with the well documented antithrombotic and antiatherogenic activity of olive oil polyphenols, indicate these antioxidants as possible therapeutic tools for the pharmacological treatment of coronary heart disease as well as in the case of cardiac surgery, including transplantation.     

Two-dimensional analysis of myocardial protein expression following myocardial ischemia and reperfusion in rabbits

Myocardial ischemia and reperfusion injury (MI/R) can be related to leukocyte activation with subsequent release of cytokines and oxygen derived free radicals. Activation of the complement system has been implicated in the pathogenesis of myocardial ischemia and reperfusion injury. Inflammatory injury will subsequently result in cellular activation and protein synthesis. In the present study we analyzed the myocardial protein expression and its pattern following myocardial ischemia and reperfusion, with and without complement inhibition with the synthetic serine protease inhibitor Futhan/nafamstat mesilate (FUT-175) known to inhibit classical and alternative complement pathway in a rabbit model of myocardial ischemia and reperfusion (60 min I+180 min R). FUT-175 significantly reduced myocardial necrosis, i.e. creatine kinase release which were analyzed for the three groups (p<0.05). Similarly, histological analysis demonstrated preservation of myocardial tissue injury and reduced leukocyte accumulation following FUT-175 treatment. Further, the myocardial protein expression was analyzed by two-dimensional gel electrophoresis following MI/R in the different groups. The protein patterns were evaluated by means of MELANIE III, a computer assisted gel analysis system. The biochemical identification of the proteins of interest was, achieved using nanohigh-performance liquid chromatography/electrospray ionization-tandem mass spectrometry. On average, 509 +/- 25 protein spots were found on the gels. A pattern of 480 spots with identical positions was found on every gel of five animals of each group. We analyzed ten spots which were significantly altered (i.e., in eight spots we observed decreased protein expression and in two spots we observed increased expression, vehicle vs. sham), by using mass spectrometry. Superoxide dismutase precursor and alphaB-crystallin were identified. We compared sham group vs. vehicle group and vehicle group vs. FUT-175 treated animals. Expression of the two identified proteins decreased by half the amount in the vehicle group when compared to sham treated animals. Treatment with FUT-175 preserved significantly superoxide dismutase precursor and alphaB-crystallin protein expression when compared to vehicle animals. The results present marked differences in myocardial protein expression after ischemia and reperfusion and following treatment with the complement inhibitor FUT-175. Our results illustrate the application of proteomics to discover possible new therapeutic targets or to detect unexpected effects of pharmacological inhibitors.