Myocardial Endoplasmic Reticulum Stress Research Articles

Sensitivity and activation of endoplasmic reticulum stress response and apoptosis in the perinatal sheep heart.

We determined in vitro sensitivity to thapsigargin in sheep cardiomyocytes from four perinatal ages. In utero cardiac activation of endoplasmic reticulum (ER) stress and apoptotic pathways was determined at these same ages. Thapsigargin-induced phosphorylation of EIF2A decreased 72% between 135 and 143 dGA (P=0.0096) and remained low at 1 dPN (P=0.0080). Conversely, thapsigargin-induced caspase cleavage was highest around the time of birth: cleaved caspase 3 was highest at 1 dPN (3.8-fold vs. 135 dGA, P=0.0380; 7.8-fold vs. 5 dPN, P=0.0118), cleaved caspase 7 and cleaved caspase 12 both increased between 135 and 143 dGA (25-fold and 6.9-fold respectively, both P<0.0001), and remained elevated at 1 dPN. Induced apoptosis, measured by TUNEL assay, was highest around the time of birth (P<0.0001). There were changes in myocardial ER stress pathway components in utero. GRP78 protein levels were high in the fetus and declined after birth (P<0.0001). EIF2A phosphorylation was profoundly depressed at 1 dPN (vs. 143 dGA, P=0.0113). There is dynamic regulation of ER proteostasis, ER stress, and the apoptosis cascade in the perinatal heart. Apoptotic signaling is more readily activated in fetal cardiomyocytes near birth, leading to widespread caspase cleavage in the newborn heart. These pathways are important for regulation of normal maturation in the healthy perinatal heart.

CARD6: Warm Ischemic Time, Cold Static Storage and Impairment of Cardiac Endoplasmic Reticular Stress in Human Hearts Donated After Circulatory Death and Brain Death

Background: Endoplasmic reticulum (ER) plays an important role in maintaining normal myocardial function at the cellular level. Overexpression of ER stress-associated proteins during ischemia impact cardiac function. Initial result of heart transplantation using donation after circulatory death (DCD) heart with short warm ischemic time (WIT, less than 30 minutes) was encouraging. However, little is known about the impact of various WIT on donor heart and with subsequent cold storage as used in normothermic regional perfusion(NRP) on the DCD hearts. In this study we investigated expression of myocardial ER stress response proteins in the DCD hearts to evaluate the impact of warm ischemic time (WIT) and subsequent cold storage. We also studied expression of myocardial ER stress response proteins in the brain death donor (DBD) hearts as a control group. Methods: A total of 23 human hearts, DCD group, n=17, with 3 WIT sub-groups [WIT 20 (n=6), 40 (n=6), ≥60 (n=5) minutes] and DBD group, n=6, no warm ischemia (control: WIT:0) were studied. All the DCD hearts received del Nido cardioplegia and all DBD hearts received Celsior solution before they were placed at cold storage for 6 hours. Left ventricular biopsies were performed at hour 0 (T0), 2 (T2), 4 (T4) and 6 (T6). Myocardial ER stress response proteins ATF6 (Activating Transcription Factor 6) and GRP78 (78-kDa glucose-regulated protein) were measured through immunoblotting after normalizing to GAPDH loading control in all cardiac biopsies. Results: Representative western blots showing the protein expressions patterns among the DBD and three different groups of DCD hearts under increasing time points (T0:0hrs, T2:2hrs, T4:4hrs, T6:6hrs) of cold static storage conditions (Fig.1A). At baseline, the proteins expression found to be comparable between DBD, DCD:20 and 40 groups, while DCD:≥60 group remained significantly higher (Fig.1B,C). We noticed differential temporal increase of ER stress proteins after 2 hours of cold storage and became prominent at hour 6 in DCD groups when compared to DBD group; while the DCD:60 group always remained highly significant throughout the cold storage compared to all other groups (Fig.1D,E). Conclusions: Our study concluded that duration of warm ischemia and subsequent cold storage condition in DCD hearts were associated with elevated ER stress response in myocardium. The length of cold storage was also found to be linked with elevated ER stress in DCD hearts. The DCD hearts with more than 60 min warm ischemic has the highest ES stress response suggesting profound alteration in ER homeostasis which may promote cardiac damage. Such findings are important in guiding clinical decision making on selecting DCD donor hearts. On the other hand the DBD hearts with up to 6 hour cold storage exerted minimal ER stress, supporting current DBD heart transplant donor preservation practice.

The key mediators involved in myocardial endoplasmic reticulum stress induced by ischaemia reperfusion injury in rats

Objective: Endoplasmic reticulum stress (ERS) occurs throughout the pathological process of myocardial ischaemia reperfusion injury (MIRI); however, the key mediators involved in the biological processes of ERS or that can influence the outcomes of ischaemia reperfusion injury (IRI) are unclear. To identify the differentially expressed genes (DEGs) encoding promising mediators in rat hearts in the context of MIRI, gene profiles were mined through transcriptome analysis. Methods: By searching the Gene Expression Omnibus (GEO) database, a suitable GEO dataset was identified in the context of MIRI with 24 h reperfusion after 45 min ischaemia in rats. DEGs were extracted and analysed with R studio, and the properties of those DEGs were elucidated by utilizing protein‒protein-interaction (PPI), Cytoscape, GO and KEGG bioinformatics techniques. The potential hub genes in the first cluster of the DEG dataset were retrieved and verified by basal experiments. Results: By searching the target dataset GSE122020, 1647 mRNAs, including 790 upregulated and 857 downregulated genes, were found in the datasets with criteria of | fold change ≥ 1.2 and p value &lt;0.05. The upregulated and downregulated genes were primarily involved in the “positive regulation of cytokine production”, “regulation of inflammatory response” and “response to pheromone” biological processes. Most of them were mainly correlated with the MAPK, Ras and calcium signalling pathways. In particular, 15 DEGs were found to be involved in the ERS signalling pathways, including Bfar, Itpr1, Srpx, Hspa1b, Usp25, Jun, Casp12, Eif2ak2, Casp4, Trim25, Ifng, Atf3, Hspa1a, Ppp1r15a, and Creb3. Through analysis with the MCODE plugin in Cytoscape, six genes, Atf3, Ppp1r15a, Casp12, Eif2ak2, Ifng, and Hspa1a, were identified as hub genes by the K-means algorithm from Cluster 1. In the in vivo MIRI model, the six genes mentioned above exhibited significant mRNA overexpression in the left anterior descending coronary artery ligation (LAD) model in rats. Conclusions: Altogether, the present study identified six potential DEGs involved in ERS induced by MIRI, which could be therapeutic targets in dealing with IRI-related ERS.

The role of ferroptosis and endoplasmic reticulum stress in intermittent hypoxia-induced myocardial injury.

Obstructive sleep apnea (OSA) is related to increased risk of cardiovascular disease. Ferroptosis is a form of programmed cell death characterized by iron overload and plays critical roles in myocardial injury. This study aimed to investigate the role of ferroptosis in intermittent hypoxia (IH)-induced myocardial injury involving endoplasmic reticulum stress (ERS). AC16 human cardiomyocytes were exposed to IH or normoxia conditions. Mice were randomly grouped as follows: normal control (NC), IH, ferrostatin-1 + IH (FIH), and N-acetylcysteine + IH (AIH). The mRNA levels of GPX4, xCT, FTH1, and FACL4 in AC16 cells were detected by qRT-PCR. The protein levels of GPX4, xCT, NOX4, ATF4, CHOP, Bcl-2, and Bax in myocardial tissue were detected by Western blot analysis. The mRNA expression levels of GPX4 and xCT in AC16 cells were significantly lower in IH group than that of NC group. In IH mice, myocardial tissues were injured accompanied by increased level of ferroptosis and ERS. Inhibition of ferroptosis and treatment of N-acetylcysteine reduced ERS and myocardial injury in mice exposed to IH. In addition, compared to ferrostatin-1, N-acetylcysteine exerted a greatereffect in relieving IH-induced myocardial damage and ERS. Ferroptosis was involved in IH-related myocardial injury accompanied by the activation of ERS. Inhibition of ferroptosis and acetylcysteine treatment alleviated IH-related myocardial injury, which may be a potential target for therapeutic approaches to OSA-induced myocardial injury.

The potential role of LCZ696 in ameliorating diabetic cardiomyopathy by inhibiting the myocardial inflammation, ER stress, and apoptosis through the RAGE/NF‐κB and PERK/CHOP signaling Pathways

The development of diabetic cardiomyopathy (DCM) is multifactorial that leads to myocardium structural and functional disruption resulting excessive activity of the renin‐angiotensin system (RAS) and finally heart failure. LCZ696 newly established as chronic heart failure drug which combines a neprilysin inhibitor (sacubitril) and an angiotensin II receptor blocker (valsartan). Present study is designed to find the effect of LCZ696 on DCM in rats. Diabetes was induced by feeding rats with a high‐fat diet for 6 weeks following a single injection of STZ (30mg/kg). Diabetic rats were randomly divided in three group (n=8), LCZ696 and valsartan treatment was started two weeks after the DM induction and continued for 8 consecutive weeks. Blood pressure of individual rats was recorded using tail‐cuff instruments. At end of the treatment echocardiography was done. In serum glucose, insulin, creatine kinase‐MB and lactate dehydrogenase (LDH). In cardiac tissues, pro‐inflammatory cytokines and advanced glycation end‐products (AGEs) formation estimated. Western blot and RT‐PCR were used to determine the apoptotic genes, endoplasmic reticulum stress (ER) parameters. LCZ696 and valsartan ameliorated the DCM progression by inhibiting of AGEs, its receptor (RAGE) at mRNA, pro‐apoptotic gene markers (BAX and caspase‐3) associated with induction of the antiapoptotic gene (Bcl‐2) at mRNA and protein expressions, NF‐κB at the mRNA and protein levels associated with restoring the elevation of pro‐inflammatory cytokines such as TNF‐α, IL‐6, and IL‐1β at the activity level. Furthermore, LCZ696 and valsartan helps in restoring the induction of ER stress parameters (GRP78, PERK, eIF2a, ATF4, and CHOP) at the mRNA and protein levels. LCZ696 and valsartan improved diastolic dysfunction and attenuated histological abnormalities. LCZ696 and valsartan attenuated DCM by inhibiting the myocardial inflammation, ER stress, and apoptosis through the RAGE/NF‐κB and PERK/CHOP signaling cascades. Collectively, present results revealed LCZ696 has more strong protective effect against DCM than valsartan.

Dietary ω-3 fatty acids reduced atrial fibrillation vulnerability via attenuating myocardial endoplasmic reticulum stress and inflammation in a canine model of atrial fibrillation

BackgroundDietary consumption of ω-3 fatty acids is correlated with a reduced incidence of cardiovascular events. Here, we investigated the effect of dietary ω-3 fatty acids on atrial fibrillation (AF) vulnerability in a canine model of AF and explored the related mechanisms. MethodsTwenty four male beagle dogs (weight, 8-10 kg) were randomly divided into four groups: (a) sham-operated group (normal chow); (b) AF+FO [AF and normal chow supplemented with fish oil (FO): 0.6 g n-3 polyunsaturated fatty acids (ω-3 PUFA) /kg/day]; (c) AF group (normal chow); (d) sham-operated FO group (chow supplemented with FO: 0.6 g ω-3 PUFA/kg/day). AF was induced by rapid atrial pacing (RAP: 400 bpm for 4 weeks). Daily oral administration of FO was initiated 1 week before surgery and continued for 4 weeks post operation. ResultsAtrial electric remodeling was significantly attenuated and AF vulnerability were significantly reduced in AF+FO group compared to AF group. Endoplasmic reticulum (ER) stress-related protein expression levels of glucose-regulated protein78, C/EBP homologous protein, cleaved-Caspase12, and phosphorylation of protein kinase R-like ER kinase as well as inflammatory cytokines interleukin-1β, interleukin-6, tumor necrosis factor-α in left atrium (LA) were significantly downregulated in AF+FO group than in AF group (all p<0.05). In addition, Masson staining revealed lower extent of LA interstitial fibrosis in AF+FO group than in AF group (p<0.01). Myocardial apoptosis was also significantly reduced in AF+FO group than in AF group (p<0.05). ConclusionsDietary ω-3 fatty acids could significantly reduce RAP-induced AF vulnerability, possibly via attenuating myocardial ER stress, inflammation, and apoptosis in this canine model of AF.

Exogenous spermine attenuates myocardial fibrosis in diabetic cardiomyopathy by inhibiting endoplasmic reticulum stress and the canonical Wnt signaling pathway.

Myocardial fibrosis is one of the main pathological manifestations of diabetic cardiomyopathy (DCM). Spermine (SPM), a product of polyamine metabolism, plays an important role in many cardiac diseases including hypertrophy, ischemia, and infarction, but its role in diabetic myocardial fibrosis has not been clarified. This study aimed to investigate the role of polyamine metabolism, specifically SPM, in diabetic myocardial fibrosis and to explore the related mechanisms. We used intraperitoneal injection of streptozotocin (STZ, 60 mg/kg) in Wistar rats and high glucose (HG, 40 mM) stimulated cardiac fibroblasts (CFs) to established a type 1 diabetes (T1D) model in vivo and in vitro, which were pretreated with exogenous SPM (5 mg/kg per day and 5 μM). The results showed that hyperglycemia induced the expression of the key polyamine synthesis enzyme ornithine decarboxylase (ODC) decreased and the key catabolic enzyme spermidine/spermine N1 -acetyltransferase (SSAT) increased compared with those in the control group. The body weight, blood insulin level, and cardiac ejection function were decreased, while blood glucose, heart weight, the ratio of heart weight to body weight, myocardial interstitial collagen deposition, and endoplasmic reticulum stress (ERS)-related protein (glucose-regulated protein-78, glucose-regulated protein-94, activating transcription factor-4, and C/EBP homology protein) expression in the T1D group were all significantly increased. HG also caused an increased expression of Wnt3, β-catenin (in cytoplasm and nucleus), while Axin2 and phosphorylated β-catenin decreased. Exogenous SPM improved the above changes caused by polyamine metabolic disorders. In conclusion, polyamine metabolism disorder occurs in the myocardial tissue of diabetic rats, causing myocardial fibrosis and ERS. Exogenous SPM plays a myocardial protective role via inhibiting of ERS and the canonical Wnt/β-catenin signaling pathway.

Anti-inflammatory and Anti-endoplasmic reticulum stress Effects of catalpol Against myocardial ischemia-reperfusion injury in streptozotocin-induced diabetic rats.

The current study was designed to investigate the effects and the mechanism of catalpol on myocardial ischemia-reperfusion (MI/R) injury in a diabetic rat model. Male Sprague-Dawley rats were divided into DM + sham, DM +I/R, and DM +I/R + C groups and diabetes was induced using single injections of streptozotocin (STZ; 70 mg/kg; i.p). After confirming the induction of diabetes, rats were administered physiological saline and catalpol (10 mg/kg; i.p.) daily for 28 days. Subsequently, rats were subjected to left anterior descending (LAD) coronary artery occlusion for 30 min followed by reperfusion for 2 h. Haemodynamic parameters were recorded throughout surgery, and following sacrifice, hearts were isolated for biochemical, histopathological, and molecular analyses. Catalpol treatment significantly ameliorated MI/R injury by improving cardiac function, normalizing myocardial enzyme activities and markers of oxidative stress, and by maintaining myocardial architecture. Furthermore, expression levels of the inflammatory cytokines TNF-α and IL-6 were decreased in biochemical and immunohistochemical studies. Additionally, the cardioprotective effects of catalpol were partly related to reductions in myocardial endoplasmic reticulum stress (ERS). In conclusion, catalpol exerts cardioprotective effects in diabetic rats by attenuating inflammation and inhibiting ERS.

Effects of curcumin on protein expression of glucose regulated protein 78 and caspase-12 of myocardial endoplasmic reticulum stress related factors in type 2 diabetes rats

To study the effect of curcumin on the expression of glucose regulated protein 78 kD(GRP78) and cysteinyl aspartate specific proteinase-12(caspase-12) of myocardial endoplasmic reticulum stress related factors in type 2 diabetes rats. Type 2 diabetes rats model was established by high-fat drink feeding and one-time intraperitoneal injecting streptozotocin(35 mg/kg). After model rats were built, rats was randomly divided into diabetic model group and low dose of curcumin group(200 mg/kg), high dose of curcumin group(400 mg/kg) and captopril group(60 mg/kg) with 10 rats in each group. The rats in each group were ig administered with corresponding drugs once a day. Continuous administration for 12 w. The levels of fasting blood glucose(FBG) and lactate dehydrogenase(LDH), electrocardiogram and heart weight index(HWI) were measured respectively. The myocardial pathological changes were observed by HE staining. The levels of collagen fiber expression in myocardial tissue were performed by Masson staining. The protein expression levels of GRP78 and caspase-12 in myocardium of rats were observed by immunohistochemistry. The result showed that compared with control group, the levels of FBG and LDH of serum were increased obviously, HWI was increased, myocardial cells were hypertrophy, the collagen fibers of intercellular space of cell were increased, the protein expressions of GRP78 and caspase-12 of myocardium were increased in rats, myocardial cell apoptosis was increased in the model group(P&lt;0. 05). Compared with model group, FBG and LDH levels and HWI were reduced, the collagen fiber of intercellular space were decreased, the protein expression levels of GRP78 and caspase-12 were lowered in high dose of curcumin group(P&lt;0. 05). It indicates that Cur defends myocardium tissue in type 2 diabetes rats, which may be related to decreasing the level of blood glucose and the protein expressions of GRP78 and caspase-12, and blocking the ERS-initiated apoptotic.

Swimming exercise inhibits myocardial ER stress in the hearts of aged mice by enhancing cGMP‑PKG signaling.

The purpose of the present study was to explore aging-associated cardiac dysfunction and the possible mechanism by which swimming exercise modulates cardiac dysfunction in aged mice. Aged mice were divided into two groups: i) Aged mice; and ii) aged mice subjected to swimming exercises. Another cohort of 4-month-old male mice served as the control group. Cardiac structure and function in mice were analyzed using hematoxylin and eosin staining, and echocardiography. The levels of oxidative stress were determined by measuring the levels of superoxide dismutase, malondialdehyde and reactive oxygen species (ROS). Levels of the endoplasmic reticulum (ER) stress-related protein PKR-like ER kinase, glucose-regulated protein 78 and C/EBP homologous protein were determined to evaluate the level of ER stress. The aged group exhibited an abnormal cardiac structure and decreased cardiac function, both of which were ameliorated by swimming exercise. The hearts of the aged mice exhibited pronounced oxidative and ER stress, which were ameliorated by exercise, and was accompanied by the reactivation of myocardial cGMP and suppression of cGMP-specific phosphodiesterase type 5 (PDE5). The inhibition of PDE5 attenuated age-induced cardiac dysfunction, blocked ROS production and suppressed ER stress. An ER stress inducer abolished the beneficial effects of the swimming exercise on cardiac function and increased ROS production. The present study suggested that exercise restored cardiac function in mice with age-induced cardiac dysfunction by inhibiting oxidative stress and ER stress, and increasing cGMP-protein kinase G signaling.

Effect of open-heart operation involving cardiopulmonary bypass on myocardial endoplasmic reticulum stress

Objective To explore the myocardial endoplasmic reticulum stress (ERS) during the open heart surgery involving cardiopulmonary bypass (CPB), and whether the reduced ERS by beating-heart technique played an important role in the myocardial protection. Methods Forty patients with rheumatic heart disease undergoing MVR were randomly divided into 2 groups. The patients in the experimental group received operation on beating-heart, while under arrested heart in the control group. Right atrial myocardial samples were taken before and after CPB. The expressions levels of mRNA and protein of GRP78 and NF-κBp65 were detected using real-time PCR and Western blotting. Results After CPB, the expression levels of mRNA and protein of GRP78 were 2.31±0.44 and 0.096±0.024, those of NF-κBp65 were 2.89±0.93 and 0.129±0.034 respectively in the control group. In the experimental group, the expression levels of mRNA and protein of GRP78 were 1.56±0.29 and 0.061±0.021, and those of NF-κBp65 were 1.73±0.48 and 0.085±0.025. As compared with the baseline values, mRNA and protein levels of GRP78 and NF-κBp65 were increased after CPB. The increasing degrees of GRP78 and NF-κBp65 were significantly lower in the experimental group than in the control group (P=0.001, 0.002; P=0.001, 0.001). Conclusion ERS could be provocated during open-heart operation. The attenuated ERS, through NF-kappaB pathway, may play an important role in myocardial protection by on-pump beating-heart technique. Key words: Open-heart operation involving cardiopulmonary bypass; Beating heart surgery; Endoplasmic reticulum stress; Inflammatory reaction

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.

Intermedin1-53 Protects Against Myocardial Fibrosis by Inhibiting Endoplasmic Reticulum Stress and Inflammation Induced by Homocysteine in Apolipoprotein E-Deficient Mice.

Aim: Endoplasmic reticulum stress (ERS) and inflammation participate in cardiac fibrosis. Importantly, a novel paracrine/autocrine peptide intermedin1–53 (IMD1–53) in the heart inhibits myocardial fibrosis in rats. However, the mechanisms are yet to be fully elucidated.Methods: Myocardial fibrosis in apolipoprotein E-deficient (ApoE -/-) mice and neonatal rat cardiac fibroblasts (CFs) were induced using homocysteine (Hcy).Results: IMD1–53 inhibited myocardial fibrosis in vivo and in vitro. Picrosirius red staining showed that IMD1–53 reduced myocardial interstitial collagen deposition in ApoE-/- mice treated with Hcy and decreased the expression of myocardial collagen I and III, which was further verified in rat CFs. IMD1–53 attenuated myocardial hypertrophy, as shown by cardiomyocyte cross-sectional area, ratio of heart weight to body weight, and mRNA levels of atrial natriuretic peptide and brain natriuretic peptide. IMD1–53 inhibited the upregulation of ERS hallmarkers such as glucose-regulated protein 78 (GRP78), GRP94, activating transcription factor 6 (ATF6), ATF4, inositol-requiring enzyme 1α, spliced-X-box-binding protein-1, protein kinase receptor-like ER kinase, and eukaryotic translation initiation factor 2α in mouse myocardium and rat CFs treated with Hcy. In addition, IMD1–53 decreased the production of inflammatory factors such as tumor necrosis factor-α, monocyte chemotactic protein-1, interleukin-6 (IL-6), and IL-1β in the mouse myocardium and rat CFs treated with Hcy. Concurrently, IMD1–53 ameliorated the expression of nuclear factor-κB, transforming growth factor-β1, and c-Jun N-terminal kinase in the mouse myocardium and rat CFs treated with Hcy.Conclusions: IMD potentially protects against myocardial fibrosis induced by Hcy in ApoE-/- mice, possibly via attenuating myocardial ERS and inflammation.

0111 : Role of endoplasmic reticulum stress in the deleterious cardiovascular consequences of chronic intermittent hypoxia. Beneficial effects of high-intensity interval training

Chronic intermittent hypoxia (IH) is described as the major detrimental factor leading to cardiovascular morbi-mortality in obstructive sleep apnea (OSA) patients. Previous animal studies demonstrated that IH increases myocardial susceptibility to ischemia-reperfusion (IR). Among mechanisms involved in the pathophysiology of IR, modulation of endoplasmic reticulum (ER) stress seems to play a major role. Exercise is known to exert beneficial effects on the cardiovascular system and a growing body of evidence demonstrates a particular efficiency of high-intensity interval training (HIT). The aim of the present study was 1) to evaluate the effects of HIT on IH-induced increased susceptibility to IR; 2) to determine whether ER stress was involved. Wistar male rats were exposed to 21 days of IH (21-5% FiO2, 60s cycle, 8h/day) or normoxia (N). After one week of IH alone, rats were daily submitted to both IH and HIT (2*24min, 15 to 30m/min). Rat hearts were either submitted to an IR protocol ex-vivo (30min-global ischemia followed by 120 min-reperfusion, Langendorff technic) or rapidly frozen to evaluate ER stress by Western Blot. IH induced a significant increase in infarct size (35.4±3.2% vs 22.7±1.7% of ventricles, in IH and N respectively, p<0.05) that was corrected by HIT (28.8±3.9 vs 21.0±5.1% of ventricles, in IH and N respectively). This was accompanied by a myocardial ER stress IH-dependent, characterized by the increased expression of the ER stress sensor (i.e. GRP78) and the activation of the proapoptotic ER stress pathway (i.e. pPERK, ATF4 and CHOP). HIT conditioning prevented this IH-induced proapoptotic ER stress. These findings show that HIT prevented the IH-dependent increased in myocardial susceptibility to IR, probably through a down-regulation of proapoptotic ER stress pathway. HIT could represent a good preventive strategy to limit myocardial ischemia reperfusion-related damages in OSA patients.

Shengmai injection improved doxorubicin-induced cardiomyopathy by alleviating myocardial endoplasmic reticulum stress and caspase-12 dependent apoptosis.

Background. Apoptosis plays vital roles in the progression of doxorubicin-induced cardiomyopathy (DOX-CM). Endoplasmic reticulum stress (ER stress) could induce specific apoptosis by caspase-12 dependent pathway. Shengmai Injection (SMI), a famous Traditional Chinese Medicine, could alleviate the heart damage via inhibiting myocardial apoptosis. However, it is unknown whether SMI can alleviate ER stress and its specific apoptosis in the setting of DOX-CM. Objective. To explore the effects of SMI on heart function, myocardial ER stress, and apoptosis of DOX-CM rats. Methods. Rats with DOX-CM were treated by SMI. Heart function was assessed by echocardiography and brain natriuretic peptide. Myocardial apoptosis was detected by TUNEL assay. ER stress was assessed by detecting the expressions of GRP78 and caspase-12. Results. At the end of eight-week, compared to control, significant heart dysfunction happened in DOX group. The ratio of apoptotic cardiomyocytes and the expressions of GRP78 and caspase-12 increased significantly (P < 0.05). Compared to DOX group, the apoptotic ratio and the expressions of GRP78 and caspase-12 significantly decreased in DOX + SMI group (P < 0.05), accompanied with improved heart function. Conclusion. SMI could alleviate myocardial ER stress and caspase-12 dependent apoptosis, which subsequently helped to improve the heart function of rats with DOX-CM.

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.

Effects of acidic reperfusate medium on apoptosis of myocardial endopasmic reticulum stress

Objective To determine the effect of acidic reperfusion solution on apoptosis of myocardial endoplasmic reticulum stress (ERS).Methods Twenty-four SD rats were randomly divided into 3 groups (n =8 each).In control group (C),the isolated heart was only reperfused 180 min with pH 7.4 HEPES-KH solution.In ischemia/reperfusion (I/R) group,the isolated heart was reperfused 20 min and then subjected to ischemia for 60 min followed by 100 min reperfusion with pH 7.4 HEPES-KH solution.In experiment group (E),the isolated heart was reperfused 20 min with pH 7.4 HEPES-KH solution and then subjected to ischemia for 60 min,followed by reperfusion with pH6.8,pH 7.1 and pH 7.4 HEPES-KH solution for 5 min,5 min,and 90 min,respectively.Cardiac troponin I (cTnI) concentration,apoptosis of myocardial cells,the expression levels of calreticulin (CRT) mRNA,the expression levels of Caspase-12 and CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) were assayed.Results As compared with C group (1.3 ±0.4,3.7 ± 1.1,0.48 ±0.04,0.12 ±0.02,0.08 ±0.01),in I/R group and E group,the cTnI concentration (12.5 ±3.2,6.7 ± 1.7),apoptosis rate (27.9 ±5.3,18.0 ± 3.9),expressions of CRT mRNA (1.03 ± 0.06,0.79 ± 0.05),Caspase-12 (0.35 ± 0.05,0.24 ± 0.03) and CHOP (0.19 ±0.04,0.13 ±0.03) were increased significantly (P ＜0.01).All the indexes in E group were significantly reduced as compared with I/R group (P ＜0.01).Conclusion Acidic reperfusion may protect the heart against I/R injury and alleviate ERS-mediated apoptosis by inhibiting excessive ERS during I/R. Key words: Myocardial protection; Acidic reperfusion fluid; Endopasmic reticulum stress; Apoptosis ; Reperfusion injury

Mechanical load-dependent cardiac ER stress in vitro and in vivo: Effects of preload and afterload

Proteins are folded in the endoplasmic reticulum (ER). ER stress initially leads to compensatory upregulation of ER chaperones and later to apoptosis, but the contribution of biomechanical load vs. neurohumoral stress to myocardial ER stress is unknown. We show that the ER chaperones Grp78 and calreticulin (CRT) are upregulated by afterload, but not by preload in vitro and in vivo. Angiotensin II upregulated ER chaperones in unloaded muscle strips, but the angiotensin receptor-1 antagonist irbesartan did not significantly blunt the induction of ER chaperones by afterload. In monocrotaline-treated rats, Grp78 and CRT were upregulated in the afterloaded right ventricle, but not in the only neurohumorally stressed left ventricle. These findings suggest that afterload but not preload induces myocardial ER stress, largely independent of angiotensin II signaling.

Sulfur dioxide inhibits excessively activated endoplasmic reticulum stress in rats with myocardial injury

It has been demonstrated that excessively activated endoplasmic reticulum stress (ERS) is related to myocardial injury. The study was designed to explore the possible role of sulfur dioxide (SO(2)) in protecting excessively activated ERS in rats with isoproterenol (ISO)-induced myocardial injury. Wistar rats were randomly divided into control, ISO, and ISO + SO(2) groups. Cardiac catheterization-derived hemodynamic parameters and myocardial enzymes in plasma were measured. Microstructure changes in myocardial tissues were examined. Cardiomyocyte apoptosis was detected by TUNEL method. Myocardial SO(2) content and aspartate amino transferase (AAT) activity were detected. Meanwhile, protein and mRNA expressions of myocardial AAT1, AAT2, and ERS markers (GRP78, caspase-12, and CHOP) were evaluated. The results showed that cardiac function was decreased, myocardial microstructure was damaged, and myocardial enzyme levels and cardiomyocyte apoptosis were increased with a downregulated endogenous AAT/SO(2) pathway, and that ERS markers were upregulated at transcriptional and translational levels in ISO-treated rats. However, the administration of an SO(2) donor, resulting in an increased SO(2) content in myocardial tissues, improved cardiac function and myocardial structure, and ameliorated myocardial enzyme levels and cardiomyocyte apoptosis associated with a downregulation of excessively activated ERS. In conclusion, the endogenous AAT/SO(2) pathway was probably responsible for the inhibition of excessively activated ERS, which might be involved in the mechanism of ISO-induced myocardial injury.

Inhibition of Endoplasm Reticulum Stress by Anisodamine Protects Against Myocardial Injury After Cardiac Arrest and Resuscitation in Rats

Anisodamine is a multi-functional bio-alkaloid with vascular activity. Our previous studies have revealed that anisodamine protects the heart from ischemia/reperfusion (I/R) injury induced by cardiac arrest (CA) and resuscitation. This study aimed to explore whether the protective effect of anisodamine is mediated by inhibition of the endoplasmic reticulum stress (ERS) response, which has been demonstrated to implicate in various I/R injuries. After 5 min of CA induced by electric stimulation, Wistar rats were randomly selected to receive cardiopulmonary resuscitation (CPR, including chest compression and epinephrine infusion) with or without anisodamine injection (n = 50/group). Hearts were harvested 24 h after the return of spontaneous circulation (ROSC). Sham-operated animals served as non-ischemic controls (n = 10). The survival rate, cardiomyocyte apoptosis, and the protein expression of ERS markers were detected. Thirty-three of the 50 rats in the Ani + CA/R group were successfully resuscitated, whereas only 18 of the 50 rats in the CA/R group gained ROSC. Survival to 24 h was significantly improved in the anisodamine treatment group (Ani + CA/R, n = 22/50) compared to the group with standard CPR (CA/R, n = 8/50). Anisodamine markedly decreased the number of apoptotic cardiomyocytes, the protein expression of GRP78, CHOP, and the active form of Caspase3 compared to the CA/R group. Our data suggest that anisodamine protects against cellular damage in rat hearts after CA and resuscitation, at least in part, by inhibiting myocardial ERS.