Proteasome mediates removal of proteins oxidized during myocardial ischemia

Numerous proteins are known to be lost following myocardial ischemia/reperfusion yet little is known about the mediating proteinases. This study examines the hypothesis that proteasome plays a significant role in the removal of proteins oxidized during myocardial ischemia. Proteasome was inhibited by perfusing isolated rat hearts with buffer containing lactacystin, 2 micromol/L, for 10 min, which resulted in 51 and 42% decreases in 20S and 26S proteasome activities that persisted for a minimum of 90 min. Lactacystin pretreatment had minor effects on postischemic recovery of isolated hearts exposed to 30 min global ischemia and 60 min reperfusion. Protein carbonyl content of lactacystin-pretreated ischemic hearts was significantly (P < 0.05) increased. One band with approximate molecular mass of 50 kDa is known to contain oxidized actin. Actin degradation was quantitated by analysis of 3-methylhistidine which was significantly (P < 0.05) decreased by 15% following 30 min ischemia and 60 min reperfusion. Pretreatment of ischemic hearts with lactacystin prevented much of the loss (-6.5%) of 3-methylhistidine. Probing immunoprecipitated actin with an antibody specific for ubiquitin revealed no bands containing ubiquitinated homologues of this protein. These observations support the conclusion that proteasome mediates removal of some of the proteins oxidized during myocardial ischemia/reperfusion, and that at least oxidized actin is removed by the 20S proteasome.     

Early activation of IKKbeta during in vivo myocardial ischemia

We have demonstrated that in vitro brief ischemia activates nuclear factor (NF)-kappaB in rat myocardium. We report in vivo ischemia-reperfusion (I/R)-induced NF-kappaB activation, IkappaB kinase -beta (IKKbeta) activity, and IkappaBalpha phosphorylation and degradation in rat myocardium. Rat hearts were subjected to occlusion of the coronary artery for up to 45 min or occlusion for 15 min followed by reperfusion for up to 3 h. Cytoplasmic and nuclear proteins were isolated from ischemic and nonischemic areas of each heart. NF-kappaB activation was increased in the ischemic area (680%) after 10 min of ischemia and in the nonischemic area (350%) after 15 min of ischemia and remained elevated during prolonged ischemia and reperfusion. IKKbeta activity was markedly increased in ischemic (1,800%) and nonischemic (860%) areas, and phosphorylated IkappaBalpha levels were significantly elevated in ischemic (180%) and nonischemic (280%) areas at 5 min of ischemia and further increased after reperfusion. IkappaBalpha levels were decreased in the ischemic (45%) and nonischemic (36%) areas after 10 min of ischemia and remained low in the ischemic area during prolonged ischemia and reperfusion. The results suggest that in vivo I/R rapidly induces IKKbeta activity and increases IkappaBalpha phosphorylation and degradation, resulting in NF-kappaB activation in the myocardium.     

Molecular mechanisms and physiological significance of autophagy during myocardial ischemia and reperfusion

Autophagy is an intracellular bulk degradation process whereby cytoplasmic proteins and organelles are degraded and recycled through lysosomes. In the heart, autophagy plays a homeostatic role at basal levels, and the absence of autophagy causes cardiac dysfunction and the development of cardiomyopathy. Autophagy is induced during myocardial ischemia and further enhanced by reperfusion. Although induction of autophagy during the ischemic phase is protective, further enhancement of autophagy during the reperfusion phase may induce cell death and appears to be detrimental. In this review we discuss the functional significance of autophagy and the underlying signaling mechanism in the heart during ischemia/reperfusion.     

Molecular mechanisms and physiological significance of autophagy during myocardial ischemia and reperfusion

Autophagy is an intracellular bulk degradation process whereby cytoplasmic proteins and organelles are degraded and recycled through lysosomes. In the heart, autophagy plays a homeostatic role at basal levels, and the absence of autophagy causes cardiac dysfunction and the development of cardiomyopathy. Autophagy is induced during myocardial ischemia and further enhanced by reperfusion. Although induction of autophagy during the ischemic phase is protective, further enhancement of autophagy during the reperfusion phase may induce cell death and appears to be detrimental. In this review we discuss the functional significance of autophagy and the underlying signaling mechanism in the heart during ischemia/reperfusion.     

Resveratrol,novel application by preconditioning to attenuate myocardial ischemia/reperfusion injury in mice through regulate AMPK pathway and autophagy level

Myocardial ischemia/reperfusion injury (MI/RI) is the main cause of deaths in the worldwide, leading to severe cardiac dysfunction. Resveratrol (RSV) is a polyphenol plant‐derived compound. Our study aimed to elucidate the underlying molecular mechanism of preconditioning RSV in protecting against MI/RI. Mice were ligated and re‐perfused by the left anterior descending branch with or without RSV (30 mg/kg·ip) for 7 days. Firstly, we found that RSV pretreatment significantly alleviated myocardial infarct size, improved cardiac function and decreased oxidative stress. Furthermore, RSV activated p‐AMPK and SIRT1, ameliorated inflammation including the level of TNF‐α and IL‐1β, and promoting autophagy level. Moreover, neonatal rat ventricular myocytes (NRVMs) and H9c2 cells with knockdown the expression of AMPK, SIRT1 or FOXO1 were used to uncover the underlying molecular mechanism for the cardio‐protection of RSV. In NRVMs, RSV increased cellular viability, decreased LDH release and reduced oxidative stress. Importantly, Compound C(CpC) and EX527 reversed the effect of RSV against MI/RI in vivo and in vitro and counteracted the autophagy level induced by RSV. Together, our study indicated that RSV could alleviate oxidative stress in cardiomyocytes through activating AMPK/SIRT1‐FOXO1 signallingpathway and enhanced autophagy level, thus presenting high potential protection on MI/RI.     

AMPK mediates a pro-survival autophagy downstream of PARP-1 activation in response to DNA alkylating agents

In this study we aim to elucidate the signaling pathway and biological function of autophagy induced by MNNG, a commonly used DNA alkylating agent. We first observed that MNNG is able to induce necrotic cell death and autophagy in Bax?/? Bak?/? double knockout MEFs. We analyzed the critical role of PARP-1 activation and ATP depletion in MNNG-mediated cell death and autophagy via AMPK activation and mTOR suppression. We provide evidence that suppression of AMPK blocks MNNG-induced autophagy and enhances cell death, suggesting the pro-survival function of autophagy in MNNG-treated cells. Taken together, data from this study reveal a novel mechanism in controlling MNNG-mediated autophagy via AMPK activation downstream of PARP-1 activation and ATP depletion.     

Nitric oxide mediates protective effect of endothelin receptor antagonism during myocardial ischemia and reperfusion

Endothelin (ET) receptor antagonism protects from ischemia-reperfusion injury. We hypothesized that the cardioprotective effect is related to nitric oxide (NO) bioavailability. Buffer-perfused rat and mouse hearts were subjected to ischemia and reperfusion. At the onset of ischemia, the rat hearts received vehicle, the dual endothelin type A/type B (ETA/ETB) receptor antagonist bosentan (10 microM), the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 100 microM), the combination of bosentan and L-NMMA or the combination of bosentan, L-NMMA, and the NO substrate L-arginine (1 mM). Hearts from wild-type and endothelial NO synthase (eNOS)-deficient mice received either vehicle or bosentan. Myocardial performance, endothelial function, NO outflow, and eNOS expression were monitored. Bosentan significantly improved myocardial function during reperfusion in rats and in wild-type mice, but not in eNOS-deficient mice. The functional protection afforded by bosentan was inhibited by L-NMMA, whereas it was restored by L-arginine. Myocardial expression of eNOS (immunoblotting) increased significantly in bosentan-treated rat hearts compared with vehicle hearts. Recovery of NO outflow during reperfusion was enhanced in the bosentan-treated rat heart. The endothelium-dependent vasodilator adenosine diphosphate increased coronary flow by 18 +/- 9% at the end of reperfusion in the bosentan group, whereas it reduced coronary flow by 7 +/- 5% in the vehicle group (P < 0.001). The response to the endothelium-independent dilator sodium nitroprusside was not different between the two groups. In conclusion, the dual ETA/ETB receptor antagonist bosentan preserved endothelial and cardiac contractile function during ischemia and reperfusion via a mechanism dependent on endothelial NO production.     

RTN1-C mediates cerebral ischemia/reperfusion injury via modulating autophagy

It has been widely accepted that autophagic cell death exacerbates the progression of cerebral ischemia/reperfusion (I/R). Our previous study revealed that overexpression of reticulon protein 1-C (RTN1-C) is involved in cerebral I/R injury. However, the underlying mechanisms have not been studied intensively. This study was designed to evaluate the effect of RTN1-C on autophagy under cerebral I/R. Using an in vitro oxygen-glucose deprivation followed by reoxygenation and a transient middle cerebral artery occlusion model in rats, we found that the expression of RTN1-C protein was significantly upregulated. We also revealed that RTN1-C knockdown suppressed overactivated autophagy both in vivo and in vitro, as indicated by decreased expressions of autophagic proteins. The number of Beclin-1/propidium iodide-positive cells was significantly less in the LV-shRTN1-C group than in the LV-shNC group. In addition, rapamycin, an activator of autophagy, aggravated cerebral I/R injury. RTN1-C knockdown reduced brain infarct volume, improved neurological deficits, and attenuated cell vulnerability to cerebral I/R injury after rapamycin treatment. Taken together, our findings demonstrated that the modulation of autophagy from RTN1-C may play vital roles in cerebral I/R injury, providing a potential therapeutic treatment for ischemic brain injury.     

The role of autophagy during ischemia/reperfusion

Ischemia (hypoxia)/reperfusion (I/R) primarily caused by atherosclerosis and thrombosis, induces tissue injury and organ malfunction, and is therefore, an important clinical problem. Understanding the cellular mechanisms of enhancing cell survival or preventing cell death during I/R could lead to development of strategies to treat this health problem.     

Role of AMPK in atherosclerosis via autophagy regulation

Atherosclerosis is characterized by the accumulation of lipids and deposition of fibrous elements in the vascular wall, which is the primary cause of cardiovascular diseases. Adenosine monophosphate-activated protein kinase (AMPK) is a metabolic sensor of energy metabolism that regulates multiple physiological processes, including lipid and glucose metabolism and the normalization of energy imbalances. Overwhelming evidence indicates that AMPK activation markedly attenuates atherosclerosis development. Autophagy inhibits cell apoptosis and inflammation and promotes cholesterol efflux and efferocytosis. Physiological autophagy is essential for maintaining normal cardiovascular function. Increasing evidence demonstrates that autophagy occurs in developing atherosclerotic plaques. Emerging evidence indicates that AMPK regulates autophagy via a downstream signaling pathway. The complex relationship between AMPK and autophagy has attracted the attention of many researchers because of this close relationship to atherosclerosis development. This review demonstrates the role of AMPK and autophagy in atherosclerosis. An improved understanding of this interrelationship will create novel preventive and therapeutic strategies for atherosclerosis.     

黄芪注射液对缺血性心肌病大鼠心肌自噬及心肌重塑的影响

目的:研究黄芪注射液对缺血性心肌病大鼠心肌重塑、网腔钙结合蛋白(calumenin)及自噬影响。方法:36只雄性SD大鼠分为正常对照组(n=12)、缺血性心肌病组(n=12)、黄芪注射液组(n=12),3组大鼠术前行心电图及心脏彩超检查后,正常对照组不做任何处理,而缺血性心肌病组和黄芪注射液组大鼠开胸结扎冠状动脉20 min后,解开结扎线行再灌注后关闭胸腔建立心肌缺血模型,黄芪注射液组术后每次注射黄芪注射液10 g/kg体重,每周注射1次,共注射4次。3组大鼠术后4周行心脏彩超检查后处死大鼠取心脏行HE染色、VG染色,观察心肌病理改变,用Western blot技术检测各组大鼠心肌细胞calumenin、LC3-Ⅰ、LC3-Ⅱ表达变化及LC3-Ⅰ/LC3-Ⅱ比值变化。结果:与缺血性心肌病组比较,黄芪注射液组大鼠心脏彩超及心肌病理得到明显改善;同时,calumenin表达增加LC3-Ⅰ/LC3-Ⅱ比值表达降低(P<0.01)。结论:黄芪注射液对缺血性心肌病大鼠心室重塑及心肌细胞自噬有明显抑制作用,该作用可能是通过calumenin所介导的。     

Actin is oxidized during myocardial ischemia

Exposure of isolated rat hearts to 30 min global ischemia followed by 60 min reperfusion resulted in a significant 80% increase (p <.05) in actin content of carbonyl groups, which was associated with significant depression (p <.05) of postischemic contractile function. This result supports the hypothesis that one mechanism of postischemic contractile dysfunction may be oxidation of contractile proteins.     

Eat your heart out: Role of autophagy in myocardial ischemia/reperfusion

Autophagy is an important process in the heart which is responsible for the normal turnover of long lived proteins and organelles. Inhibition of autophagy leads to the accumulation of protein aggregates and dysfunctional organelles which can cause cell death. Autophagy occurs at low basal levels under normal conditions in the heart, but is rapidly upregulated in response to stress such as nutrient deprivation, hypoxia, and pressure overload. Autophagy is a prominent feature of myocardial ischemia and reperfusion. Although enhanced autophagy is often seen in dying cardiac myocytes, the functional significance of autophagy under these conditions is not clear. Upregulation of autophagy has been reported to protect cardiac cells against death as well as be the cause of it. Here, we review the evidence that autophagy can have both beneficial and detrimental roles in the myocardium, and discuss potential mechanisms by which autophagy provides protection in cells.     

Eat your heart out: Role of autophagy in myocardial ischemia/reperfusion

Autophagy is an important process in the heart which is responsible for the normal turnover of long lived proteins and organelles. Inhibition of autophagy leads to the accumulation of protein aggregates and dysfunctional organelles which can cause cell death. Autophagy occurs at low basal levels under normal conditions in the heart, but is rapidly upregulated in response to stress such as nutrient deprivation, hypoxia, and pressure overload. Autophagy is a prominent feature of myocardial ischemia and reperfusion. Although enhanced autophagy is often seen in dying cardiac myocytes, the functional significance of autophagy under these conditions is not clear. Upregulation of autophagy has been reported to protect cardiac cells against death as well as be the cause of it. Here, we review the evidence that autophagy can have both beneficial and detrimental roles in the myocardium, and discuss potential mechanisms by which autophagy provides protection in cells.     

Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy

Ischemia and reperfusion (I/R) injury is associated with extensive loss of cardiac myocytes. Bnip3 is a mitochondrial pro-apoptotic Bcl-2 protein which is expressed in the adult myocardium. To investigate if Bnip3 plays a role in I/R injury, we generated a TAT-fusion protein encoding the carboxyl terminal transmembrane deletion mutant of Bnip3 (TAT-Bnip3DeltaTM) which has been shown to act as a dominant negative to block Bnip3-induced cell death. Perfusion with TAT-Bnip3DeltaTM conferred protection against I/R injury, improved cardiac function, and protected mitochondrial integrity. Moreover, Bnip3 induced extensive fragmentation of the mitochondrial network and increased autophagy in HL-1 myocytes. 3D rendering of confocal images revealed fragmented mitochondria inside autophagosomes. Enhancement of autophagy by ATG5 protected against Bnip3-mediated cell death, whereas inhibition of autophagy by ATG5K130R enhanced cell death. These results suggest that Bnip3 contributes to I/R injury which triggers a protective stress response with upregulation of autophagy and removal of damaged mitochondria.     

Enhancing AMPK activation during ischemia protects the diabetic heart against reperfusion injury

AMPK activation during ischemia helps the myocardium to cope with the deficit of energy production. As AMPK activity is considered to be impaired in diabetes, we hypothesized that enhancing AMPK activation during ischemia above physiological levels would protect the ischemic diabetic heart through AMPK activation and subsequent inhibition of mitochondrial permeability transition pore (mPTP) opening. Isolated perfused hearts from normoglycemic Wistar or diabetic Goto-Kakizaki (GK) rats (n ≥ 6/group) were subjected to 35 min of ischemia in the presence of 10, 20, and 40 μM of A-769662, a known activator of AMPK, followed by 120 min of reperfusion with normal buffer. Myocardial infarction and AMPK phosphorylation were assessed. The effect of A-769662 on mPTP opening in adult cardiomyocytes isolated from both strains was also determined. A-769662 at 20 μM reduced infarct size in both Wistar (30.5 ± 2.7 vs. 51.8 ± 3.9% vehicle; P < 0.001) and GK hearts (22.7 ± 3.0 vs. 48.5 ± 4.7% vehicle; P < 0.001). This protection was accompanied by a significant increase in AMPK and GSK-3β phosphorylation. In addition, A-769662 significantly inhibited mPTP opening in both Wistar and GK cardiomyocytes subjected to oxidative stress. We demonstrate that AMPK activation during ischemia via A-769662 reduces myocardial infarct size in both the nondiabetic and diabetic rat heart. Furthermore, this cardioprotective effect appears to be mediated through inhibition of mPTP opening. Our findings suggest that improving AMPK activation during ischemia can be another mechanism for protecting the ischemic heart.