Inhibition of endoplasmic reticulum stress by neuregulin-1 protects against myocardial ischemia/reperfusion injury

Neuregulin-1 (NRG-1), an endogenously produced polypeptide, is the ligand of cardiomyocyte ErbB receptors, with cardiovascular protective effects. In the present study, we explored whether the cardioprotective effect of NRG-1 against I/R injury is mediated by inhibiting myocardial endoplasmic reticulum (ER) stress. In vitro, NRG-1 directly inhibited the upregulation of ER stress markers such as glucose-regulated protein 78, CCAAT/enhancer binding protein homologous protein and cleaved caspase-12 induced by the ER stress inducers tunicamycin or dithiothreitol in both neonatal and adult ventricular myocytes. Attenuating ErbB signals by an ErbB inhibitor AG1478 or ErbB4 knockdown and preincubation with phosphoinositide 3-kinase inhibitors all reversed the effect of NRG-1 inhibiting ER stress in cultured neonatal rat cardiomyocytes. Concurrently, cardiomyocyte ER stress and apoptosis induced by hypoxia-reoxygenation were decreased by NRG-1 treatment in vitro. Furthermore, in an in vivo rat model of myocardium ischemia/reperfusion (I/R), intravenous NRG-1 administration significantly decreased ER stress and myocardial infarct size induced by I/R. NRG-1 could protect the heart against I/R injury by inhibiting myocardial ER stress, which might be mediated by the phosphoinositide 3-kinase/Akt signaling pathway.     

RISK and SAFE Signaling Pathway Involvement in Apolipoprotein A-I-Induced Cardioprotection

Recent findings indicate that apolipoprotein A-I (ApoA-I) may be a protective humoral mediator involved in remote ischemic preconditioning (RIPC). This study sought to determine if ApoA-I mediates its protective effects via the RISK and SAFE signaling pathways implicated in RIPC. Wistar rats were allocated to one of the following groups. Control: rats were subjected to myocardial ischemia/reperfusion (I/R) without any further intervention; RIPC: four cycles of limb I/R were applied prior to myocardial ischemia; ApoA-I: 10 mg/Kg of ApoA-I were intravenously injected prior to myocardial ischemia; ApoA-I + inhibitor: pharmacological inhibitors of RISK/SAFE pro-survival kinase (Akt, ERK1/2 and STAT-3) were administered prior to ApoA-I injection. Infarct size was significantly reduced in the RIPC group compared to Control. Similarly, ApoA-I injection efficiently protected the heart, recapitulating RIPC-induced cardioprotection. The ApoA-I protective effect was associated with Akt and GSK-3β phosphorylation and substantially inhibited by pretreatment with Akt and ERK1/2 inhibitors. Pretreatment with ApoA-I in a rat model of I/R recapitulates RIPC-induced cardioprotection and shares some similar molecular mechanisms with those of RIPC-involved protection of the heart.     

Acute treatment with Danshen-Gegen decoction protects the myocardium against ischemia/reperfusion injury via the redox-sensitive PKC?/mKATP pathway in rats

Danshen-Gegen (DG) decoction, an herbal formulation comprising Radix Salvia Miltiorrhiza and Radix Puerariae Lobatae, is prescribed for the treatment of coronary heart disease in Chinese medicine. Experimental and clinical studies have demonstrated that DG decoction can reduce the extent of atherosclerosis. In the present study, using an ex vivo rat model of myocardial ischemia/reperfusion (I/R) injury, we investigated the myocardial preconditioning effect of an aqueous DG extract prepared from an optimized weight-to-weight ratio of Danshen and Gegen. Short-term treatment with DG extract at a daily dose of 1 g/kg and 2 g/kg for 3 days protected against myocardial I/R injury in rats. The cardioprotection afforded by DG pretreatment was paralleled by enhancements in mitochondrial antioxidant status and membrane structural integrity, as well as a decrease in the sensitivity of mitochondria to Ca²⁺-stimulated permeability transition in vitro, particularly under I/R conditions. Short-term treatment with the DG extract also enhanced the translocation of PKC? from the cytosol to mitochondria in rat myocardium, and this translocation was inhibited by α-tocopherol co-treatment with DG extract in rats. Short-term DG treatment may precondition the myocardium via a redox-sensitive PKC?/mK_ATP pathway, with resultant inhibition of the mitochondrial permeability transition through the opening of mitochondrial K_ATP channels. Our results suggest that clinical studies examining the effectiveness of DG extract given prophylactically in affording protection against myocardial I/R injury would be warranted.     

bFGF attenuates endoplasmic reticulum stress and mitochondrial injury on myocardial ischaemia/reperfusion via activation of PI3K/Akt/ERK1/2 pathway

Extensive research focused on finding effective strategies to prevent or improve recovery from myocardial ischaemia/reperfusion (I/R) injury. Basic fibroblast growth factor (bFGF) has been shown to have therapeutic potential in some heart disorders, including ischaemic injury. In this study, we demonstrate that bFGF administration can inhibit the endoplasmic reticulum (ER) stress and mitochondrial dysfunction induced in the heart in a mouse model of I/R injury. In vitro, bFGF exerts a protective effect by inhibiting the ER stress response and mitochondrial dysfunction proteins that are induced by tert‐Butyl hydroperoxide (TBHP) treatment. Both of these in vivo and in vitro effects are related to the activation of two downstream signalling pathways, PI3K/Akt and ERK1/2. Inhibition of these PI3K/Akt and ERK1/2 pathways by specific inhibitors, LY294002 and PD98059, partially reduces the protective effect of bFGF. Taken together, our results indicate that the cardioprotective role of bFGF involves the suppression of ER stress and mitochondrial dysfunction in ischaemic oxidative damage models and oxidative stress‐induced H9C2 cell injury; furthermore, these effects underlie the activation of the PI3K/Akt and ERK1/2 signalling pathways.     

Preconditioning the hyperlipidemic myocardium: fact or fantasy?

Ischemic heart disease is a leading cause of death worldwide. Myocardial ischemia results in reduced coronary flow, followed by diminished oxygen and nutrient supply to the heart. Reperfusion to an ischemic myocardium often augments the ischemic damage, known as ischemia-reperfusion (I/R) injury. Number of studies demonstrated that the hyperlipidemic myocardium is rather sensitive and more vulnerable to I/R-induced myocardial injury. Repeated brief ischemia and reperfusion cycles, termed as ischemic preconditioning, given before a sustained ischemia is known to reduce myocardial damage occur as a result of I/R. A plethora of evidence supports the fact that preconditioning is one of the promising interventional strategies having an ability to limit I/R-induced myocardial injury. Despite this fact, the preconditioning-mediated cardioprotection is blunted in chronic hyperlipidemic condition. This suggests that preconditioning is moderately a ‘healthy heart protective phenomenon’. The mechanisms by which chronic hyperlipidemia abrogates cardioprotective effects of preconditioning are uncertain and are not completely understood. The impaired opening of mitochondrial-K_ATP channels, eNOS uncoupling and excessive generation of superoxides in the hyperlipidemic myocardium could play a role in attenuating preconditioning-mediated myocardial protection against I/R injury. Moreover, hyperlipidemia-induced loss of cardioprotective effect of preconditioning is associated with redistribution of both sarcolemmal and mitochondrial Connexin 43. We addressed, in this review, the potential mechanisms involved in hyperlipidemia-induced impairment of myocardial preconditioning. Additionally, novel pharmacologic interventions to attenuate hyperlipidemia-associated exaggerated I/R-induced myocardial injury have been discussed.     

Quercetin attenuates renal ischemia/reperfusion injury via an activation of AMP-activated protein kinase-regulated autophagy pathway

Renal ischemia/reperfusion (I/R) is a major cause of acute renal failure. Quercetin, a flavonoid antioxidant, presents in many kinds of food. The molecular mechanism of quercetin on renal protection during I/R is still unclear. Here, we investigated the role of AMP-activated protein kinase (AMPK)-regulated autophagy in renal protection by quercetin. To investigate whether quercetin protects renal cells from I/R-induced cell injury, an in vitro model of I/R and an in vivo I/R model were used. Cell apoptosis was determined by propidium iodide/annexin V staining. Western blotting and immunofluorescence were used to determine the autophagy. AMPK expression was inhibited with appropriate short hairpin RNA (shRNA). In cultured renal tubular cell I/R model, quercetin decreased the cell injury, up-regulated the AMPK phosphorylation, down-regulated the mammalian target of rapamycin (mTOR) phosphorylation and activated autophagy during I/R. Knockdown of AMPK by shRNA transfection decreased the quercetin-induced autophagy but did not affect the mTOR phosphorylation. In I/R mouse model, quercetin decreased the increased serum creatinine level and altered renal histological score. Quercetin also increased AMPK phosphorylation, inhibited the mTOR phosphorylation and activated autophagy in the kidneys of I/R mice. These results suggest that quercetin activates an AMPK-regulated autophagy signaling pathway, which offers a protective effect in renal I/R injury.     

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α₂-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α₂-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α₂-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α₂-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α₂-adrenergic receptor stimulation.     

TLR4 inhibits mesenchymal stem cell (MSC) STAT3 activation and thereby exerts deleterious effects on MSC-mediated cardioprotection

Background

Bone marrow-derived mesenchymal stem cells (MSC) improve myocardial recovery after ischemia/reperfusion (I/R) injury. These effects are mediated in part by the paracrine secretion of angiogenic and tissue growth-promoting factors. Toll-like receptor 4 (TLR4) is expressed by MSC and induces apoptosis and inhibits proliferation in neuronal progenitors as well as many other cell types. It is unknown whether knock-out (KO) of TLR4 will change the paracrine properties of MSC and in turn improve MSC-associated myocardial protection.

Methodology/Principal Findings

This study explored the effect of MSC TLR4 on the secretion of angiogenic factors and chemokines in vitro by using ELISA and cytokine array assays and investigated the role of TLR4 on MSC-mediated myocardial recovery after I/R injury in an isolated rat heart model. We observed that MSC isolated from TLR4 KO mice exhibited a greater degree of cardioprotection in a rat model of myocardial I/R injury. This enhanced protection was associated with increased angiogenic factor production, proliferation and differentiation. TLR4-dificiency was also associated with decreased phosphorylation of PI-3K and AKT, but increased activation of STAT3. siRNA targeting of STAT3 resulted in attenuation of the enhanced cardioprotection of TLR4-deficient MSC.

Conclusions/Significance

This study indicates that TLR4 exerts deleterious effects on MSC-derived cardioprotection following I/R by a STAT3 inhibitory mechanism.     

ATPase inhibitory factor 1 protects the heart from acute myocardial ischemia/reperfusion injury through activating AMPK signaling pathway

Rationale: Myocardial ischemia/reperfusion (I/R) injury is a common clinic scenario that occurs in the context of reperfusion therapy for acute myocardial infarction (AMI). The mitochondrial F1Fo-ATPase inhibitory factor 1 (IF1) blocks the reversal of the F1Fo-ATP synthase to prevent detrimental consumption of cellular ATP and associated demise. In the present study, we study the role and mechanism of IF1 in myocardial I/R injury.Methods: Mice were ligated the left anterior descending coronary artery to build the I/R model in vivo. Rat hearts were isolated and perfused with constant pressure according to Langendorff. Also, neonatal cardiomyocytes hypoxia-reoxygenation (H/R) model was also used. Myocardial infarction area, cardiac function, cellular function, and cell viability was conducted and compared.Results: Our data revealed that IF1 is upregulated in hearts after I/R and cardiomyocytes with hypoxia/re-oxygenation (H/R). IF1 delivered with adenovirus and adeno-associated virus serotype 9 (AAV9) ameliorated cardiac dysfunction and pathological development induced by I/R ex vivo and in vivo. Mechanistically, IF1 stimulates glucose uptake and glycolysis activity and stimulates AMPK activation during in vivo basal and I/R and in vitro OGD/R conditions, and activation of AMPK by IF1 is responsible for its cardioprotective effects against H/R-induced injury.Conclusions: These results suggest that increased IF1 in the I/R heart confer cardioprotective effects via activating AMPK signaling. Therefore, IF1 can be used as a potential therapeutic target for the treatment of pathological ischemic injury and heart failure.     

Impaired autophagy contributes to hepatocellular damage during ischemia/reperfusion: Heme oxygenase-1 as a possible regulator

Liver ischemia and reperfusion (I/R) injury is characterized by oxidative stress that is accompanied by alterations of the endogenous defensive system. Emerging evidence suggests a protective role for autophagy induced by multiple stressors including reactive oxygen species. Meanwhile, heme oxygenase-1 (HO-1) has long been implicated in cytoprotection against oxidative stress in vitro and in vivo. Therefore, we investigated the impact of autophagy in the pathogenesis of liver I/R and its molecular mechanisms, particularly its linkage to HO-1. By using transmission electron microscopic analysis and biochemical autophagic flux assays with microtubule-associated protein 1 light chain 3-II, and beclin-1, representative autophagy markers, and p62, a selective substrate for autophagy, we found that reperfusion reduced autophagy both in the rat liver and in primary cultured hepatocytes. When autophagy was further inhibited with chloroquine or wortmannin, I/R-induced hepatocellular injury was aggravated. While livers that underwent I/R showed increased levels of mammalian target of rapamaycin and calpain 1 and 2, inhibition of calpain 1 and 2 induced an autophagic response in hepatocytes subjected to hypoxia/reoxygenation. HO-1 increased autophagy, and HO-1 reduced I/R-induced calcium overload in hepatocytes and prevented calpain 2 activation both in vivo and in vitro. Taken together, these findings suggest that the impaired autophagy during liver I/R, which is mediated by calcium overload and calpain activation, contributes to hepatocellular damage and the HO-1 system protects the liver from I/R injury through enhancing autophagy.     

Protective Role of AMP-Activated Protein Kinase-Evoked Autophagy on an In Vitro Model of Ischemia/Reperfusion-Induced Renal Tubular Cell Injury

Ischemia/reperfusion (I/R) injury is a common cause of injury to target organs such as brain, heart, and kidneys. Renal injury from I/R, which may occur in renal transplantation, surgery, trauma, or sepsis, is known to be an important cause of acute kidney injury. The detailed molecular mechanism of renal I/R injury is still not fully clear. Here, we investigate the role of AMP-activated protein kinase (AMPK)-evoked autophagy in the renal proximal tubular cell death in an in vitro I/R injury model. To mimic in vivo renal I/R injury, LLC-PK1 cells, a renal tubular cell line derived from pig kidney, were treated with antimycin A and 2-deoxyglucose to mimic ischemia injury followed by reperfusion with growth medium. This I/R injury model markedly induced apoptosis and autophagy in LLC-PK1 cells in a time-dependent manner. Autophagy inhibitor 3-methyladenine (3MA) significantly enhanced I/R injury-induced apoptosis. I/R could also up-regulate the phosphorylation of AMPK and down-regulate the phosphorylation of mammalian target of rapamycin (mTOR). Cells transfected with small hairpin RNA (shRNA) for AMPK significantly increased the phosphorylation of mTOR as well as decreased the induction of autophagy followed by enhancing cell apoptosis during I/R. Moreover, the mTOR inhibitor RAD001 significantly enhanced autophagy and attenuated cell apoptosis during I/R. Taken together, these findings suggest that autophagy induction protects renal tubular cell injury via an AMPK-regulated mTOR pathway in an in vitro I/R injury model. AMPK-evoked autophagy may be as a potential target for therapeutic intervention in I/R renal injury.     

七氟烷对糖尿病心肌缺血/再灌注损伤的影响

糖尿病是一种常见病、多发病,严重威胁着人类的健康。现已明确,糖尿病是冠心病发病的一个重要因素。心肌缺血/再灌注(ischemia/reperfusion,I/R)损伤是临床常见的病理过程,同时是冠心病发病及心肌血运重建治疗过程中的核心环节,如何减轻I/R损伤一直是国际研究热点之一。糖尿病与I/R损伤对心肌都有损害作用,相关研究证明糖尿病能够进一步恶化I/R损伤对心肌的损伤作用。研究表明,缺血预处理(ischemia preconditioning,IPC)可以延缓或减轻心肌I/R损伤,同时,麻醉药预处理(anesthetic induced preconditioning,APC)也具有IPC样的心肌保护作用。其中,七氟烷作为现阶段临床较常用的吸入麻醉药,同样对心肌I/R损伤具有保护作用。本文就七氟烷对糖尿病心肌I/R损伤的影响及其机制做一综述。     

Protective roles of cornuside in acute myocardial ischemia and reperfusion injury in rats

Cornuside is a secoiridoid glucoside isolated from the fruit of Cornus officinalis SIEB. et ZUCC. In this study, we investigated the anti-myocardial ischemia and reperfusion (I/R) injury effects of cornuside in vivo and elucidated the potential mechanism. Rat models of myocardial I/R were induced by coronary occlusion followed by reperfusion or by Isoproterenol (ISO), treatment of rats with cornuside (20 and 40 mg/kg, i.v.) protected the animals from myocardial I/R injury as indicated by a decrease in infarct volume, improvement in hemodynamics and reduction of myocardial damage severity. Treatment with cornuside also attenuated polymorphonuclear leukocytes (PMNs) infiltration, decreased myeloperoxidase (MPO) activity in the heart, lowered serum levels of pro-inflammatory factors and reduced phosphorylated IκB-α and nuclear factor kappa B (NF-κB) levels in the heart. Additionally, cornuside was shown to have remarkable antioxidant activity and inhibited ISO-induced myocardial cell necrosis. Thus, cornuside appeared to protect the rat from myocardial I/R injury by acting as an anti-inflammatory agent. These findings suggested that cornuside may be used therapeutically in the setting of myocardial I/R where inflammation and oxidant injury are prominent.     

Ghrelin protects heart against ERS-induced injury and apoptosis by activating AMP-activated protein kinase

Ghrelin, the endogenous ligand of growth hormone secretagogue receptor (GHS-R), is a cardioprotective peptide. In our previous work, we have revealed that ghrelin could protect heart against ischemia/reperfusion (I/R) injury by inhibiting endoplasmic reticulum stress (ERS), which contributes to many heart diseases. In current study, using both in vivo and in vitro models, we investigated how ghrelin inhibits myocardial ERS. In the in vivo rat heart injury model induced by isoproterenol (ISO), we found that exogenous ghrelin could alleviate heart dysfunction, reduce myocardial injury and apoptosis and inhibit the excessive myocardial ERS induced by ISO. More importantly, the activation of AMP-activated protein kinase (AMPK) was observed. To explore the role of AMPK activation in ERS inhibition by ghrelin, we set up two in vitro ERS models by exposing cultured rat cardiomyocytes to tunicamycin(Tm) or dithiothreitol (DTT). In both models, compared with Tm or DTT treatment alone, pre-incubation cardiomyocytes with ghrelin significantly activated AMPK, reversed the upregulation of the ERS markers, C/EBP-homologous protein (CHOP) and cleaved caspase-12, and reduced apoptosis of cardiomyocytes. Further, we found that the ERS inhibitory and anti-apoptotic actions induced by ghrelin were blocked by an AMPK inhibitor. To investigate how ghrelin activates AMPK, selective antagonist of GHS-R1a and inhibitor of Ca²⁺/Calmodulin-dependent protein kinase kinase (CaMKK) were added, respectively, before ghrelin pre-incubation, and we found that AMPK activation was prevented and the ERS inhibitory and anti-apoptotic actions of ghrelin were blocked. In conclusion, ghrelin could protect heart against ERS-induced injury and apoptosis, at least partially through a GHS-R1a/CaMKK/AMPK pathway.     

Both ischemic preconditioning and ghrelin administration protect hippocampus from ischemia/reperfusion and upregulate uncoupling protein-2

Background  

A major endogenous protective mechanism in many organs against ischemia/reperfusion (I/R) injury is ischemic preconditioning (IPC). By moderately uncoupling the mitochondrial respiratory chain and decreasing production of reactive oxygen species (ROS), IPC reduces apoptosis induced by I/R by reducing cytochrome c release from the mitochondria. One element believed to contribute to reduce ROS production is the uncoupling protein UCP2 (and UCP3 in the heart). Although its implication in IPC in the brain has been shown in vitro, no in vivo study of protein has shown its upregulation. Our first goal was to determine in rat hippocampus whether UCP2 protein upregulation was associated with IPC-induced protection and increased ROS production. The second goal was to determine whether the peptide ghrelin, which possesses anti-oxidant and protective properties, alters UCP2 mRNA levels in the same way as IPC during protection.     

Protection against myocardial ischemia/reperfusion injury in TLR4-deficient mice is mediated through a phosphoinositide 3-kinase-dependent mechanism

TLRs play a critical role in the induction of innate and adaptive immunity. However, TLRs have also been reported to mediate the pathophysiology of organ damage following ischemia/reperfusion (I/R) injury. We have reported that TLR4(-/-) mice show decreased myocardial injury following I/R; however, the protective mechanisms have not been elucidated. We examined the role of the PI3K/Akt signaling pathway in TLR4(-/-) cardioprotection following I/R injury. TLR4(-/-) and age-matched wild-type (WT) mice were subjected to myocardial ischemia for 45 min, followed by reperfusion for 4 h. Pharmacologic inhibitors of PI3K (wortmannin or LY294002) were administered 1 h before myocardial I/R. Myocardial infarct size/area at risk was reduced by 51.2% in TLR4(-/-) vs WT mice. Cardiac myocyte apoptosis was also increased in WT vs TLR4(-/-) mice following I/R. Pharmacologic blockade of PI3K abrogated myocardial protection in TLR4(-/-) mice following I/R. Specifically, heart infarct size/area at risk was increased by 98% in wortmannin and 101% in LY294002-treated TLR4(-/-) mice, when compared with control TLR4(-/-) mice. These data indicate that protection against myocardial I/R injury in TLR4(-/-) mice is mediated through a PI3K/Akt-dependent mechanism. The mechanisms by which PI3K/Akt are increased in the TLR4(-/-) myocardium may involve increased phosphorylation/inactivation of myocardial phosphatase and tensin homolog deleted on chromosome 10 as well as increased phosphorylation/inactivation of myocardial glycogen synthase kinase-3beta. These data implicate innate immune signaling pathways in the pathology of acute myocardial I/R injury. These data also suggest that modulation of TLR4/PI3K/Akt-dependent signaling pathways may be a viable strategy for reducing myocardial I/R injury.     

Lycopene Protects against Hypoxia/Reoxygenation Injury by Alleviating ER Stress Induced Apoptosis in Neonatal Mouse Cardiomyocytes

Endoplasmic reticulum (ER) stress induced apoptosis plays a pivotal role in myocardial ischemia/reperfusion (I/R)-injury. Inhibiting ER stress is a major therapeutic target/strategy in treating cardiovascular diseases. Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear. In the present study, using neonatal mouse cardiomyocytes to establish an in vitro model of hypoxia/reoxygenation (H/R) to mimic myocardium I/R in vivo, we aimed to explore the hypothesis that lycopene could alleviate the ER stress and ER stress-induced apoptosis in H/R-injury. We observed that lycopene alleviated the H/R injury as revealed by improving cell viability and reducing apoptosis, suppressed reactive oxygen species (ROS) generation and improved the phosphorylated AMPK expression, attenuated ER stress as evidenced by decreasing the expression of GRP78, ATF6 mRNA, sXbp-1 mRNA, eIF2α mRNA and eIF2α phosphorylation, alleviated ER stress-induced apoptosis as manifested by reducing CHOP/GADD153 expression, the ratio of Bax/Bcl-2, caspase-12 and caspase-3 activity in H/R-treated cardiomyocytes. Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes. Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes. These results suggest that the protective effects of lycopene on H/R-injury are, at least in part, through alleviating ER stress and ER stress-induced apoptosis in neonatal mouse cardiomyocytes.     

Ischemic Postconditioning-Mediated miRNA-21 Protects against Cardiac ischemia/reperfusion Injury via PTEN/Akt Pathway

Background

Ischemic postconditioning (IPost) protects the reperfused heart from infarction which has drawn much attention recently. However, studies to date have rarely investigated the role of microRNAs (miRNAs) in IPost. The aims of this study were to investigate whether miR-21 is involved in the protective effect of IPost against myocardial ischemia-reperfusion (I/R) injury and disclose the potential molecular mechanisms involved.

Methods and Results

We found that miR-21 was remarkably up-regulated in mouse hearts after IPost. To determine the protective role of IPost-induced miR-21 up-regulation, the mice were divided into the following four groups: I/R group; I/R+IPost group (I/R mice treated with IPost); Antagomir-21+IPost+I/R group (I/R mice treated with anagomir-21 and IPost); Scramble+IPost+I/R group (I/R mice treated with scramble and IPost). The results showed IPost could reduce I/R injury-induced infarct size of the left ventricle, improve cardiac function, and prevent myocardial apoptosis, while knockdown of miR-21 with antagomir-21 could reverse these protective effects of IPost against mouse I/R injury. Furthermore, we confirmed that miR-21 plays a protective role in myocardial apoptosis through PTEN/Akt signaling pathway, which was abrogated by the PI3K inhibitor LY294002. The protective effect of miR-21 on myocardial apoptosis was further revealed in mouse hearts after IPost treatment in vivo.

Conclusions

Our data clearly demonstrate that miR-21 is involved in IPost-mediated cardiac protection against I/R injury and dysfunction through the PTEN/Akt signaling pathway in vivo. Identifying the beneficial roles of IPost-regulated miRNAs in cardiac protection, which may be a rational target selection for ischemic cardioprotection.     

The Interplay between the Renin Angiotensin System and Pacing Postconditioning Induced Cardiac Protection

BackgroundAccumulating evidence suggests a cardioprotective role of pacing postconditioning (PPC) maneuvers in animal models and more recently in humans. The procedure however remains to be optimized and its interaction with physiological systems remains to be further explored. The renin angiotensin system (RAS) plays a dual role in ischemia/reperfusion (I/R) injury. The interaction between RAS and PPC induced cardiac protection is however not clearly understood. We have recently demonstrated that angiotensin (1–7) via Mas receptor played a significant role in PPC mediated cardiac protection against I/R injury.ObjectiveThe objective of this study was to investigate the role of angiotensin converting enzyme (ACE)—chymase—angiotensin II (Ang II)—angiotensin receptor 1 (AT1) axes of RAS in PPC mediated cardiac protection.MethodsIsolated rat hearts were subjected to I/R (control) or PPC in the presence or absence of Ang II, chymostatin (inhibitor of locally produced Ang II), ACE blocker (captopril) or AT1 antagonist (irbesartan). Hemodynamics data was computed digitally and infarct size was determined histologically using TTC staining and biochemically by measuring creatine kinase (CK) and lactate dehydrogenase levels.ResultsCardiac hemodynamics were significantly (P<0.001) improved and infarct size and cardiac enzymes were significantly (P<0.001) reduced in hearts subjected to PPC relative to hearts subjected to I/R injury. Exogenous administration of Ang II did not affect I/R injury or PPC mediated protection. Nonetheless inhibition of endogenously synthesized Ang II protected against I/R induced cardiac damage yet did not block or augment the protective effects of PPC. The administration of AT1 antagonist did not alleviate I/R induced damage. Interestingly it abrogated PPC induced cardiac protection in isolated rat hearts. Finally, PPC induced protection and blockade of locally produced Ang II involved enhanced activation of ERK1/2 and Akt components of the reperfusion injury salvage kinase (RISK) pathway.ConclusionsThis study demonstrate a novel role of endogenously produced Ang II in mediating I/R injury and highlights the significance of AT1 signaling in PPC mediated cardiac protection in isolated rodents hearts ex vivo. The interaction between Ang II-AT1 and PPC appears to involve alterations in the activation state of ERK1/2 and Akt components of the RISK pathway.     

HDAC Inhibition Elicits Myocardial Protective Effect through Modulation of MKK3/Akt-1

We and others have demonstrated that HDAC inhibition protects the heart against myocardial injury. It is known that Akt-1 and MAP kinase play an essential role in modulation of myocardial protection and cardiac preconditioning. Our recent observations have shown that Akt-1 was activated in post-myocardial infarction following HDAC inhibition. However, it remains unknown whether MKK3 and Akt-1 are involved in HDAC inhibition-induced myocardial protection in acute myocardial ischemia and reperfusion injury. We sought to investigate whether the genetic disruption of Akt-1 and MKK3 eliminate cardioprotection elicited by HDAC inhibition and whether Akt-1 is associated with MKK3 to ultimately achieve protective effects. Adult wild type and MKK3^−/−, Akt-1^−/− mice received intraperitoneal injections of trichostatin A (0.1mg/kg), a potent inhibitor of HDACs. The hearts were subjected to 30 min myocardial ischemia/30 min reperfusion in the Langendorff perfused heart after twenty four hours to elicit pharmacologic preconditioning. Left ventricular function was measured, and infarct size was determined. Acetylation and phosphorylation of MKK3 were detected and disruption of Akt-1 abolished both acetylation and phosphorylation of MKK3. HDAC inhibition produces an improvement in left ventricular functional recovery, but these effects were abrogated by disruption of either Akt-1 or MKK3. Disruption of Akt-1 or MKK3 abolished the effects of HDAC inhibition-induced reduction of infarct size. Trichostatin A treatment resulted in an increase in MKK3 phosphorylation or acetylation in myocardium. Taken together, these results indicate that stimulation of the MKK3 and Akt-1 pathway is a novel approach to HDAC inhibition -induced cardioprotection.