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.

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.

Abstract 13914: Ischemic Preconditioning Induces MG53 Secretion From the Heart Through H 2 O 2 /PKC-delta Signaling

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Dan Shan ◽  
Yan Zhang ◽  
Rui-ping Xiao
Keyword(s):  
Ischemic Preconditioning ◽  
Cell Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Embryonic Stem ◽  
Underlying Mechanism ◽  
Neonatal Rat ◽  
Control Group ◽  
Systemic Delivery

Introduction: Ischemic heart disease is the leading cause of morbidity and mortality worldwide. Ischemic preconditioning (IPC) is the most powerful intrinsic protection against cardiac ischemia/reperfusion (I/R) injury. Previous studies have shown that a multifunctional TRIM family protein, MG53 (or TRIM72), not only plays an essential role in IPC-mediated cardioprotection, but also as a myokine/cardiokine, can be secreted from the heart and skeletal muscle in response to metabolic stress in addition to its intracellular actions. Hypothesis: We hypothesized that IPC-mediated cardioprotection is causally related to MG53 secretion and figured out the underlying mechanism. Methods and Results: Using proteomic analysis in conjunction with genetic and pharmacological approaches, we examined MG53 secretion in response to IPC and explored the underlying mechanism using rodents in in vivo , isolated perfused hearts, and cultured neonatal rat ventricular cardiomyocytes. IPC profoundly increased perfusate MG53 levels in mouse hearts by 5.50 ± 0.32 and 4.26 ± 0.40 folds from baseline over 0-60 and 60-120 min of reperfusion, respectively. Mechanistically, IPC-induced MG53 secretion is dependent on H 2 O 2 -evoked, Src-mediated phosphorylation of PKC-δ-Y311. Functionally, systemic delivery of recombinant human MG53 proteins (rhMG53) to mimic elevated circulating MG53 not only restored IPC function in MG53-deficient mice, but also protected rodent hearts from I/R injury even in the absence of IPC. Treatment of rhMG53 overtly decreased the infarct size (IF/AAR) induced by I/R compared to the BSA-treated control group (11.9 ± 1.8% vs 27.3 ± 2.0%, P <0.01), and reduced the mortality from 44.7% to 5.3% in rats. Moreover, H 2 O 2 augmented MG53 secretion, and MG53 knockdown exacerbated H 2 O 2 -induced cell injury in human embryonic stem cell-derived cardiomyocytes. Conclusions: In conclusion, IPC and oxidative stress can trigger MG53 secretion from the heart via an H 2 O 2 -PKC-δ-dependent mechanism, and secreted MG53 acts as an essential factor conveying IPC-induced cardioprotection against ischemia/reperfusion injury. Recombinant MG53 proteins can be developed into a novel treatment for various diseases of human heart in which the endogenous MG53 is low.

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.

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.

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 Inhibition of GPR4 attenuates SH-SY5Y cell injury in cerebral ischemia/reperfusion via anti-apoptotic pathways

2021 ◽  
Author(s):  
Chunli Xing ◽  
Guizhen Yan ◽  
Qishuai Liu
Keyword(s):  
Cerebral Ischemia ◽  
Cell Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Ph Sensor ◽  
Cell Activity ◽  
Endothelial Permeability ◽  
Glucose Deprivation ◽  
Cerebral Ischemia Reperfusion ◽  
Ldh Release

Cerebral ischemia/reperfusion injury (CIRI) can lead to increased vascular endothelial permeability and blood-brain barrier damage in patients with stroke. G protein-coupled receptor 4 (GPR4) is a functional pH sensor that plays a key role in renal ischemia-reperfusion-induced apoptosis. However, whether GPR4 has a role in cerebral ischemia remains to be further studied. Our study found that after oxygen-glucose deprivation/reoxygenation (OGD/R) treatment, the levels of GPR4 and CHOP in SH-SY5Y cells were significantly increased, which was accompanied by a decrease in cell viability, and an increase in LDH release and apoptosis. After knockdown of GPR4 using shRNA, CHOP levels in SH-SY5Y cells were also decreased, which unexpectedly increased cell activity and decreased LDH release and apoptosis rate. Interestingly, CHOP overexpression reversed the effect of GPR4 knockdown, suggesting that OGD/R-induced CIRI may involve endoplasmic reticulum stress-related apoptosis. In conclusion, our study provided a basis for further research on the mechanism of CIRI.

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

2002 ◽  
Vol 282 (3) ◽  
pp. H935-H941 ◽  
Author(s):  
Richard S. Vander Heide
Keyword(s):  
Cell Injury ◽  
Ventricular Myocytes ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Left Ventricular ◽  
Foreign Protein ◽  
Large Animal ◽  
Human Form ◽  
Adult Rat ◽  
Simulated Ischemia

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

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.

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