Myristoylated methionine sulfoxide reductase A protects the heart from ischemia-reperfusion injury

Methionine sulfoxide reductase A (MsrA) catalytically scavenges reactive oxygen species and also repairs oxidized methionines in proteins. Increasing MsrA protects cells and organs from a variety of oxidative stresses while decreasing MsrA enhances damage, but the mechanisms of action have not been elucidated. A single gene encodes MsrA of which ～25% is targeted to the mitochondria, a major site of reactive oxygen species production. The other ～75% is targeted to the cytosol and is posttranslationally modified by myristoylation. To determine the relative importance of MsrA in each compartment in protecting against ischemia-reperfusion damage, we created a series of transgenic mice overexpressing MsrA targeted to the mitochondria or the cytosol. We used a Langendorff model of ischemia-reperfusion and assayed both the rate pressure product and infarct size following ischemia and reperfusion as measures of injury. While the mitochondrially targeted MsrA was expected to be protective, it was not. Notably, the cytosolic form was protective but only if myristoylated. The nonmyristoylated, cytosolic form offered no protection against injury. We conclude that cytosolic MsrA protects the heart from ischemia-reperfusion damage. The requirement for myristoylation suggests that MsrA must interact with a hydrophobic domain to provide protection.     

Cardiac mTOR protects the heart against ischemia-reperfusion injury

Cardiac mammalian target of rapamycin (mTOR) is necessary and sufficient to prevent cardiac dysfunction in pathological hypertrophy. However, the role of cardiac mTOR in heart failure after ischemic injury remains undefined. To address this question, we used transgenic (Tg) mice with cardiac-specific overexpression of mTOR (mTOR-Tg mice) to study ischemia-reperfusion (I/R) injury in two animal models: 1) in vivo I/R injury with transient coronary artery ligation and 2) ex vivo I/R injury in Langendorff-perfused hearts with transient global ischemia. At 28 days after I/R, mortality was lower in mTOR-Tg mice than littermate control mice [wild-type (WT) mice]. Echocardiography and MRI demonstrated that global cardiac function in mTOR-Tg mice was preserved, whereas WT mice exhibited significant cardiac dysfunction. Masson's trichrome staining showed that 28 days after I/R, the area of interstitial fibrosis was smaller in mTOR-Tg mice compared with WT mice, suggesting that adverse left ventricular remodeling is inhibited in mTOR-Tg mice. In the ex vivo I/R model, mTOR-Tg hearts demonstrated improved functional recovery compared with WT hearts. Perfusion with Evans blue after ex vivo I/R yielded less staining in mTOR-Tg hearts than WT hearts, indicating that mTOR overexpression inhibited necrosis during I/R injury. Expression of proinflammatory cytokines, including IL-6 and TNF-α, in mTOR-Tg hearts was lower than in WT hearts. Consistent with this, IL-6 in the effluent post-I/R injury was lower in mTOR-Tg hearts than in WT hearts. These findings suggest that cardiac mTOR overexpression in the heart is sufficient to provide substantial cardioprotection against I/R injury and suppress the inflammatory response.     

Activation of estrogen receptor-alpha protects the in vivo rabbit heart from ischemia-reperfusion injury

The estrogen receptor (ER) mediates estrogenic activity in a variety of organs, including those in the reproductive, cardiovascular, immune, and central nervous systems. Experimental studies have demonstrated that 17beta-estradiol (E2) protects the heart from ischemia-reperfusion injury. Two estrogen receptors, ER alpha and ER beta, mediate the actions of estrogen; however, it is not certain which ER mediates the cardioprotective effects of E2. In the present study, the ER-selective agonists 4,4',4'-[4-propyl-(1H)-pyrazole-1,3,5-triyl]tris-phenol (PPT; ER alpha) and 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; ER beta) were assessed for their cardioprotective potential in an in vivo rabbit model of ischemia-reperfusion injury. Anesthetized female rabbits were administered PPT (3 mg/kg), DPN (3 mg/kg), E2 (20 microg/rabbit), or vehicle intravenously 30 min before a 30-min occlusion of the left anterior descending coronary artery followed by 4 h of reperfusion. Acute treatment with E2 (17.7 +/- 2.9%; P < 0.001) and PPT (18.1 +/- 2.9%; P < 0.001), but not DPN (45.3 +/- 2.4%) significantly decreased infarct size as a percent of area at risk compared with vehicle (45.3 +/- 2.4%). Coadministration of PPT or E2 with the ER antagonist ICI-182,780 limited the infarct size-sparing effect of the compounds (43.8 +/- 6.6% and 40.6 +/- 5.7% respectively, expressed as a percentage of risk region). PPT reduced the release of cardiac-specific troponin-I and reduced the tissue deposition of the membrane attack complex and C-reactive protein similar to that of E2. The results indicate that activation of ER alpha, but not ER beta, is required for the observed cardioprotective effects of E2.     

Hydrogen sulfide protects rat lung from ischemia-reperfusion injury

Recent studies have indicated that hydrogen sulfide (H(2)S) is capable of modulating many physiological processes, which prompted us to investigate the potential of H(2)S as a lung protective agent. To explore changes in the generation of endogenous H(2)S and the role of H(2)S in the pathogenesis of pulmonary ischemia-reperfusion (I/R) injury in rats, we built an isolated rat lung I/R model. Lungs were subjected to 45 min ischemia followed by reperfusion (45 min) and were pretreated with H(2)S (50 micromol/l or 100 micromol/l) or an irreversible inhibitor of cystathionine-gamma-lyase (CSE), propargylglycine (PPG; 2 mmol/l). We examined indices of lung injury: lung histological change, perfusion flow rate, ratio of lung wet weight to dry weight (w/d), and lung compliance. H(2)S content and CSE protein expression in lung tissues were measured. Malondialdehyde (MDA) content, activities of superoxide dismutase (SOD) and catalase (CAT), and restraint of superoxide anion (O(2)(-)) production in lung tissues were measured to reflect oxidative stress. In the current study, we demonstrated that H(2)S content and CSE activity in lungs after I/R were significantly higher than those in the control group. Preperfusion with H(2)S attenuated the lung I/R injury while preperfusion with PPG aggravated the lung I/R injury. H(2)S preperfusion reduced I/R-induced MDA production and potentiated SOD and CAT activities and the restraint of O(2)(-) production in the lungs under I/R, which attenuated lung oxidative injury. These findings suggest that endogenous CSE/H(2)S pathway might be involved in the pathogenesis of lung I/R injury and that administration of H(2)S might be of clinical benefit in lung I/R injury.     

Calpain inhibitor-1 protects the rat heart from ischemia-reperfusion injury: analysis by mechanical work and energetics

We hypothesized that calpain inhibitor-1 protected left ventricular (LV) function from ischemia-reperfusion injury by inhibiting the proteolysis of alpha-fodrin. To test this hypothesis, we investigated the effect of calpain inhibitor-1 on LV mechanical work and energetics in the cross-circulated rat hearts that underwent 15-min global ischemia and 60-min reperfusion (n = 9). After ischemia-reperfusion with calpain inhibitor-1, mean end-systolic pressure at midrange LV volume and systolic pressure-volume area (PVA) at midrange LV volume (total mechanical energy per beat) were hardly changed, although they were significantly (P < 0.01) decreased after ischemia-reperfusion without calpain inhibitor-1. Mean myocardial oxygen consumption per beat (Vo(2)) intercepts (PVA-independent Vo(2); Vo(2) for the total Ca(2+) handling in excitation-contraction coupling and basal metabolism) of Vo(2)-PVA linear relations were also unchanged after ischemia-reperfusion with calpain inhibitor-1, although they were significantly (P < 0.01) decreased after ischemia-reperfusion without calpain inhibitor-1. There were no significant differences in O(2) costs of LV PVA and contractility among the hearts in control (or normal) postischemia-reperfusion and postischemia-reperfusion with calpain inhibitor-1. Western blot analysis of alpha-fodrin and the immunostaining of 150-kDa products of alpha-fodrin confirmed that calpain inhibitor-1 almost completely protected the proteolysis of alpha-fodrin. Our results indicate that calpain inhibitor-1 prevents the heart from ischemia-reperfusion injury associated with the impairment of total Ca(2+) handling by directly inhibiting the proteolysis of alpha-fodrin.     

Carbamylated erythropoietin protects the kidneys from ischemia-reperfusion injury without stimulating erythropoiesis

Several studies have shown that erythropoietin (EPO) can protect the kidneys from ischemia-reperfusion injury and can raise the hemoglobin (Hb) concentration. Recently, the EPO molecule modified by carbamylation (CEPO) has been identified and was demonstrated to be able to protect several organs without increasing the Hb concentration. We hypothesized that treatment with CEPO would protect the kidneys from tubular apoptosis and inhibit subsequent tubulointerstitial injury without erythropoiesis. The therapeutic effect of CEPO was evaluated using a rat ischemia-reperfusion injury model. Saline-treated kidneys exhibited increased tubular apoptosis with interstitial expression of alpha-smooth muscle actin (alpha-SMA), while EPO treatment inhibited tubular apoptosis and alpha-SMA expression to some extent. On the other hand, CEPO-treated kidneys showed minimal tubular apoptosis with limited expression of alpha-SMA. Moreover, CEPO significantly promoted tubular epithelial cell proliferation without erythropoiesis. In conclusion, we identified a new therapeutic approach using CEPO to protect kidneys from ischemia-reperfusion injury.     

Protective effects of melatonin against ischemia-reperfusion injury in the isolated rat heart

There has been increased interest in melatonin recently, since it was shown to be a potent scavenger of toxic free radicals. Melatonin has been found to be effective in protecting against pathological states due to reactive oxygen species release. The present study was performed in order to determine whether melatonin or 5-methoxy-carbonylamino-N-acetyl-tryptamine (5-MCA-NAT), a structurally related indole compound, protect against ischemia-reperfusion injury in the isolated rat heart. Wistar rats were treated in vivo with either melatonin (1 or 10 mg/kg, i.p.) or 5-MCA-NAT (10 mg/kg, i.p.) or their vehicle, 30 min before their hearts were excised and perfused according to the Langendorff technique. Two different protocols were then applied. In the first one, a regional ischemia (5 min)-reperfusion (30 min) sequence was performed in order to record incidence and duration of reperfusion arrhythmias. In the second one, infarct size was assessed after a regional ischemia (30 min)-reperfusion (120 min) sequence. Results show a spectacular protection against ischemia-reperfusion injuries (on arrhythmias as well as on infarct size) in rats pre-treated with 10 mg/kg of melatonin or 5-MCA-NAT. In conclusion, both melatonin and its structural analog, 5-MCA-NAT, appear to confer protection against ischemia-reperfusion injury in the isolated rat heart. This observation suggests that melatonin could have a potential clinical application in the treatment of myocardial ischemia, even if the mechanisms underlying this protection remain to be determined.     

Na+/Ca2+ exchange inhibition protects the rat heart from ischemia-reperfusion injury by blocking energy-wasting processes

We have recently reported that exposure of rat hearts to high Ca(2+) produces a Ca(2+) overload-induced contractile failure in rat hearts, which was associated with proteolysis of alpha-fodrin. We hypothesized that contractile failure after ischemia-reperfusion (I/R) is similar to that after high Ca(2+) infusion. To test this hypothesis, we investigated left ventricular (LV) mechanical work and energetics in the cross-circulated rat hearts, which were subjected to 15 min global ischemia and 60 min reperfusion. Sixty minutes after I/R, mean systolic pressure-volume area (PVA; a total mechanical energy per beat) at midrange LV volume (mLVV) (PVA(mLVV)) was significantly decreased from 5.89 +/- 1.55 to 3.83 +/- 1.16 mmHg.ml.beat(-1).g(-1) (n = 6). Mean myocardial oxygen consumption per beat (Vo(2)) intercept of (Vo(2)-PVA linear relation was significantly decreased from 0.21 +/- 0.05 to 0.15 +/- 0.03 microl O(2).beat(-1).g(-1) without change in its slope. Initial 30-min reperfusion with a Na(+)/Ca(2+) exchanger (NCX) inhibitor KB-R7943 (KBR; 10 micromol/l) significantly reduced the decrease in mean PVA(mLVV) and Vo(2) intercept (n = 6). Although Vo(2) for the Ca(2+) handling was finally decreased, it transiently but significantly increased from the control for 10-15 min after I/R. This increase in Vo(2) for the Ca(2+) handling was completely blocked by KBR, suggesting an inhibition of reverse-mode NCX by KBR. alpha-Fodrin proteolysis, which was significantly increased after I/R, was also significantly reduced by KBR. Our study shows that the contractile failure after I/R is similar to that after high Ca(2+) infusion, although the contribution of reverse-mode NCX to the contractile failure is different. An inhibition of reverse-mode NCX during initial reperfusion protects the heart against reperfusion injury.     

Extracellular calcium protects isolated rat hepatocytes from injury

The incubation of isolated rat hepatocytes in calcium-free medium resulted in a pronounced increase in lipid peroxidation, mitochondrial and cytoplasmic glutathione depletion, glutathione disulfide formation and efflux of reduced glutathione as compared with hepatocytes incubated in calcium containing medium. These data suggest that extracellular calcium ions serve a protective role in isolated rat hepatocytes against cell injury.     

Alpha-melanocyte-stimulating hormone protects against mesenteric ischemia-reperfusion injury

Mesenteric ischemia-reperfusion (I/R) injury to the intestine is a common and often devastating clinical occurrence for which there are few therapeutic options. alpha-Melanocyte-stimulating hormone (alpha-MSH) is a tridecapeptide released by the pituitary gland and immunocompetent cells that exerts anti-inflammatory actions and abrogates postischemic injury to the kidneys and brainstem of rodents. To test the hypothesis that alpha-MSH would afford similar protection in the postischemic small intestine, we analyzed the effects of this peptide on intestinal transit, histology, myeloperoxidase activity, and nuclear factor-kappaB (NF-kappaB) activation after 45 min of superior mesenteric artery occlusion and 相似文献   

Local hypothermia during early reperfusion protects skeletal muscle from ischemia-reperfusion injury

Amputated tissue maintained in a hypothermic environment can endure prolonged ischemia and improve replantation success. The authors hypothesized that local tissue hypothermia during the early reperfusion period may provide a protective effect against ischemia-reperfusion injury similar to that seen when hypothermia is provided during the ischemic period. A rat gracilis muscle flap model was used to assess the protective effects of exposing skeletal muscle to local hypothermia during ischemia only (p = 18), reperfusion only (p = 18), and both ischemia and reperfusion (p = 18). Gracilis muscles were isolated and exposed to hypothermia of 10 degrees C during 4 hours of ischemia, the initial 3 hours of reperfusion, or both periods. Ischemia-reperfusion outcome measures used to evaluate muscle flap injury included muscle viability (percent nitroblue tetrazolium staining), local edema (wet-to-dry weight ratio), neutrophil infiltration (intramuscular neutrophil density per high-power field), neutrophil integrin expression (CD11b mean fluorescence intensity), and neutrophil oxidative potential (dihydro-rhodamine oxidation mean fluorescence intensity) after 24 hours of reperfusion. Nitroblue tetrazolium staining demonstrated improved muscle viability in the experimental groups (ischemia-only: 78.8 +/- 3.5 percent, p < 0.001; reperfusion-only: 80.2 +/- 5.2 percent, p < 0.001; and ischemia-reperfusion: 79.6 +/- 7.6 percent, p < 0.001) when compared with the nonhypothermic control group (50.7 +/- 9.3 percent). The experimental groups demonstrated decreased local muscle edema (4.09 +/- 0.30, 4.10 +/- 0.19, and 4.04 +/- 0.31 wet-to-dry weight ratios, respectively) when compared with the nonhypothermic control group (5.24 +/- 0.31 wet-to-dry weight ratio; p < 0.001, p < 0.001, and p < 0.001, respectively). CD11b expression was significantly decreased in the reperfusion-only (32.65 +/- 8.75 mean fluorescence intensity, p < 0.001) and ischemia-reperfusion groups (25.26 +/- 5.32, p < 0.001) compared with the nonhypothermic control group (62.69 +/- 16.93). There was not a significant decrease in neutrophil CD11b expression in the ischemia-only group (50.72 +/- 11.7 mean fluorescence intensity, p = 0.281). Neutrophil infiltration was significantly decreased in the reperfusion-only (20 +/- 11 counts per high-power field, p = 0.025) and ischemia-reperfusion groups (23 +/- 3 counts, p = 0.041) compared with the nonhypothermic control group (51 +/- 28 counts). No decrease in neutrophil density was observed in the ischemia-only group (40 +/- 15 counts per high-power field, p = 0.672) when compared with the nonhypothermic control group (51 +/- 28 counts). Finally, dihydrorhodamine oxidation was significantly decreased in the reperfusion-only group (45.83 +/- 11.89 mean fluorescence intensity, p = 0.021) and ischemia-reperfusion group (44.30 +/- 11.80, p = 0.018) when compared with the nonhypothermic control group (71.74 +/- 20.83), whereas no decrease in dihydrorhodamine oxidation was observed in the ischemia-only group (65.93 +/- 10.3, p = 0.982). The findings suggest a protective effect of local hypothermia during early reperfusion to skeletal muscle after an ischemic insult. Inhibition of CD11b expression and subsequent neutrophil infiltration and depression of neutrophil oxidative potential may represent independent protective mechanisms isolated to local tissue hypothermia during the early reperfusion period (reperfusion-only and ischemia-reperfusion groups). This study provides evidence for the potential clinical utility of administering local hypothermia to ischemic muscle tissue during the early reperfusion period.     

Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein-associated O-GlcNAc

Increased levels of protein O-linked N-acetylglucosamine (O-GlcNAc) have been shown to increase cell survival following stress. Therefore, the goal of this study was to determine whether in isolated neonatal rat ventricular myocytes (NRVMs) an increase in protein O-GlcNAcylation resulted in improved survival and viability following ischemia-reperfusion (I/R). NRVMs were exposed to 4 h of ischemia and 16 h of reperfusion, and cell viability, necrosis, apoptosis, and O-GlcNAc levels were assessed. Treatment of cells with glucosamine, hyperglycemia, or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate(PUGNAc), an inhibitor of O-GlcNAcase, significantly increased O-GlcNAc levels and improved cell viability, as well as reducing both necrosis and apoptosis compared with untreated cells following I/R. Alloxan, an inhibitor of O-GlcNAc transferase, markedly reduced O-GlcNAc levels and exacerbated I/R injury. The improved survival with hyperglycemia was attenuated by azaserine, which inhibits glucose metabolism via the hexosamine biosynthesis pathway. Reperfusion in the absence of glucose reduced O-GlcNAc levels on reperfusion compared with normal glucose conditions and decreased cell viability. O-GlcNAc levels significantly correlated with cell viability during reperfusion. The effects of glucosamine and PUGNAc on cellular viability were associated with reduced calcineurin activation as measured by translocation of nuclear factor of activated T cells, suggesting that increased O-GlcNAc levels may attenuate I/R induced increase in cytosolic Ca²⁺. These data support the concept that activation of metabolic pathways leading to an increase in O-GlcNAc levels is an endogenous stress-activated response and that augmentation of this response improves cell survival. Thus strategies designed to activate these pathways may represent novel interventions for inducing cardioprotection. hexosamine biosynthesis; calcium; protein O-glycosylation     

Melatonin protects diabetic heart against ischemia-reperfusion injury,role of membrane receptor-dependent cGMP-PKG activation

It has been demonstrated that the anti-oxidative and cardioprotective effects of melatonin are, at least in part, mediated by its membrane receptors. However, the direct downstream signaling remains unknown. We previously found that melatonin ameliorated myocardial ischemia-reperfusion (MI/R) injury in diabetic animals, although the underlying mechanisms are also incompletely understood. This study was designed to determine the role of melatonin membrane receptors in melatonin's cardioprotective actions against diabetic MI/R injury with a focus on cGMP and its downstream effector PKG. Streptozotocin-induced diabetic Sprague-Dawley rats and high-glucose medium-incubated H9c2 cardiomyoblasts were utilized to determine the effects of melatonin against MI/R injury. Melatonin treatment preserved cardiac function and reduced oxidative damage and apoptosis. Additionally, melatonin increased intracellular cGMP level, PKGIα expression, p-VASP/VASP ratio and further modulated myocardial Nrf-2-HO-1 and MAPK signaling. However, these effects were blunted by KT5823 (a selective inhibitor of PKG) or PKGIα siRNA except that intracellular cGMP level did not changed significantly. Additionally, our in vitro study showed that luzindole (a nonselective melatonin membrane receptor antagonist) or 4P–PDOT (a selective MT₂ receptor antagonist) not only blocked the cytoprotective effect of melatonin, but also attenuated the stimulatory effect of melatonin on cGMP-PKGIα signaling and its modulatory effect on Nrf-2-HO-1 and MAPK signaling. This study showed that melatonin ameliorated diabetic MI/R injury by modulating Nrf-2-HO-1 and MAPK signaling, thus reducing myocardial apoptosis and oxidative stress and preserving cardiac function. Importantly, melatonin membrane receptors (especially MT₂ receptor)-dependent cGMP-PKGIα signaling played a critical role in this process.     

Increased expression of HSP27 protects canine myocytes from simulated ischemia-reperfusion injury

Previous studies have shown that adult rat myocytes can be protected from simulated ischemia-reperfusion (I/R) injury by small heat shock proteins (sHSPs). However, to date the cardioprotective effect of sHSPs has not been confirmed in adult myocytes from a large animal species. Left ventricular myocytes from adult dogs were cultured and infected with a replication-deficient adenovirus designed to increase expression of the human form of HSP27. The response to simulated I/R injury was compared using morphologic criteria. Virus-infected myocytes expressed two- to threefold more HSP27 and sustained less injury in response to simulated I/R than control cells (P < 0.001; paired t-test). Canine myocytes can be isolated, cultured, and induced to increase the expression of a foreign protein without significant effects on differentiation and/or viability. Increased expression of HSP27 provides significant protection from simulated I/R injury in adult canine myocytes. Determining the mechanism by which sHSPs protect from lethal cell injury will provide important new insights into the mechanism of irreversible cell injury in adult myocardium.     

Propyl paraben inhibits voltage-dependent sodium channels and protects cardiomyocytes from ischemia-reperfusion injury

The effects of propyl paraben, an antimicrobial preservative, on voltage-dependent sodium current and myocardial ischemia-reperfusion injury were investigated in isolated adult rat cardiomyocytes. Whole cell voltage-clamp recording showed that propyl paraben reversibly blocked the voltage-gated sodium channel both in concentration- and voltage-dependent manners. Propyl paraben (500 microM but not 100 microM) significantly shifted the steady-state inactivation of the sodium channel toward the hyperpolarizing direction at the V(1/2) point. Consistent with the above result, the propidium iodide (PI) uptake test revealed that pretreatment with 500 microM but not 100 microM of propyl paraben significantly reduced cell death induced by 45 min of sustained ischemia followed by 15 h of reperfusion (42.37 +/- 7.01% of cell viability in control and 71.05 +/- 7.06% in the propyl paraben group), suggesting that propyl paraben can protect myocytes from ischemia-reperfusion injury. These results indicate a possible correlation between the inhibition of sodium current and cardioprotection against ischemia-reperfusion injury.