Attenuation of antioxidative capacity enhances reperfusion injury in aged rat myocardium after MI/R

Mortality due to ischemic cardiovascular diseases is significantly higher in elderly than in young adults. Myocardial ischemia-reperfusion (MI/R) can induce oxidative stress and an inflammatory response. We hypothesized that increased vulnerability of aged myocardium to reperfusion injury could be caused by decreased antioxidative capacity, rather than increased oxidant production, after MI/R. Aged (20-mo-old) and young (4-mo-old) male F344BN rats were subjected to 30 min of myocardial ischemia by ligation of the left main coronary artery followed by release of the ligature and 4 h of reperfusion. Four experimental groups were studied: young sham-operated rats, aged sham-operated rats, young rats subjected to MI/R, and aged rats subjected to MI/R. MI/R significantly increased infiltrated leukocyte number and myeloperoxidase (MPO) activity in perinecrotic areas of hearts of young rats compared with aged MI/R rats. These changes in infiltrated leukocyte number and MPO activity were associated with an increase in superoxide generation in perinecrotic areas from hearts of young rats compared with aged rats. Plasma levels of TNF-alpha and IL-1beta were significantly higher in young than in aged MI/R rats. However, plasma 8-hydroxy-2'-deoxyguanosine levels and creatine kinase activity were increased in aged compared with young MI/R rats. Increased reperfusion damage in aged rats was associated with a significant decrease in plasma ratio of GSH to GSSG. Our results suggest that enhanced ischemia-reperfusion injury in aged rat hearts may be related to reduced antioxidative capacity, rather than increased reactive oxygen species production. These findings contribute to a better understanding of effects of aging on oxidative stress and inflammatory responses of the heart after MI/R.     

A new model of congestive heart failure in rats

Current rodent models of ischemia/infarct or pressure-volume overload are not fully representative of human heart failure. We developed a new model of congestive heart failure (CHF) with both ischemic and stress injuries combined with fibrosis in the remote myocardium. Sprague-Dawley male rats were used. Ascending aortic banding (Ab) was performed to induce hypertrophy. Two months post-Ab, ischemia-reperfusion (I/R) injury was induced by ligating the left anterior descending (LAD) artery for 30 min. Permanent LAD ligation served as positive controls. A debanding (DeAb) procedure was performed after Ab or Ab + I/R to restore left ventricular (LV) loading properties. Cardiac function was assessed by echocardiography and in vivo hemodynamic analysis. Myocardial infarction (MI) size and myocardial fibrosis were assessed. LV hypertrophy was observed 4 mo post-Ab; however, systolic function was preserved. LV hypertrophy regressed within 1 mo after DeAb. I/R for 2 mo induced a small to moderate MI with mild impairment of LV function. Permanent LAD ligation for 2 mo induced large MI and significant cardiac dysfunction. Ab for 2 mo followed by I/R for 2 mo (Ab + I/R) resulted in moderate MI with significantly reduced ejection fraction (EF). DeAb post Ab + I/R to reduce afterload could not restore cardiac function. Perivascular fibrosis in remote myocardium after Ab + I/R + DeAb was associated with decreased cardiac function. We conclude that Ab plus I/R injury with aortic DeAb represents a novel model of CHF with increased fibrosis in remote myocardium. This model will allow the investigation of vascular and fibrotic mechanisms in CHF characterized by low EF, dilated LV, moderate infarction, near-normal aortic diameter, and reperfused coronary arteries.     

Cardioprotection by HO-4038, a novel verapamil derivative, targeted against ischemia and reperfusion-mediated acute myocardial infarction

Many cardiac interventional procedures, such as coronary angioplasty, stenting, and thrombolysis, attempt to reintroduce blood flow (reperfusion) to an ischemic region of myocardium. However, the reperfusion is accompanied by a complex cascade of cellular and molecular events resulting in oxidative damage, termed myocardial ischemia-reperfusion (I/R) injury. In this study, we evaluated the ability of HO-4038, an N-hydroxypiperidine derivative of verapamil, on the modulation of myocardial tissue oxygenation (Po(2)), I/R injury, and key signaling molecules involved in cardioprotection in an in vivo rat model of acute myocardial infarction (MI). MI was created in rats by ligating the left anterior descending coronary artery (LAD) for 30 min followed by 24 h of reperfusion. Verapamil or HO-4038 was infused through the jugular vein 10 min before the induction of ischemia. Myocardial Po(2) and the free-radical scavenging ability of HO-4038 were measured using electron paramagnetic resonance spectroscopy. HO-4038 showed a significantly better scavenging ability of reactive oxygen radicals compared with verapamil. The cardiac contractile functions in the I/R hearts were significantly higher recovery in HO-4038 compared with the verapamil group. A significant decrease in the plasma levels of creatine kinase and lactate dehydrogenase was observed in the HO-4038 group compared with the verapamil or untreated I/R groups. The left ventricular infarct size was significantly less in the HO-4038 (23 +/- 2%) compared with the untreated I/R (36 +/- 4%) group. HO-4038 significantly attenuated the hyperoxygenation (36 +/- 1 mmHg) during reperfusion compared with the untreated I/R group (44 +/- 2 mmHg). The HO-4038-treated group also markedly attenuated superoxide production, increased nitric oxide generation, and enhanced Akt and Bcl-2 levels in the reperfused myocardium. Overall, the results demonstrated that HO-4038 significantly protected hearts against I/R-induced cardiac dysfunction and damage through the combined beneficial actions of calcium-channel blocking, antioxidant, and prosurvival signaling activities.     

Toll-like receptor 3 plays a role in myocardial infarction and ischemia/reperfusion injury

Innate immune and inflammatory responses mediated by Toll like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. This study examined the role of TLR3 in myocardial injury induced by two models, namely, myocardial infarction (MI) and I/R. First, we examined the role of TLR3 in MI. TLR3 deficient (TLR3^−/−) and wild type (WT) mice were subjected to MI induced by permanent ligation of the left anterior descending (LAD) coronary artery for 21 days. Cardiac function was measured by echocardiography. Next, we examined whether TLR3 contributes to myocardial I/R injury. TLR3^−/− and WT mice were subjected to myocardial ischemia (45 min) followed by reperfusion for up to 3 days. Cardiac function and myocardial infarct size were examined. We also examined the effect of TLR3 deficiency on I/R-induced myocardial apoptosis and inflammatory cytokine production. TLR3^−/− mice showed significant attenuation of cardiac dysfunction after MI or I/R. Myocardial infarct size and myocardial apoptosis induced by I/R injury were significantly attenuated in TLR3^−/− mice. TLR3 deficiency increases B-cell lymphoma 2 (BCL2) levels and attenuates I/R-increased Fas, Fas ligand or CD95L (FasL), Fas-Associated protein with Death Domain (FADD), Bax and Bak levels in the myocardium. TLR3 deficiency also attenuates I/R-induced myocardial nuclear factor KappaB (NF-κB) binding activity, Tumor necrosis factor alpha (TNF-α) and Interleukin-1 beta (IL-1β) production as well as I/R-induced infiltration of neutrophils and macrophages into the myocardium. TLR3 plays an important role in myocardial injury induced by MI or I/R. The mechanisms involve activation of apoptotic signaling and NF-κB binding activity. Modulation of TLR3 may be an effective approach for ameliorating heart injury in heart attack patients.     

Inhaled nitric oxide decreases infarction size and improves left ventricular function in a murine model of myocardial ischemia-reperfusion injury

To learn whether nitric oxide (NO) inhalation can decrease myocardial ischemia-reperfusion (I/R) injury, we studied a murine model of myocardial infarction (MI). Anesthetized mice underwent left anterior descending coronary artery ligation for 30, 60, or 120 min followed by reperfusion. Mice breathed NO beginning 20 min before reperfusion and continuing thereafter for 24 h. MI size and area at risk were measured, and left ventricular (LV) function was evaluated using echocardiography and invasive hemodynamic measurements. Inhalation of 40 or 80 ppm, but not 20 ppm, NO decreased the ratio of MI size to area at risk. NO inhalation improved LV systolic function, as assessed by echocardiography 24 h after reperfusion, and systolic and diastolic function, as evaluated by hemodynamic measurements 72 h after reperfusion. Myocardial neutrophil infiltration was reduced in mice breathing NO, and neutrophil depletion prevented inhaled NO from reducing myocardial I/R injury. NO inhalation increased arterial nitrite levels but did not change myocardial cGMP levels. Breathing 40 or 80 ppm NO markedly and significantly decreased MI size and improved LV function after ischemia and reperfusion in mice. NO inhalation may represent a novel method to salvage myocardium at risk of I/R injury.     

The polysulfide diallyl trisulfide protects the ischemic myocardium by preservation of endogenous hydrogen sulfide and increasing nitric oxide bioavailability

Diallyl trisulfide (DATS), a polysulfide constituent found in garlic oil, is capable of the release of hydrogen sulfide (H(2)S). H(2)S is a known cardioprotective agent that protects the heart via antioxidant, antiapoptotic, anti-inflammatory, and mitochondrial actions. Here, we investigated DATS as a stable donor of H(2)S during myocardial ischemia-reperfusion (MI/R) injury in vivo. We investigated endogenous H(2)S levels, infarct size, postischemic left ventricular function, mitochondrial respiration and coupling, endothelial nitric oxide (NO) synthase (eNOS) activation, and nuclear E2-related factor (Nrf2) translocation after DATS treatment. Mice were anesthetized and subjected to a surgical model of MI/R injury with and without DATS treatment (200 μg/kg). Both circulating and myocardial H(2)S levels were determined using chemiluminescent gas chromatography. Infarct size was measured after 45 min of ischemia and 24 h of reperfusion. Troponin I release was measured at 2, 4, and 24 h after reperfusion. Cardiac function was measured at baseline and 72 h after reperfusion by echocardiography. Cardiac mitochondria were isolated after MI/R, and mitochondrial respiration was investigated. NO metabolites, eNOS phosphorylation, and Nrf2 translocation were determined 30 min and 2 h after DATS administration. Myocardial H(2)S levels markedly decreased after I/R injury but were rescued by DATS treatment (P < 0.05). DATS administration significantly reduced infarct size per area at risk and per left ventricular area compared with control (P < 0.001) as well as circulating troponin I levels at 4 and 24 h (P < 0.05). Myocardial contractile function was significantly better in DATS-treated hearts compared with vehicle treatment (P < 0.05) 72 h after reperfusion. DATS reduced mitochondrial respiration in a concentration-dependent manner and significantly improved mitochondrial coupling after reperfusion (P < 0.01). DATS activated eNOS (P < 0.05) and increased NO metabolites (P < 0.05). DATS did not appear to significantly induce the Nrf2 pathway. Taken together, these data suggest that DATS is a donor of H(2)S that can be used as a cardioprotective agent to treat MI/R injury.     

Cardioprotection of the aged rat heart by GSK-3beta inhibitor is attenuated: age-related changes in mitochondrial permeability transition pore modulation

It is well established that inhibition of glycogen synthase kinase (GSK)-3β in the young adult myocardium protects against ischemia-reperfusion (I/R) injury through inhibition of mitochondrial permeability transition pore (mPTP) opening. Here, we investigated age-associated differences in the ability of GSK-3β inhibitor [SB-216763 (SB)] to protect the heart and to modulate mPTP opening during I/R injury. Fischer 344 male rats were assigned from their respective young or old age groups. Animals were subjected to 30 min ischemia following 120 min reperfusion to determine myocardial infarction (MI) size in vivo. Ischemic tissues were collected 10 min after reperfusion for nicotinamide adenine dinucleotide (NAD(+)) measurements and immunoblotting. In parallel experiments, ventricular myocytes isolated from young or old rats were exposed to oxidative stress through generation of reactive oxygen species (ROS), and mPTP opening times were measured by using confocal microscopy. Our results showed that SB decreased MI in young SB-treated rats compared with young untreated I/R animals, whereas SB failed to significantly affect MI in the old animals. SB also significantly increased GSK-3β phosphorylation in young rats, but phosphorylation levels were already highly elevated in old control groups. There were no significant differences observed between SB-treated and untreated old animals. NAD(+) levels were better maintained in young SB-treated animals compared with the young untreated group during I/R, but this relative improvement was not observed in old animals. SB also significantly prolonged the time to mPTP opening induced by ROS in young cardiomyocytes, but not in aged cardiomyocytes. These results demonstrate that this GSK-3β inhibitor fails to protect the aged myocardium in response to I/R injury or prevent mPTP opening following a rise in ROS and suggest that healthy aging alters mPTP regulation by GSK-3β.     

Oxidative stress and calcium signaling in the adverse effects of environmental particles (PM10)

This review focuses on the potential role that oxidative stress plays in the adverse effects of PM(10). The central hypothesis is that the ability of PM(10) to cause oxidative stress underlies the association between increased exposure to PM(10) and both exacerbations of lung disease and lung cancer. Pulmonary inflammation may also underlie the cardiovascular effects seen following increased PM(10), although the mechanisms of the cardiovascular effects of PM(10) are not well understood. PM(10) is a complex mix of various particle types and several of the components of PM(10) are likely to be involved in the induction of oxidative stress. The most likely of these are transition metals, ultrafine particle surfaces, and organic compounds. In support of this hypothesis, oxidative stress arising from PM(10) has been shown to activate a number of redox-responsive signaling pathways in lung target cells. These pathways are involved in expression of genes that play a role in responses relevant to inflammation and pathological change, including MAPKs, NF-kappaB, AP-1, and histone acetylation. Oxidative stress from particles is also likely to play an important role in the carcinogenic effects associated with PM(10) and hydroxyl radicals from PM(10) cause DNA damage in vitro.     

Lipopolysaccharide pretreatment protects against ischemia/reperfusion injury via increase of HSP70 and inhibition of NF-κB

It has been reported that pretreatment of rats with lipopolysaccharide (LPS) increases myocardial functional recovery in ischemia/reperfusion (I/R) hearts. However, the mechanisms by which LPS induces cardioprotection against I/R injury have not been fully elucidated. In this study, we pretreated rats with LPS (1.0 mg/kg) 24 h before they were subjected to I/R injury, and then examined the roles of heat shock protein-70 (HSP70) and nucleus factor-κB (NF-κB) in LPS-induced cardioprotection. We observed that pretreatment with low-dose LPS resulted in significantly increased levels of HSP70 in the myocardium, which could dramatically inhibit NF-κB translocation and reduce degradation of inhibitory κB. Inhibition of NF-κB, in turn, attenuated release of inflammatory cytokines (tumor necrosis factor-α, interleukin (IL)-1β, and IL-6) and reduced apoptosis of myocardium and infarct area following I/R injury. Moreover, HSP70 could ameliorate oxidative stress following I/R injury. To further investigate whether increase of HSP70 might be responsible for protection of the myocardium against I/R injury, we co-administered the HSP70 inhibitor, quercetin, with LPS before I/R injury. We found that LPS-induced cardioprotection was attenuated by co-administration with quercetin. Herein, we concluded that increased levels of HSP70 through LPS pretreatment led to inhibition of NF-κB activity in the myocardium after I/R injury. Our results indicated that LPS-induced cardioprotection was mediated partly through inhibition of NF-κB via increase of HSP70, and LPS pretreatment could provide a means of reducing myocardial I/R injury.     

Mammalian target of rapamycin inhibition attenuates myocardial ischaemia–reperfusion injury in hypertrophic heart

Pathological cardiac hypertrophy aggravated myocardial infarction and is causally related to autophagy dysfunction and increased oxidative stress. Rapamycin is an inhibitor of serine/threonine kinase mammalian target of rapamycin (mTOR) involved in the regulation of autophagy as well as oxidative/nitrative stress. Here, we demonstrated that rapamycin ameliorates myocardial ischaemia reperfusion injury by rescuing the defective cytoprotective mechanisms in hypertrophic heart. Our results showed that chronic rapamycin treatment markedly reduced the phosphorylated mTOR and ribosomal protein S6 expression, but not Akt in both normal and aortic‐banded mice. Moreover, chronic rapamycin treatment significantly mitigated TAC‐induced autophagy dysfunction demonstrated by prompted Beclin‐1 activation, elevated LC3‐II/LC3‐I ratio and increased autophagosome abundance. Most importantly, we found that MI/R‐induced myocardial injury was markedly reduced by rapamycin treatment manifested by the inhibition of myocardial apoptosis, the reduction of myocardial infarct size and the improvement of cardiac function in hypertrophic heart. Mechanically, rapamycin reduced the MI/R‐induced iNOS/gp91^phox protein expression and decreased the generation of NO and superoxide, as well as the cytotoxic peroxynitrite. Moreover, rapamycin significantly mitigated MI/R‐induced endoplasmic reticulum stress and mitochondrial impairment demonstrated by reduced Caspase‐12 activity, inhibited CHOP activation, decreased cytoplasmic Cyto‐C release and preserved intact mitochondria. In addition, inhibition of mTOR also enhanced the phosphorylated ERK and eNOS, and inactivated GSK3β, a pivotal downstream target of Akt and ERK signallings. Taken together, these results suggest that mTOR signalling protects against MI/R injury through autophagy induction and ERK‐mediated antioxidative and anti‐nitrative stress in mice with hypertrophic myocardium.     

Nebivolol Protects against Myocardial Infarction Injury via Stimulation of Beta 3-Adrenergic Receptors and Nitric Oxide Signaling

Nebivolol, third-generation β-blocker, may activate β3-adrenergic receptor (AR), which has been emerged as a novel and potential therapeutic targets for cardiovascular diseases. However, it is not known whether nebivolol administration plays a cardioprotective effect against myocardial infarction (MI) injury. Therefore, the present study was designed to clarify the effects of nebivolol on MI injury and to elucidate the underlying mechanism. MI model was constructed by left anterior descending (LAD) artery ligation. Nebivolol, β3-AR antagonist (SR59230A), Nitro-L-arginine methylester (L-NAME) or vehicle was administered for 4 weeks after MI operation. Cardiac function was monitored by echocardiography. Moreover, the fibrosis and the apoptosis of myocardium were assessed by Masson''s trichrome stain and TUNEL assay respectively 4 weeks after MI. Nebivolol administration reduced scar area by 68% compared with MI group (p<0.05). Meanwhile, nebivolol also decreased the myocardial apoptosis and improved the heart function after MI (p<0.05 vs. MI). These effects were associated with increased β3-AR expression. Moreover, nebivolol treatment significantly increased the phosphorylation of endothelial NOS (eNOS) and the expression of neuronal NOS (nNOS). Conversely, the cardiac protective effects of nebivolol were abolished by SR and L-NAME. These results indicate that nebivolol protects against MI injury. Furthermore, the cardioprotective effects of nebivolol may be mediated by β3-AR-eNOS/nNOS pathway.     

Acute hyperglycaemia enhances oxidative stress and aggravates myocardial ischaemia/reperfusion injury: role of thioredoxin‐interacting protein

Hyperglycaemia during acute myocardial infarction is common and associated with increased mortality. Thioredoxin‐interacting protein (Txnip) is a modulator of cellular redox state and contributes to cell apoptosis. This study aimed to investigate whether or not hyperglycaemia enhances Txnip expression in myocardial ischaemia/reperfusion (MI/R) and consequently exacerbates MI/R injury. Rats were subjected to 30 min. of left coronary artery ligation followed by 4 hrs of reperfusion and treated with saline or high glucose (HG, 500 g/l, 4 ml/kg/h intravenously). In vitro study was performed on cultured rat cardiomyocytes subjected to simulated ischaemia/reperfusion (SI/R) and incubated with HG (25 mM) or normal glucose (5.6 mM) medium. In vivo HG infusion during MI/R significantly impaired cardiac function, aggravated myocardial injury and increased cardiac oxidative stress. Meanwhile, Txnip expression was enhanced whereas thioredoxin activity was inhibited following HG treatment in ischaemia/reperfusion (I/R) hearts. In addition, HG activated p38 MAPK and inhibited Akt in I/R hearts. In cultured cardiomyocytes subjected to SI/R, HG incubation stimulated Txnip expression and reduced thioredoxin activity. Overexpression of Txnip enhanced HG‐induced superoxide generation and aggravated cardiomyocyte apoptosis, whereas Txnip RNAi significantly blunted the deleterious effects of HG. Moreover, inhibition of p38 MAPK or activation of Akt markedly blocked HG‐induced Txnip expression in I/R cardiomyocytes. Most importantly, intramyocardial injection of Txnip siRNA markedly decreased Txnip expression and alleviated MI/R injury in HG‐treated rats. Hyperglycaemia enhances myocardial Txnip expression, possibly through reciprocally modulating p38 MAPK and Akt activation, leading to aggravated oxidative stress and subsequently, amplification of cardiac injury following MI/R.     

Gypenosides alleviate myocardial ischemia-reperfusion injury via attenuation of oxidative stress and preservation of mitochondrial function in rat heart

Gypenosides (GP) are the predominant components of Gynostemma pentaphyllum, a Chinese herb medicine that has been widely used for the treatment of chronic inflammation, hyperlipidemia, and cardiovascular disease. GP has been demonstrated to exert protective effects on the liver and brain against ischemia-reperfusion (I/R) injury, yet whether it is beneficial to the heart during myocardial I/R is unclear. In this study, we demonstrate that pre-treatment with GP dose-dependently limits infarct size, alleviates I/R-induced pathological changes in the myocardium, and preserves left ventricular function in a rat model of cardiac I/R injury. In addition, GP pre-treatment reduces oxidative stress and protects the intracellular antioxidant machinery in the myocardium. Further, we show that the cardioprotective effect of GP is associated with the preservation of mitochondrial function in the cardiomyocytes, as indicated by ATP level, enzymatic activities of complex I, II, and IV on the mitochondrial respiration chain, and the activity of citrate synthase in the citric acid cycle for energy generation. Moreover, GP maintains mitochondrial membrane integrity and inhibits the release of cytochrome c from the mitochondria to the cytosol. The cytoprotective effect of GP is further confirmed in vitro in H9c2 cardiomyoblast cell line with oxygen-glucose deprivation and reperfusion (OGD/R), and the results indicate that GP protects cell viability, reduces oxidative stress, and preserves mitochondrial function. In conclusion, our study suggests that GP may be of clinical value in cytoprotection during acute myocardial infarction and reperfusion.     

Regulation of metabolism by mitochondrial enzyme acetylation in cardiac ischemia-reperfusion injury

Ischemia reperfusion injury (I/R injury) contributes significantly to morbidity and mortality following myocardial infarction (MI). Although rapid reperfusion of the ischemic myocardium was established decades ago as a highly beneficial therapy for MI, significant cell death still occurs after the onset of reperfusion. Mitochondrial dysfunction is closely associated with I/R injury, resulting in the uncontrolled production of reactive oxygen species (ROS). Considerable efforts have gone into understanding the metabolic perturbations elicited by I/R injury. Recent work has identified the critical role of reversible protein acetylation in maintaining normal mitochondrial biologic function and energy metabolism both in the normal heart and during I/R injury. Several studies have shown that modification of class I HDAC and/or Sirtuin (Sirt) activity is cardioprotective in the setting of I/R injury. A better understanding of the role of these metabolic pathways in reperfusion injury and their regulation by reversible protein acetylation presents a promising way forward in improving the treatment of cardiac reperfusion injury. Here we briefly review some of what is known about how acetylation regulates mitochondrial metabolism and how it relates to I/R injury.     

罗布麻通过抑制氧化应激反应减轻心肌缺血再灌注损伤

目的:观察罗布麻叶提取物(apocynum venetum leaf extract AVLE)预处理对心肌缺血再灌注(MI/R)损伤的影响。方法:采用SD大鼠MI/R模型,随机分为sham(假手术)组、MI/R组、AVLE预处理+MI/R组,检测血流动力学,采用氯化三苯基四氮唑和伊文思蓝双染法检测心梗面积、以血浆肌酸激酶(CK)和乳酸脱氢酶(LDH)活性检测心肌损伤情况、以超氧化物、丙二醛(MAD)和超氧化物歧化酶(SOD)含量检测心肌氧化应激以及Western blot方法检测gp91phox的表达。结果:与MI/R组相比,AVLE预处理组左室压上升、下降最大速率(±LVdp/dtmax)升高(P0.05),心肌梗死面积减少,两组分别为41.5±4.5%和32.0±3.5%(P0.05),血浆CK和LDH活性分别降低到1653±62 U/L和2461±152 U/L(P0.05),减少了心肌组织超氧化物的含量(P0.05)。AVLE治疗显着降低gp91phox的表达(P0.05),使SOD活性增加(P0.05),MDA水平显著降低(P0.05)。结论:AVLE通过抑制I/R心肌的氧化应激发挥心脏保护作用。     

High mobility box 1 mediates neutrophil recruitment in myocardial ischemia-reperfusion injury through toll like receptor 4-related pathway

This study aimed to explore the role of high mobility box 1 (HMGB1) and its receptor toll like receptor 4 (TLR4) on neutrophils in myocardial ischemia reperfusion (I/R) injury. We constructed TLR4-mutant (C3H/HeJ) and control (C3H/HeN) mouse models of myocardial I/R injury and subjected the mice to 30min of ischemia and 6h of reperfusion. Light microscope was used to observe structural changes in the myocardium. HMGB1 levels were measured using quantitative real-time PCR and immunohistochemistry. Neutrophil accumulation, TNF-a expression and IL-8 levels were analyzed via myeloperoxidase (MPO) biochemical studies, quantitative real-time PCR and ELISA, respectively. The results demonstrated that fewer neutrophils infiltrated in the myocardium of TLR4-mutant mice after myocardial I/R and that TLR4 deficiency markedly decreased the ischemic injury caused by ischemia/reperfusion, and inhibited the expression of HMGB1, TNF-a, and IL-8, all of which were up-regulated by ischemia/reperfusion. These findings suggest that HMGB1 plays a central role in recruiting neutrophils during myocardial I/R leading to worsened myocardial I/R injury. This recruitment mechanism is possibly due to its inflammatory and chemokine functions based on the TLR4-dependent pathway.     

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.     

Overexpression of extracellular superoxide dismutase decreases lung injury after exposure to oil fly ash

The mechanism of tissue injury after exposure to air pollution particles is not known. The biological effect has been postulated to be mediated via an oxidative stress catalyzed by metals present in particulate matter (PM). We utilized a transgenic (Tg) mouse model that overexpresses extracellular superoxide dismutase (EC-SOD) to test the hypothesis that lung injury after exposure to PM results from an oxidative stress in the lower respiratory tract. Wild-type (Wt) and Tg mice were intratracheally instilled with either saline or 50 microg of residual oil fly ash (ROFA). Twenty-four hours later, specimens were obtained and included bronchoalveolar lavage (BAL) and lung for both homogenization and light histopathology. After ROFA exposure, EC-SOD Tg mice showed a significant reduction in BAL total cell counts (composed primarily of neutrophils) and BAL total protein compared with Wt. EC-SOD animals also demonstrated diminished concentrations of inflammatory mediators in BAL. There was no statistically significant difference in BAL lipid peroxidation; however, EC-SOD mice had lower concentrations of oxidized glutathione in the BAL. We conclude that enhanced EC-SOD expression decreased both lung inflammation and damage after exposure to ROFA. This supports a participation of oxidative stress in the inflammatory injury after PM exposure rather than reflecting a response to metals alone.     

TNF-α inhibitor protects against myocardial ischemia/reperfusion injury via Notch1-mediated suppression of oxidative/nitrative stress

TNF-α inhibitor reportedly protects against myocardial ischemia/reperfusion (MI/R) injury. It can also increase Notch1 expression in inflammatory bowel disease, revealing the regulation of Notch1 signaling by TNF-α inhibitor. However, the interaction between TNF-α inhibitor and Notch1 signaling in MI/R remains unclear. This study aimed to determine the involvement of TNF-α inhibitor with Notch1 in MI/R and delineate the related mechanism. Notch1-specific small interfering RNA (20 μg) or Jagged1 (a Notch ligand, 12 μg) was delivered through intramyocardial injection. Forty-eight hours after injection, mice received 30 min of myocardial ischemia followed by 3 h (for cell apoptosis and oxidative/nitrative stress) or 24 h (for infarct size and cardiac function) of reperfusion. Ten minutes before reperfusion, mice randomly received an intraperitoneal injection of vehicle, etanercept, diphenyleneiodonium, 1400W, or EUK134. Finally, downregulation of Notch1 significantly reversed the alleviation of MI/R injury induced by etanercept, as evidenced by enlarged myocardial infarct size, suppressed cardiac function, and increased myocardial apoptosis. Moreover, Notch1 blockade increased the expression of inducible NO synthase (iNOS) and gp^91phox, enhanced NO and superoxide production, and accelerated their cytotoxic reaction product, peroxynitrite. Furthermore, NADPH inhibition with diphenyleneiodonium or iNOS suppression with 1400W mitigated the aggravation of MI/R injury induced by Notch1 downregulation in mice treated with etanercept. Additionally, either Notch1 activation with Jagged1 or peroxynitrite decomposition with EUK134 reduced nitrotyrosine content and attenuated MI/R injury. These data indicate that MI/R injury can be attenuated by TNF-α inhibitor, partly via Notch1 signaling-mediated suppression of oxidative/nitrative stress.     

Acute hyperglycemia exacerbates myocardial ischemia/reperfusion injury and blunts cardioprotective effect of GIK

There is a close association between hyperglycemia and increased risk of mortality after acute myocardial infarction (AMI). However, whether acute hyperglycemia exacerbates myocardial ischemia/reperfusion (MI/R) injury remains unclear. We observed the effects of acute hyperglycemia on MI/R injury and on the cardioprotective effect of glucose-insulin-potassium (GIK). Male rats were subjected to 30 min of myocardial ischemia and 6 h of reperfusion. Rats were randomly received one of the following treatments (at 4 ml.kg(-1).h(-1) iv): Vehicle, GIK (GIK during reperfusion; glucose: 200g/l, insulin: 60 U/l, KCL: 60 mmol/l), HG (high glucose during ischemia; glucose:500 g/l), GIK + HG (HG during I and GIK during R) or GIK + wortmannin (GIK during R and wortmannin 15 min before R). Blood glucose, plasma insulin concentration and left ventricular pressure (LVP) were monitored throughout the experiments. Hyperglycemia during ischemia not only significantly increased myocardial apoptosis (23.6 +/- 1.7% vs. 18.8 +/- 1.4%, P < 0.05 vs. vehicle), increased infarct size (IS) (45.6 +/- 3.0% vs. 37.6 +/- 2.0%, P < 0.05 vs. vehicle), decreased Akt and GSK-3beta phosphorylations (0.5 +/- 0.2 and 0.6 +/- 0.1% fold of vehicle, respectively, P < 0.05 vs. vehicle) following MI/R, but almost completely blocked the cardioprotective effect afforded by GIK, as evidenced by significantly increased apoptotic index (19.1 +/- 2.0 vs. 10.3 +/- 1.2%, P < 0.01 vs. GIK), increased myocardial IS (39.2 +/- 2.8 vs. 27.2 +/- 2.1%, P < 0.01 vs. GIK), decreased Akt phosphorylation (1.1 +/- 0.1 vs. 1.7 +/- 0.2%, P < 0.01 vs. GIK) and GSK-3beta phosphorylation (1.4 +/- 0.2 vs. 2.3 +/- 0.2%, P < 0.05 vs. GIK). Hyperglycemia significantly exacerbates MI/R injury and blocks the cardioprotective effect afforded by GIK, which is, at least in part, due to hyperglycemia-induced decrease of myocardial Akt activation.