Effect of Endoplasmic Reticulum Stress on Resveratrol-Induced Cardioprotection

To investigate if resveratrol prevent the mitochondrial permeability transition pore (mPTP) opening through inhibition of endoplasmic reticulum stress (ERS). Methods: Rat heart tissue-derived cardiac H9c2 myoblast cell line was cultured. Fluorescence images of mitochondrial membrane potential were obtained with confocal microscopy. Western blotting analyzes the ERS marker protein GRP78 expression. Transmission electron microscopy detects the subcellular structure. Results: Exposure of cardiac H9c2 cells to 100 μM 2-DG, the ERS inducer, for 20 min caused a marked decrease in mitochondrial specific tetramethylrhodamine ethyl ester (TMRE) fluorescence. Resveratrol significantly prevented the loss of TMRE fluorescence. Western blotting revealed that resveratrol decreased GRP78 expression. Experiments with transmission electron microscopy revealed that resveratrol prevented 2-DG-induced swelling of endoplasmic reticulum and mitochondrial damages. Conclusions: These data suggest that inhibition of ERS leads to the prevention of mPTP opening. Resveratrol prevents the mPTP opening through inhibition of ERS.

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Roles of Endoplasmic Reticulum Stress in NECA-Induced Cardioprotection against Ischemia/Reperfusion Injury

Oxidative Medicine and Cellular Longevity ◽

10.1155/2017/2490501 ◽

2017 ◽

Vol 2017 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Fengmei Xing ◽

Hui Han ◽

Yonggui He ◽

Yidong Zhang ◽

Liwei Jing ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Mitochondrial Permeability Transition ◽

Ischemia Reperfusion Injury ◽

Glycogen Synthase ◽

Ischemia Reperfusion ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Mptp Opening ◽

Adenosine Receptor Agonist

Objective. This study aimed to investigate whether the nonselective A2 adenosine receptor agonist NECA induces cardioprotection against myocardial ischemia/reperfusion (I/R) injury via glycogen synthase kinase 3β (GSK-3β) and the mitochondrial permeability transition pore (mPTP) through inhibition of endoplasmic reticulum stress (ERS). Methods and Results. H9c2 cells were exposed to H2O2 for 20 minutes. NECA significantly prevented H2O2-induced TMRE fluorescence reduction, indicating that NECA inhibited the mPTP opening. NECA blocked H2O2-induced GSK-3β phosphorylation and GRP94 expression. NECA increased GSK-3β phosphorylation and decreased GRP94 expression, which were prevented by both ERS inductor 2-DG and PKG inhibitor KT5823, suggesting that NECA may induce cardioprotection through GSK-3β and cGMP/PKG via ERS. In isolated rat hearts, both NECA and the ERS inhibitor TUDCA decreased myocardial infarction, increased GSK-3β phosphorylation, and reversed GRP94 expression at reperfusion, suggesting that NECA protected the heart by inhibiting GSK-3β and ERS. Transmission electron microscopy showed that NECA and TUDCA reduced mitochondrial swelling and endoplasmic reticulum expansion, further supporting that NECA protected the heart by preventing the mPTP opening and ERS. Conclusion. These data suggest that NECA prevents the mPTP opening through inactivation of GSK-3β via ERS inhibition. The cGMP/PKG signaling pathway is responsible for GSK-3β inactivation by NECA.

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Abstract 14997: Zn2+ and mPTP Mediate ERS Inhibition-induced Cardioprotection Against Ischemia/Reperfusion Injury

Circulation ◽

10.1161/circ.130.suppl_2.14997 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Jinkun Xi ◽

Yonggui He ◽

Shuo Fei ◽

Huan Zheng ◽

Zhelong Xu

Keyword(s):

Endoplasmic Reticulum ◽

Reperfusion Injury ◽

Mitochondrial Permeability Transition ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Mptp Opening ◽

Role Of Zn

While the role of endoplasmic reticulum stress (ERS) in myocardial ischemia/reperfusion (I/R) injury has been extensively investigated, the precise mechanism by which inhibition of ERS induces cardioprotection remains unclear. We aimed to explore the mechanism of ERS inhibition-induced cardioprotection against I/R injury, focusing on the role of Zn 2+ and the mitochondrial permeability transition pore (mPTP). Exposure of H9c2 cells to 800 μM H 2 O 2 for 20 min increased GRP78 and GRP94 expressions (296.2 ± 41.0 and 150.6 ± 13.5 %, respectively), suggesting that H 2 O 2 can induce ERS. Cells treated with H 2 O 2 showed a significant decrease (40.6 ± 7.4 %) in TMRE fluorescence compared to the normal group (92.6 ± 0.1 %), indicating that H 2 O 2 can induce the mPTP opening. In contrast, ERS inhibitor TUDCA (30 μM) prevented the loss of TMRE fluorescence (77.8 ± 6.8 %), implying that inhibition of ERS can prevent the mPTP opening. This effect of TUDCA was blocked by zinc chelator TPEN (37.7 ± 13.0 %), indicating a role of Zn 2+ in the action of TUDCA on the mPTP opening. In support, TUDCA increased intracellular free zinc, as indicated by a marked increase in Newport Green DCF fluorescence intensity. In isolated rat hearts, GRP78 expression was not increased during ischemia but was increased upon reperfusion (1.3, 1.5, 1.9, and 1.6-fold increases at 10, 30, 60, and 120 min of reperfusion). Hearts treated with TUDCA showed a significant reduction of GRP78 expression 30 and 60 min after the onset of reperfusion, an effect that was reversed by TPEN. The immunofluorescence study also showed that the effect of TUDCA on GRP78 expression was reversed by TPEN. TUDCA reduced infarct size, and this was reversed by the mPTP opener atractyloside, indicating that ERS inhibition may protect the heart by modulating the mPTP opening. Experiments with transmission electron microscopy and hematoxylin-eosin staining revealed that TUDCA prevented endoplasmic reticulum and mitochondrial damages at reperfusion, which was blocked by TPEN. In conclusion, reperfusion but not ischemia initiates ERS and inhibition of ERS protects the heart from reperfusion injury through prevention of the mPTP opening. Increased intracellular free Zn 2+ accounts for the cardioprotective effect of ERS inhibition.

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Mitochondrial depolarization underlies delay in permeability transition by preconditioning with isoflurane: roles of ROS and Ca2+

AJP Cell Physiology ◽

10.1152/ajpcell.00006.2010 ◽

2010 ◽

Vol 299 (2) ◽

pp. C506-C515 ◽

Cited By ~ 45

Author(s):

Filip Sedlic ◽

Ana Sepac ◽

Danijel Pravdic ◽

Amadou K. S. Camara ◽

Martin Bienengraeber ◽

...

Keyword(s):

Cell Survival ◽

Mitochondrial Permeability Transition ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Underlying Mechanism ◽

Ros Production ◽

Trypan Blue Exclusion ◽

Mptp Opening ◽

Mitochondrial Ros ◽

Cardioprotective Effects

During reperfusion, the interplay between excess reactive oxygen species (ROS) production, mitochondrial Ca2+ overload, and mitochondrial permeability transition pore (mPTP) opening, as the crucial mechanism of cardiomyocyte injury, remains intriguing. Here, we investigated whether an induction of a partial decrease in mitochondrial membrane potential (ΔΨm) is an underlying mechanism of protection by anesthetic-induced preconditioning (APC) with isoflurane, specifically addressing the interplay between ROS, Ca2+, and mPTP opening. The magnitude of APC-induced decrease in ΔΨm was mimicked with the protonophore 2,4-dinitrophenol (DNP), and the addition of pyruvate was used to reverse APC- and DNP-induced decrease in ΔΨm. In cardiomyocytes, ΔΨm, ROS, mPTP opening, and cytosolic and mitochondrial Ca2+ were measured using confocal microscope, and cardiomyocyte survival was assessed by Trypan blue exclusion. In isolated cardiac mitochondria, antimycin A-induced ROS production and Ca2+ uptake were determined spectrofluorometrically. In cells exposed to oxidative stress, APC and DNP increased cell survival, delayed mPTP opening, and attenuated ROS production, which was reversed by mitochondrial repolarization with pyruvate. In isolated mitochondria, depolarization by APC and DNP attenuated ROS production, but not Ca2+ uptake. However, in stressed cardiomyocytes, a similar decrease in ΔΨm attenuated both cytosolic and mitochondrial Ca2+ accumulation. In conclusion, a partial decrease in ΔΨm underlies cardioprotective effects of APC by attenuating excess ROS production, resulting in a delay in mPTP opening and an increase in cell survival. Such decrease in ΔΨm primarily attenuates mitochondrial ROS production, with consequential decrease in mitochondrial Ca2+ uptake.

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Abstract 168: Resveratrol Translocates Gsk-3β To Mitochondria And Protects The Heart At Reperfusion By Targeting The Mitochondrial Permeability Transition Pore

Circulation ◽

10.1161/circ.116.suppl_16.ii_11-b ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Jinkun Xi ◽

Huihua Wang ◽

Guillaume Chanoit ◽

Guang Cheng ◽

Robert A Mueller ◽

...

Keyword(s):

Reperfusion Injury ◽

Mitochondrial Permeability Transition Pore ◽

Mitochondrial Permeability Transition ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Cyclophilin D ◽

Risk Zone ◽

Mitochondrial Permeability ◽

Mptp Opening ◽

Gsk 3Β

Although resveratrol has been demonstrated to be cardioprotective, the detailed cellular and molecular mechanisms that mediate the protection remain elusive. We aimed to determine if resveratrol protects the heart at reperfusion by modulating the mitochondrial permeability transition pore (mPTP) opening through glycogen synthase kinase 3β (GSK-3β). Resveratrol (10μM) given at reperfusion reduced infarct size (12.2 ± 2.5 % of risk zone vs. 37.9 ± 3.1 % of risk zone in control, n = 6) in isolated rat hearts subjected to 30 min regional ischemia followed by 2 h of reperfusion, an effect that was abrogated by the mPTP opener atractyloside (30.9 ± 8.1 % of risk zone), implying that resveratrol may protect the heart at reperfusion by modulating the mPTP opening. To define the signaling mechanism underlying the action of resveratrol, we determined GSK-3β activity by measuring its phosphorylation at Ser 9 . Resveratrol significantly enhanced GSK-3β phosphorylation upon reperfusion (225.2 ± 30.0 % of control at 5 min of reperfusion). Further experiments showed that resveratrol induces translocation of GSK-3β to mitochondria and translocated GSK-3β interacts with the mPTP component cyclophilin D but not VDAC (the voltage-dependent anion channel) or ANT (the adenine nucleotide translocator) in cardiac mitochondria. Taken together, these data suggest that resveratrol prevents myocardial reperfusion injury by targeting the mPTP opening via GSK-3β. Translocation of GSK-3β to mitochondria and its interaction with the mPTP component cyclophilin D may serve as an essential mechanism that mediates the protective effect of resveratrol on reperfusion injury.

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Pollen, Tapetum, and Orbicule Development inColletia paradoxaandDiscaria americana(Rhamnaceae)

The Scientific World JOURNAL ◽

10.1100/2012/948469 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 9

Author(s):

M. Gotelli ◽

B. Galati ◽

D. Medan

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Endoplasmic Reticulum ◽

Pollen Grains ◽

Pollen Grain ◽

Ultrastructural Changes ◽

Transmission Electron ◽

The Family ◽

Tapetal Cells ◽

Grain Formation

Tapetum, orbicule, and pollen grain ontogeny inColletia paradoxaandDiscaria americanawere studied with transmission electron microscopy (TEM). The ultrastructural changes observed during the different stages of development in the tapetal cells and related to orbicule and pollen grain formation are described. The proorbicules have the appearance of lipid globule, and their formation is related to the endoplasmic reticulum of rough type (ERr). This is the first report on the presence of orbicules in the family Rhamnaceae. Pollen grains are shed at the bicellular stage.

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Enigmatic origin of the poxvirus membrane from the endoplasmic reticulum shown by 3D imaging of vaccinia virus assembly mutants

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1716255114 ◽

2017 ◽

Vol 114 (51) ◽

pp. E11001-E11009 ◽

Cited By ~ 10

Author(s):

Andrea S. Weisberg ◽

Liliana Maruri-Avidal ◽

Himani Bisht ◽

Bryan T. Hansen ◽

Cindi L. Schwartz ◽

...

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Endoplasmic Reticulum ◽

Vaccinia Virus ◽

Virus Assembly ◽

Transmembrane Protein ◽

Membrane Dynamics ◽

Deletion Mutants ◽

Viral Membrane ◽

Transmission Electron

The long-standing inability to visualize connections between poxvirus membranes and cellular organelles has led to uncertainty regarding the origin of the viral membrane. Indeed, there has been speculation that viral membranes form de novo in cytoplasmic factories. Another possibility, that the connections are too short-lived to be captured by microscopy during a normal infection, motivated us to identify and characterize virus mutants that are arrested in assembly. Five conserved vaccinia virus proteins, referred to as Viral Membrane Assembly Proteins (VMAPs), that are necessary for formation of immature virions were found. Transmission electron microscopy studies of two VMAP deletion mutants had suggested retention of connections between viral membranes and the endoplasmic reticulum (ER). We now analyzed cells infected with each of the five VMAP deletion mutants by electron tomography, which is necessary to validate membrane continuity, in addition to conventional transmission electron microscopy. In all cases, connections between the ER and viral membranes were demonstrated by 3D reconstructions, supporting a role for the VMAPs in creating and/or stabilizing membrane scissions. Furthermore, coexpression of the viral reticulon-like transmembrane protein A17 and the capsid-like scaffold protein D13 was sufficient to form similar ER-associated viral structures in the absence of other major virion proteins. Determination of the mechanism of ER disruption during a normal VACV infection and the likely participation of both viral and cell proteins in this process may provide important insights into membrane dynamics.

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Protectors of the mitochondrial permeability transition pore activated by iron and doxorubicin

Current Cancer Drug Targets ◽

10.2174/1568009621999210120192558 ◽

2021 ◽

Vol 21 ◽

Author(s):

Tatiana A. Fedotcheva ◽

Nadezhda I. Fedotcheva

Keyword(s):

Mitochondrial Permeability Transition Pore ◽

Mitochondrial Permeability Transition ◽

Membrane Damage ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Selective Electrode ◽

Mitochondrial Permeability ◽

Isolated Mitochondria ◽

Mptp Opening ◽

Dependent Cell

Aim: The study of action of iron, DOX, and their complex on the mitochondrial permeability transition pore (MPTP) opening and the detection of possible protectors of MPTP in the conditions close to mitochondria-dependent ferroptosis. Background: The toxicity of doxorubicin (DOX) is mainly associated with the free iron accumulation and mitochondrial dysfunction. DOX can provoke ferroptosis, iron-dependent cell death driven by the membrane damage. The mitochondrial permeability transition pore (MPTP) is considered as a common pathway leading to the development of apoptosis, necrosis, and, possibly, ferroptosis. The influence of DOX on the Ca2+ -induced opening of MPTP in the presence of iron has not yet been studied. Objective: The study was conducted on isolated liver and heart mitochondria. MPTP and succinate-ubiquinone oxidoreductase were studied as targets of DOX in mitochondria-dependent ferroptosis. Methods: The study was conducted on isolated mitochondria of the liver and heart. Changes of threshold calcium concentrations required for MPTP opening were measured by a Ca2+ selective electrode, mitochondrial membrane potential was registered by tetraphenylphosphonium (TPP+)-selective electrode, and mitochondrial swelling was recorded as a decrease in absorbance at 540 nm. The activity of succinate dehydrogenase (SDH) was determined by the reduction of the electron acceptor DCPIP. Conclusion: MPTP and the respiratory complex II are identified as the main targets of the iron-dependent action of DOX on the isolated mitochondria. All MPTP protectors tested abolished or weakened the effect of iron and a complex of iron with DOX on Ca2+ -induced MPTP opening, acting in different stages of MPTP activation. These data open new approaches to the modulation of the toxic influence of DOX on mitochondria with the aim to reduce their dysfunction

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Abstract 17188: Cryoem Analysis in Cardiac Isolated Mitochondria Reveals New Insights for CsA and mPTP Activation

Circulation ◽

10.1161/circ.138.suppl_1.17188 ◽

2018 ◽

Vol 138 (Suppl_1) ◽

Author(s):

Jasiel O Strubbe ◽

Jason Schrad ◽

James F Conway ◽

Kristin N Parent ◽

Jason N Bazil

Keyword(s):

Time Course ◽

Mitochondrial Permeability Transition ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Myocardial Necrosis ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Granule Formation ◽

Membrane Rupture ◽

Mptp Opening

Excessive Ca 2+ accumulation is the main source of cardiac tissue and cell death during myocardial ischemia-reperfusion injury (IR injury) and myocardial infarction. Calcium dysregulation and overload leads to mitochondrial dysfunction, excessive reactive oxygen species (ROS) production, catastrophic energy failure, and opening of the cyclosporine A-sensitive mitochondrial permeability transition pore (mPTP). Mitochondrial Ca 2+ accumulation also results in the formation of amorphous Ca 2+ -phosphate granules localized in the mitochondrial matrix. These amorphous electron-dense granules are main components of the mitochondrial Ca 2+ sequestration and buffering system by mechanisms not yet well understood. The two aims of the present study are to test the relationship of Ca 2+ -phosphate granule size and number in cardiac mitochondria 1) exposed to a bolus calcium sufficient to elicit permeabilization and 2) whether CsA-treated mitochondria alters granule formation and size. A time course series of CryoEM images was analyzed to follow the permeabilization process. CryoEM results showed that mitochondrial incubated for longer time-courses have increased number of small granules (40 - 110 nm), swelling, membrane rupture and induction of mPTP opening. Conversely, shorter incubation time resulted in less granules per mitochondrion yet of similar size (35 - 90 nm). CsA- treated mitochondria, on the other hand, showed bigger phosphate granules (120 - 160 nm), and both lower granules per mitochondria and mPTP opening susceptibility. These results suggest a novel mechanism for CsA in which Ca 2+ -phosphate granule sizes are enhanced while maintaining fewer per mitochondrion. This effect may explain why CsA-treated mitochondria have higher calcium tolerance, delayed Ca 2+ -dependent opening of the mPTP, and protects against reperfusion-induced myocardial necrosis.

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Abstract 321: Obesity Causes Enhanced Sensitivity Of The Mitochondrial Permeability Transition Pore

Circulation Research ◽

10.1161/res.115.suppl_1.321 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Judith Bernal-Ramírez ◽

Adriana Riojas-Hernández ◽

Flor E Morales-Marroquín ◽

Elvía M Domínguez-Barragán ◽

David Rodríguez-Mier ◽

...

Keyword(s):

Cell Death ◽

Cardiac Dysfunction ◽

Mitochondrial Permeability Transition Pore ◽

Mitochondrial Permeability Transition ◽

Permeability Transition Pore ◽

Permeability Transition ◽

H2o2 Production ◽

Mitochondrial Permeability ◽

Removal Capacity ◽

Mptp Opening

Several mechanisms have been implicated in heart failure (HF) development due to obesity, including altered Ca2+ homeostasis and mitochondrial increased reactive oxygen species (ROS). Besides their metabolic role, mitochondria are important cell death regulators, since their disruption induces apoptosis. The mitochondrial permeability transition pore (MPTP) formation is key in this process. Ca2+ and ROS are known inducers of MPTP, and mitochondria are the main ROS generators. However, it has not been demonstrated that MPTP formation is involved in cardiac cell death due to obesity. Therefore, the aim of this work was to determine whether Ca2+ alterations and/or MPTP opening underlie cardiac dysfunction. We used obese Zucker fa/fa rats (32 weeks old), displaying concentric hypertrophy and cardiac dysfunction. We measured: i) Systolic and diastolic Ca2+ signaling in isolated myocytes, in basal conditions and upon β-adrenergic stimulation (β-AS), and ii) in vitro mitochondrial function: respiration, ROS production and MPTP opening. We found that the main alteration in Ca2+ signaling in fa/fa myocytes was a decrease in SERCA Ca2+ removal capacity, since Ca2+ transient amplitude and spark frequency were unchanged. Furthermore, in fa/fa myocytes, β-AS response was preserved. On the other hand, fa/fa mitochondria respiration, in state 3 decreased, but was unchanged in state 4, when glutamate/malate were used as substrate, resulting in an small decrease in respiratory control. In addition, fa/fa mitochondria were more sensitive to MPTP opening, induced by Ca2+ and carboxyatractiloside (CAT). Moreover, fa/fa mitochondria showed increased H2O2 production, and in exposed thiol groups in the adenine nucleotide translocase, a regulatory MPTP component. Since Ca2+ signaling is relatively normal in fa/fa cells, it does not seem to be the main contributor to the cardiac contractile dysfunction. However, given that fa/fa mitochondria showed decrease respiratory performance, were more susceptible to MPTP opening, and showed enhanced H2O2 production. We conclude that fa/fa mitochondria were more vulnerable to enhanced oxidative stress, causing MPTP opening, which could be exacerbated by SERCA slower Ca2+ removal capacity, leading to myocyte apoptosis.

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