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

2007 ◽  
Vol 292 (1) ◽  
pp. C178-C187 ◽  
Author(s):  
Voraratt Champattanachai ◽  
Richard B. Marchase ◽  
John C. Chatham
Keyword(s):  
Cell Viability ◽  
Cell Survival ◽  
Ventricular Myocytes ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Activated T Cells ◽  
Neonatal Cardiomyocytes ◽  
Hexosamine Biosynthesis ◽  
Glcnac Transferase

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.

scholarly journals Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein O-GlcNAc and increased mitochondrial Bcl-2

2008 ◽  
Vol 294 (6) ◽  
pp. C1509-C1520 ◽  
Author(s):  
Voraratt Champattanachai ◽  
Richard B. Marchase ◽  
John C. Chatham
Keyword(s):  
Protective Effect ◽  
Ventricular Myocytes ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Cellular Injury ◽  
Protective Effects ◽  
Neonatal Cardiomyocytes ◽  
Glcnac Transferase ◽  
Neonatal Rat Ventricular Myocytes

We have previously reported that glucosamine protected neonatal rat ventricular myocytes against ischemia-reperfusion (I/R) injury, and this was associated with an increase in protein O-linked- N-acetylglucosamine ( O-GlcNAc) levels. However, the protective effect of glucosamine could be mediated via pathways other that O-GlcNAc formation; thus the initial goal of the present study was to determine whether increasing O-GlcNAc transferase (OGT) expression, which catalyzes the formation of O-GlcNAc, had a protective effect similar to that of glucosamine. To better understand the potential mechanism underlying O-GlcNAc-mediated cytoprotection, we examined whether increased O-GlcNAc levels altered the expression and translocation of members of the Bcl-2 protein family. Both glucosamine (5 mM) and OGT overexpression increased basal and I/R-induced O-GlcNAc levels, significantly decreased cellular injury, and attenuated loss of cytochrome c. Both interventions also attenuated the loss of mitochondrial membrane potential induced by H2O2 and were also associated with an increase in mitochondrial Bcl-2 levels but had no effect on Bad or Bax levels. Compared with glucosamine and OGT overexpression, NButGT (100 μM), an inhibitor of O-GlcNAcase, was less protective against I/R and H2O2 and did not affect Bcl-2 expression, despite a 5- to 10-fold greater increase in overall O-GlcNAc levels. Decreased OGT expression resulted in lower basal O-GlcNAc levels, prevented the I/R-induced increase in O-GlcNAc and mitochondrial Bcl-2, and increased cellular injury. These results demonstrate that the protective effects of glucosamine are mediated via increased formation of O-GlcNAc and suggest that this is due, in part, to enhanced mitochondrial Bcl-2 translocation.

Glucosamine inhibits angiotensin II-induced cytoplasmic Ca2+ elevation in neonatal cardiomyocytes via protein-associated O-linked N-acetylglucosamine

2006 ◽  
Vol 290 (1) ◽  
pp. C57-C65 ◽  
Author(s):  
Tamas Nagy ◽  
Voraratt Champattanachai ◽  
Richard B. Marchase ◽  
John C. Chatham
Keyword(s):  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Ang Ii ◽  
Neonatal Cardiomyocytes ◽  
Hexosamine Biosynthesis ◽  
Intracellular Ca ◽  
Glcnac Transferase ◽  
Neonatal Rat Ventricular Myocytes ◽  
The Relationship ◽  
Hexosamine Biosynthesis Pathway

We previously reported that glucosamine and hyperglycemia attenuate the response of cardiomyocytes to inositol 1,4,5-trisphosphate-generating agonists such as ANG II. This appears to be related to an increase in flux through the hexosamine biosynthesis pathway (HBP) and decreased Ca2+ entry into the cells; however, a direct link between HBP and intracellular Ca2+ homeostasis has not been established. Therefore, using neonatal rat ventricular myocytes, we investigated the relationship between glucosamine treatment; the concentration of UDP- N-acetylglucosamine (UDP-GlcNAc), an end product of the HBP; and the level of protein O-linked N-acetylglucosamine ( O-GlcNAc) on ANG II-mediated changes in intracellular free Ca2+ concentration ([Ca2+]i). We found that glucosamine blocked ANG II-induced [Ca2+]i increase and that this phenomenon was associated with a significant increase in UDP-GlcNAc and O-GlcNAc levels. O-(2-acetamido-2-deoxy-d-glucopyranosylidene)-amino- N-phenylcarbamate, an inhibitor of O-GlcNAcase that increased O-GlcNAc levels without changing UDP-GlcNAc concentrations, mimicked the effect of glucosamine on the ANG II-induced increase in [Ca2+]i. An inhibitor of O-GlcNAc-transferase, alloxan, prevented the glucosamine-induced increase in O-GlcNAc but not the increase in UDP-GlcNAc; however, alloxan abrogated the inhibition of the ANG II-induced increase in [Ca2+]i. These data support the notion that changes in O-GlcNAc levels mediated via increased HBP flux may be involved in the regulation of [Ca2+]i homeostasis in the heart.

Abstract 123: Adenoviral RhoA Regulation of Dynamin-related Protein 1 and Cardiac Mitochondrial Fission

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Cameron S Brand ◽  
Valerie P Tan-Sah ◽  
Joan Heller Brown ◽  
Shigeki Miyamoto
Keyword(s):  
G Protein ◽  
Ventricular Myocytes ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Mitochondrial Fission ◽  
G Protein Coupled Receptors ◽  
Neonatal Rat ◽  
Related Protein ◽  
Rhoa Signaling ◽  
G Protein Coupled

G protein coupled receptors can signal downstream through various pathways, including activation of the small G protein RhoA. In cardiomyocytes, RhoA signaling is protective against ischemia/reperfusion injury. We have previously shown that this is mediated through downstream activation of Protein Kinase D (PKD), increased phosphorylation of cofilin, and diminished translocation of pro-apoptotic proteins to the mitochondria (Xiang et al, Sci. Signaling 2013). Mitophagy, a process that removes damaged mitochondria and limits mitochondrial death signaling, has also been suggested to be a cardioprotective response to oxidative stress. A step considered to be preliminary to clearance of damaged mitochondria via mitophagy is mitochondrial fission, and we hypothesized that RhoA signaling increases mitochondrial fission in cardiomyocytes. Constitutively active RhoA expressed in neonatal rat ventricular myocytes (NRVMs) was found to accumulate at the mitochondria. This was associated with an increase in small, fragmented mitochondria as observed by fluorescent confocal microscopy and electron microscopy, indicative of increased mitochondrial fission. The main protein involved in mitochondrial fission, dynamin-related protein 1 (Drp1), translocates from the cytosol to the mitochondria when activated. We used a tagged adenoviral Drp1 construct to determine whether expression of active RhoA changes Drp1 levels at the mitochondria. Mitochondrial Drp1 increased within 12 hours of adenoviral expression of active RhoA. Adenoviral RhoA expression also increased phosphorylation of Drp1 at serine-616 in NRVMs. In summary, we show that in cardiomyocytes, RhoA associates with mitochondria, can increase Drp1 phosphorylation and Drp1 mitochondrial localization, and can induce mitochondrial fission. The relationship between these mitochondrial signaling events and the protein kinases that are involved are currently under investigation. We suggest that G protein coupled receptors that stimulate RhoA can induce Drp1 accumulation and mitochondrial fission, which contributes to their cardioprotective effect.

Effect of increased expression of cytoskeletal protein vinculin on ischemia-reperfusion injury in ventricular myocytes

2003 ◽  
Vol 284 (3) ◽  
pp. H911-H918 ◽  
Author(s):  
Hongguang Wei ◽  
Thomas L'Ecuyer ◽  
Richard S. Vander Heide
Keyword(s):  
Cell Injury ◽  
Ventricular Myocytes ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Rational Approach ◽  
Cytoskeletal Protein ◽  
Chemical Inhibitors ◽  
Rat Myocytes ◽  
Ldh Release

The transition from reversible to irreversible ischemic injury (ischemia-reperfusion, I/R) occurs coincident with the loss of vinculin, a cytoskeletal protein involved in the attachment of the myofibrils to the sarcolemmal membrane. If the loss of vinculin were critical to the development of I/R, then increased levels of vinculin would be predicted to delay the onset of irreversible injury assuming that the protein is functional and localized to the proper subcellular site. The present study determined whether increased expression of vinculin, specifically in the cytoskeletal compartment, would provide protection from I/R injury. Neonatal rat myocytes were cultured and infected with a newly created replication-deficient adenovirus driving the expression of vinculin. I/R was induced with chemical inhibitors of glycolysis and mitochondrial respiration. Irreversible cell injury was assessed with lactate dehydrogenase (LDH) release. Virus-infected myocytes expressed significantly more vinculin in the cytoskeletal fraction and increased the expression of paxillin but sustained the same amount of injury in response to simulated I/R as control cells ( n = 4; P = not significant, paired t-test). Hypothermic I/R (ischemia at 25°C) resulted in a significant reduction in LDH release ( P ≤ 0.02; n = 4). Virus-mediated overexpression of vinculin does not appear to represent a rational approach to overcoming I/R injury.

scholarly journals Involvement of PKCε in Cardioprotection Induced by Adaptation to Chronic Continuous Hypoxia

2015 ◽  
pp. 191-201 ◽  
Author(s):  
K. HOLZEROVÁ ◽  
M. HLAVÁČKOVÁ ◽  
J. ŽURMANOVÁ ◽  
G. BORCHERT ◽  
J. NECKÁŘ ◽  
...  
Keyword(s):  
Cell Viability ◽  
Ventricular Myocytes ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Left Ventricular ◽  
Acute Ischemia ◽  
Mrna Levels ◽  
Inhibitory Peptide ◽  
Male Wistar Rats ◽  
Ldh Release

Continuous normobaric hypoxia (CNH) renders the heart more tolerant to acute ischemia/reperfusion injury. Protein kinase C (PKC) is an important component of the protective signaling pathway, but the contribution of individual PKC isoforms under different hypoxic conditions is poorly understood. The aim of this study was to analyze the expression of PKCε after the adaptation to CNH and to clarify its role in increased cardiac ischemic tolerance with the use of PKCε inhibitory peptide KP-1633. Adult male Wistar rats were exposed to CNH (10 % O2, 3 weeks) or kept under normoxic conditions. The protein level of PKCε and its phosphorylated form was analyzed by Western blot in homogenate, cytosolic and particulate fractions; the expression of PKCε mRNA was measured by RT-PCR. The effect of KP-1633 on cell viability and lactate dehydrogenase (LDH) release was analyzed after 25-min metabolic inhibition followed by 30-min re-energization in freshly isolated left ventricular myocytes. Adaptation to CNH increased myocardial PKCε at protein and mRNA levels. The application of KP-1633 blunted the hypoxia-induced salutary effects on cell viability and LDH release, while control peptide KP-1723 had no effect. This study indicates that PKCε is involved in the cardioprotective mechanism induced by CNH.

Abstract 318: Targeting of Tp53inp1 by Mir-221 to Reduce P62-mediated Autophagy is Cardioprotective in Ischemia / Reperfusion Injury

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Peipei Wang ◽  
Qiying Chen ◽  
Arthur M Richards
Keyword(s):  
Protective Effect ◽  
Cell Count ◽  
Cell Injury ◽  
Ventricular Myocytes ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Autophagosome Formation ◽  
Direct Targeting ◽  
Neonatal Rat Ventricular Myocytes

Purpose: Tumor protein 53-induced nuclear protein 1 (Tp53inp1) acts as a tumor suppressor by inducing cell death. Tp53inp1 mRNA is a predicted target of miR-221. Whether targeting Tp53inp1 plays a role in miR-221-mediated cardioprotection has not been investigated. We hypothesized that miRNA-221 directly targets Tp53inp1 to reduce ischemia/reperfusion (I/R)-induced autophagy. Method: Myoblast H9c2 cells underwent 16 hours 0.2% O 2 hypoxia followed by 2 hours re-oxygenation (H-R, simulating I/R). H9c2 were transfected with miRNA-221 mimic (25 nmol) and scrambled mimic control (miR-221 and MC). Cell count/viability, WST assay, cell injury-induced LDH release, and GFP-LC3 labeled autophagosome formation were measured. Cells were collected for RT-qPCR and western blot (WB) analyses. pCMV-Myc-Tp53inp1 and pcDNA3.1-Flag-p62 plasmids were cloned and transfected into H9c2 for recovery and immuno-precipitation (IP) studies. The effects of miRNA-221 inhibitor in H9c2 were also assessed. Results: miR-221 significantly reduced H-R injury as indicated by higher cell count/viability and WST activity, and reduced LDH (miR-221 vs. MC p<0.05). qPCR confirmed that (1) miRNA-221 expression was reduced in H-R; (2) RISC-loaded (IP pull-down Ago-2) miRNA-221 increased by ~80 fold and reduced by 95% following mimic and inhibitor transfection respectively; (3) Increased Tp53inp1 following H-R was reversed by miR-221. miR-221 inhibited H-R induced autophagosome formation (GFP-LC3). WB indicated (1) increase of LC3-I/II ratio and p62, indicators of reduced autophagy, and (2) decrease of Tp53inp1 by miR-221. IP pull-down Myc-Tp53inp1 indicated the formation of p62-Tp53inp1 complex. The protective effect of miR-221 was abolished by Tp53inp1 overexpression (pCMV-Myc-Tp53inp1 and miRNA-221 mimic co-transfection). The protective effect was corroborated in neonatal rat ventricular myocytes (NRVM). MiRNA-221 inhibitor induced reverse effects. Conclusion: The cardioprotection of miR-221 entails direct targeting of Tp53inp1 which reducing p62-Tp53inp1 complex formation and inhibiting H-R-induced autophagy.

Interleukin 35 protects cardiomyocytes following ischemia/reperfusion-induced apoptosis via activation of mitochondrial STAT3

2021 ◽  
Author(s):  
Fengyun Zhou ◽  
Ting Feng ◽  
Xiangqi Lu ◽  
Huicheng Wang ◽  
Yangping Chen ◽  
...  
Keyword(s):  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Protective Role ◽  
Cytochrome C Release ◽  
Neonatal Cardiomyocytes ◽  
Myocardial Ischemia Reperfusion Injury ◽  
Myocardial Ischemia Reperfusion ◽  
Underlying Mechanisms ◽  
Induced Apoptosis

Abstract Mitochondrial reactive oxygen species (mtROS)-induced apoptosis has been suggested to contribute to myocardial ischemia/reperfusion injury. Interleukin 35 (IL-35), a novel anti-inflammatory cytokine, has been shown to protect the myocardium and inhibit mtROS production. However, its effect on cardiomyocytes upon exposure to hypoxia/reoxygenation (H/R) damage has not yet been elucidated. The present study aimed to investigate the potential protective role and underlying mechanisms of IL-35 in H/R-induced mouse neonatal cardiomyocyte injury. Mouse neonatal cardiomyocytes were challenged to H/R in the presence of IL-35, and we found that IL-35 dose dependently promotes cell viability, diminishes mtROS, maintains mitochondrial membrane potential, and decreases the number of apoptotic cardiomyocytes. Meanwhile, IL-35 remarkably activates mitochondrial STAT3 (mitoSTAT3) signaling, inhibits cytochrome c release, and reduces apoptosis signaling. Furthermore, co-treatment of the cardiomyocytes with the STAT3 inhibitor AG490 abrogates the IL-35-induced cardioprotective effects. Our study identified the protective role of IL-35 in cardiomyocytes following H/R damage and revealed that IL-35 protects cardiomyocytes against mtROS-induced apoptosis through the mitoSTAT3 signaling pathway during H/R.

scholarly journals LncRNA LSINCT5/miR-222 regulates myocardial ischemia‑reperfusion injury through PI3K/AKT pathway

2021 ◽  
Author(s):  
Xueying Tong ◽  
Jiajuan Chen ◽  
Wei Liu ◽  
Hui Liang ◽  
Hezhong Zhu
Keyword(s):  
Myocardial Ischemia ◽  
Cell Viability ◽  
Reperfusion Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Akt Signaling ◽  
Akt Pathway ◽  
Akt Signaling Pathway ◽  
Myocardial Ischemia Reperfusion Injury ◽  
Myocardial Ischemia Reperfusion

AbstractCardiovascular diseases rank the top cause of morbidity and mortality worldwide and are usually associated with blood reperfusion after myocardial ischemia/reperfusion injury (MIRI), which often causes severe pathological damages and cardiomyocyte apoptosis. LSINCT5 expression in the plasma of MI patients (n = 53), healthy controls (n = 42) and hypoxia-reoxygenation (HR)-treated cardiomyocyte AC16 cells was examined using qRT-PCR. The effects of LSINCT5 on cell viability and apoptosis were detected by MTT and flow cytometry, respectively. The expression of apoptosis-related proteins Bcl2, Bax and caspase 3 were tested by Western blot. The interaction between LSINCT5 and miR-222 was predicted by bioinformatic analysis. Moreover, changes in viability and apoptosis of AC16 cells co-transfected with siLSINCT5 and miR-222 inhibitor after HR treatment were examined. At last, the expression of proteins in PI3K/AKT pathway, namely PTEN, PI3K and AKT, was examined to analyze the possible pathway participating in LSINCT5-mediated MI/RI. Our study showed that LSINCT5 expression was upregulated in the plasma of MI patients and HR-treated AC16 cells. LSINCT5 overexpression significantly decreased cell viability and apoptosis. Luciferase reporter gene assay and RNA pulldown assay showed that LSINCT5 was a molecular sponge of miR-222. MiR-222 silencing in AC16 cells simulated the phenotypes of MIRI patients and HR-treated cells, indicating that LSINCT5 functions via miR-222 to regulate proliferation and apoptosis of HR-treated AC16 cells. We also showed that proteins of PI3K/AKT signaling pathway were affected in HR-treated AC16 cells, and LSINTC5 knockdown rescued these effects. LncRNA LSINCT5 was upregulated during MI pathogenesis, and LSINCT5 regulated MIRI possibly via a potential LSINCT5/miR-222 axis and PI3K/AKT signaling pathway. Our findings may provide novel evidence for MIRI prevention.

Necrostatin-1 Analog DIMO Exerts Cardioprotective Effect against Ischemia Reperfusion Injury by Suppressing Necroptosis via Autophagic Pathway in Rats

Pharmacology ◽  
2021 ◽  
Vol 106 (3-4) ◽  
pp. 189-201
Author(s):  
Shigang Qiao ◽  
Wen-jie Zhao ◽  
Huan-qiu Li ◽  
Gui-zhen Ao ◽  
Jian-zhong An ◽  
...  
Keyword(s):  
Cell Death ◽  
Ischemia Reperfusion Injury ◽  
Myocardial Infarct ◽  
Ischemia Reperfusion ◽  
Glucose Deprivation ◽  
Neonatal Rat ◽  
Ligand Docking ◽  
Autophagic Flux ◽  
Myocardial Infarct Size ◽  
Rat Cardiomyocytes

Aim: It has been reported that necrostatin-1 (Nec-1) is a specific necroptosis inhibitor that could attenuate programmed cell death induced by myocardial ischemia/reperfusion (I/R) injury. This study aimed to observe the effect and mechanism of novel Nec-1 analog (Z)-5-(3,5-dimethoxybenzyl)-2-imine-1-methylimidazolin-4-1 (DIMO) on myocardial I/R injury. Methods: Male SD rats underwent I/R injury with or without different doses of DIMO (1, 2, or 4 mg/kg) treatment. Isolated neonatal rat cardiomyocytes were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) treatment with or without DIMO (0.1, 1, 10, or 100 μM). Myocardial infarction was measured by TTC staining. Cardiomyocyte injury was assessed by lactate dehydrogenase assay (LDH) and flow cytometry. Receptor-interacting protein 1 kinase (RIP1K) and autophagic markers were detected by co-immunoprecipitation and Western blotting analysis. Molecular docking of DIMO into the ATP binding site of RIP1K was performed using GLIDE. Results: DIMO at doses of 1 or 2 mg/kg improved myocardial infarct size. However, the DIMO 4 mg/kg dose was ineffective. DIMO at the dose of 0.1 μM decreased LDH leakage and the ratio of PI-positive cells followed by OGD/R treatment. I/R or OGD/R increased RIP1K expression and in its interaction with RIP3K, as well as impaired myocardial autophagic flux evidenced by an increase in LC3-II/I ratio, upregulated P62 and Beclin-1, and activated cathepsin B and L. In contrast, DIMO treatment reduced myocardial cell death and reversed the above mentioned changes in RIP1K and autophagic flux caused by I/R and OGD/R. DIMO binds to RIP1K and inhibits RIP1K expression in a homology modeling and ligand docking. Conclusion: DIMO exerts cardioprotection against I/R- or OGD/R-induced injury, and its mechanisms may be associated with the reduction in RIP1K activation and restoration impaired autophagic flux.

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