scholarly journals Sevoflurane postconditioning attenuates cardiomyocyte hypoxia/reoxygenation injury via restoring mitochondrial morphology

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2659 ◽  
Author(s):  
Jin Yu ◽  
Jianjiang Wu ◽  
Peng Xie ◽  
Yiliyaer Maimaitili ◽  
Jiang Wang ◽  
...  
Keyword(s):  
Cell Death ◽  
Membrane Potential ◽  
Cell Viability ◽  
Mitochondrial Membrane ◽  
Mitochondrial Fusion ◽  
Mitochondrial Morphology ◽  
Mptp Opening ◽  
Reoxygenation Injury ◽  
Sevoflurane Postconditioning ◽  
Hypoxia Reoxygenation

Background Anesthetic postconditioning is a cellular protective approach whereby exposure to a volatile anesthetic renders a tissue more resistant to subsequent ischemic/reperfusion event. Sevoflurane postconditioning (SPostC) has been shown to exert cardioprotection against ischemia/reperfusion injury, but the underlying mechanism is unclear. We hypothesized that SPostC protects cardiomyocytes against hypoxia/reoxygenation (H/R) injury by maintaining/restoring mitochondrial morphological integrity, a critical determinant of cell fate. Methods Primary cultures of neonatal rat cardiomyocytes (NCMs) were subjected to H/R injury (3 h of hypoxia followed by 3 h reoxygenation). Intervention with SPostC (2.4% sevoflurane) was administered for 15 min upon the onset of reoxygenation. Cell viability, Lactate dehydrogenase (LDH) level, cell death, mitochondrial morphology, mitochondrial membrane potential and mitochondrial permeability transition pore (mPTP) opening were assessed after intervention. Mitochondrial fusion and fission regulating proteins (Drp1, Fis1, Mfn1, Mfn2 and Opa1) were assessed by immunofluorescence staining and western blotting was performed to determine the level of protein expression. Results Cardiomyocyte H/R injury resulted in significant increases in LDH release and cell death that were concomitant with reduced cell viability and reduced mitochondrial interconnectivity (mean area/perimeter ratio) and mitochondrial elongation, and with reduced mitochondrial membrane potential and increased mPTP opening. All the above changes were significantly attenuated by SPostC. Furthermore, H/R resulted in significant reductions in mitochondrial fusion proteins Mfn1, Mfn2 and Opa1 and significant enhancement of fission proteins Drp1 and Fis1. SPostC significantly enhanced Mfn2 and Opa1 and reduced Drp1, without significant impact on Mfn1 and Fis1. Conclusions Sevoflurane postconditioning attenuates cardiomyocytes hypoxia/reoxygenation injury (HRI) by restoring mitochondrial fusion/fission balance and morphology.

scholarly journals Sevoflurane Postconditioning Attenuates Hypoxia/Reoxygenation Injury of Cardiomyocytes Under High Glucose by Regulating HIF-1α/MIF/AMPK Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Haiping Ma ◽  
Yongjie Li ◽  
Tianliang Hou ◽  
Jing Li ◽  
Long Yang ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Protective Effect ◽  
Cell Viability ◽  
Mrna Expression ◽  
High Glucose ◽  
Mitochondrial Membrane ◽  
Ampk Signaling ◽  
H9c2 Cardiomyocytes ◽  
Reoxygenation Injury ◽  
Hypoxia Reoxygenation

Subject: Cardiovascular disease, as a very common and serious coexisting disease in diabetic patients, and is one of the risk factors that seriously affect the prognosis and complications of surgical patients. Previous studies have shown that sevoflurane post-conditioning (SPostC) exerts a protective effect against myocardial ischemia/reperfusion injury by HIF-1α, but the protective effect is weakened or even disappeared under hyperglycemia. This study aims to explore whether regulating the HIF-1α/MIF/AMPK signaling pathway can restore the protective effect and reveal the mechanism of SPostC on cardiomyocyte hypoxia/reoxygenation injury under high glucose conditions.Methods: H9c2 cardiomyocytes were cultured in normal and high-concentration glucose medium to establish a hypoxia/reoxygenation (H/R) injury model of cardiomyocytes. SPostC was performed with 2.4% sevoflurane for 15 min before reoxygenation. Cell damage was determined by measuring cell viability, lactate dehydrogenase activity, and apoptosis; Testing cell energy metabolism by detecting reactive oxygen species (ROS) generation, ATP content and mitochondrial membrane potential; Analysis of the change of HIF-1α, MIF and AMPKα mRNA expression by RT-PCR. Western blotting was used to examine the expression of HIF-1α, MIF, AMPKα and p-AMPKα proteins. HIF-1α and MIF inhibitors and agonists were administered 40 min before hypoxia.Results: 1) SPostC exerts a protective effect by increasing cell viability, reducing LDH levels and cell apoptosis under low glucose (5 μM) after undergoing H/R injury; 2) High glucose concentration (35 μM) eliminated the cardioprotective effect of SPostC, which is manifested by a significantly decrease in the protein and mRNA expression level of the HIF-1α/MIF/AMPK signaling pathway, accompanied by decreased cell viability, increased LDH levels and apoptosis, increased ROS production, decreased ATP synthesis, and decreased mitochondrial membrane potential; 3. Under high glucose (35 μM), the expression levels of HIF-1α and MIF were up-regulated by using agonists, which can significantly increase the level of p-AMPKα protein, and the cardioprotective effect of SPostC was restored.Conclusion: The signal pathway of HIF-1α/MIF/AMPK of H9c2 cardiomyocytes may be the key point of SPostC against H/R injure. The cardioprotective of SPostC could be restored by upregulating the protein expression of HIF-1α and MIF under hyperglycemia.

scholarly journals Naringin Effectively Protects Cardiomyocytes Against Hypoxia/Reoxygenation Injury

2021 ◽  
Vol 37 (3) ◽  
Author(s):  
Vu Thi Thu ◽  
Ngo Thi Hai Yen
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Cell Group ◽  
H9c2 Cell ◽  
H9c2 Cells ◽  
H9c2 Cardiomyocytes ◽  
Reoxygenation Injury ◽  
Hypoxia Reoxygenation

This study was conducted to evaluate the protective effect of Naringin (NAR) on H9C2 cardiomyocytes in hypoxia/reoxygenation (HR) injury in vitro induced by the hypoxia chamber. Methods: H9C2 cells were grown under normal (control) and HR conditions. The viability, cardiolipin content and mitochondrial membrane potential of H9C2 cells in experimental groups were analyzed by using suitable kits. Results: The obtained results showed that the addition of Naringin (16÷160 µM) significantly increased the survival rate of H9C2 cells under HR conditions. In particular, NAR had the highest efficiency in preserving mitochondrial function at concentrations of 80 µM and 160 µM. In HR-exposed H9C2 cell group, the cardiolipin content and mitochondrial membrane potential values of H9C2 cells were decreased sharply with that of control (71,64±1,37% and 68,12±2,78%, p<0,05). Interestingly, mitochondrial cardiolipin contents were signigicantly increased in H9C2 cells post-hypoxic treated wtih NAR at dose of 80 µM 160 µM to 87,76±1,89% and 81,09±1,21%. Additionally, post-hypoxic supplementation of NAR at concentration of 80 µM and 160 µM effectively increased mitochondrial membrane potential values. Conclusion: The obtained results are preliminary data on the effects of NAR in protecting mitochondrial-targeted cardiomyocytes against HR injury.

Bax translocates from cytosol to mitochondria in cardiac cells during apoptosis: development of a GFP-Bax-stable H9c2 cell line for apoptosis analysis

2005 ◽  
Vol 289 (1) ◽  
pp. H477-H487 ◽  
Author(s):  
Qi Hou ◽  
Yi-Te Hsu
Keyword(s):  
Cell Death ◽  
Membrane Potential ◽  
Cell Line ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
H9c2 Cell ◽  
Serum Withdrawal ◽  
Induction Of Apoptosis ◽  
H9c2 Cell Line ◽  
Hypoxia Reoxygenation

The proapoptotic protein Bax plays an important role in cardiomyocytic cell death. Ablation of this protein has been shown to diminish cardiac damage in Bax-knockout mice during ischemia-reperfusion. Presently, studies of Bax-mediated cardiac cell death examined primarily the expression levels of Bax and its prosurvival factor Bcl-2 rather than the localization of this protein, which dictates its function. Using immunofluorescence labeling, we have shown that in neonatal rat cardiomyocytes and in H9c2 cardiomyoblasts, Bax translocates from cytosol to mitochondria upon the induction of apoptosis by hypoxia-reoxygenation-serum withdrawal and by the presence of the free-radical inducer menadione. Also, we found that Bax translocation to mitochondria was associated with the exposure of an NH2-terminal epitope, and that this translocation could be partially blocked by the prosurvival factors Bcl-2 and Bcl-XL. To visualize the translocation of Bax in living cells, we have developed an H9c2 cell line that stably expresses green fluorescent protein (GFP)-tagged Bax. This cell line has GFP-Bax localized primarily in the cytosol in the absence of apoptotic inducers. Upon induction of apoptosis by a number of stimuli, including menadione, staurosporine, sodium nitroprusside, and hypoxia-reoxygenation-serum withdrawal, we could observe the translocation of Bax from cytosol to mitochondria. This translocation was not affected by retinoic acid-induced differentiation of H9c2 cells. Additionally, this translocation was associated with loss of mitochondrial membrane potential, release of cytochrome c, and fragmentation of nuclei. Finally, using a tetramethylrhodamine-based dye, we have shown that a rapid screening process based on the loss of mitochondrial membrane potential could be developed to monitor GFP-Bax translocation to mitochondria. Overall, the GFP-Bax-stable H9c2 cell line that we have developed represents a unique tool for examining Bax-mediated apoptosis, and it could be of great importance in screening therapeutic compounds that could block Bax translocation to mitochondria to attenuate apoptosis.

scholarly journals Sevoflurane postconditioning alleviates hypoxia-reoxygenation injury of cardiomyocytes by promoting mitochondrial autophagy through the HIF-1/BNIP3 signaling pathway

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7165 ◽  
Author(s):  
Long Yang ◽  
Jianjiang Wu ◽  
Peng Xie ◽  
Jin Yu ◽  
Xin Li ◽  
...  
Keyword(s):  
Cell Viability ◽  
Signaling Pathway ◽  
Cell Damage ◽  
Hypoxia Inducible Factor ◽  
Protective Effects ◽  
Interacting Protein ◽  
Lactate Dehydrogenase Activity ◽  
Reoxygenation Injury ◽  
Sevoflurane Postconditioning ◽  
Hypoxia Reoxygenation

Background Sevoflurane postconditioning (SpostC) can alleviate hypoxia-reoxygenation injury of cardiomyocytes; however, the specific mechanism remains unclear. This study aimed to investigate whether SpostC promotes mitochondrial autophagy through the hypoxia-inducible factor-1 (HIF-1)/BCL2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) signaling pathway to attenuate hypoxia-reoxygenation injury in cardiomyocytes. Methods The H9C2 cardiomyocyte hypoxia/reoxygenation model was established and treated with 2.4% sevoflurane at the beginning of reoxygenation. Cell damage was determined by measuring cell viability, lactate dehydrogenase activity, and apoptosis. Mitochondrial ultrastructural and autophagosomes were observed by transmission electron microscope. Western blotting was used to examine the expression of HIF-1, BNIP3, and Beclin-1 proteins. The effects of BNIP3 on promoting autophagy were determined using interfering RNA technology to silence BNIP3. Results Hypoxia-reoxygenation injury led to accumulation of autophagosomes in cardiomyocytes, and cell viability was significantly reduced, which seriously damaged cells. Sevoflurane postconditioning could upregulate HIF-1α and BNIP3 protein expression, promote autophagosome clearance, and reduce cell damage. However, these protective effects were inhibited by 2-methoxyestradiol or sinBNIP3. Conclusion Sevoflurane postconditioning can alleviate hypoxia-reoxygenation injury in cardiomyocytes, and this effect may be achieved by promoting mitochondrial autophagy through the HIF-1/BNIP3 signaling pathway.

scholarly journals SESAMOL ANTAGONIZES ROTENONE-INDUCED CELL DEATH IN SH-SY5Y NEURONAL CELLS

2016 ◽  
Vol 8 (12) ◽  
pp. 72
Author(s):  
Rohini D. ◽  
Vijayalakshmi K.
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Death ◽  
Superoxide Dismutase ◽  
Membrane Potential ◽  
Glutathione Peroxidase ◽  
Cell Viability ◽  
Mitochondrial Membrane ◽  
Thiobarbituric Acid ◽  
Oxygen Species ◽  
Thiobarbituric Acid Reactive Substances

<p><strong>Objective: </strong>To investigate the neuroprotective effect of sesamol against rotenone-induced cell death in SH-SY5Y cells associated with Parkinsonism.</p><p><strong>Methods: </strong>SH-SY5Y cells were maintained in Dulbecco’s modified Eagle’s medium. After differentiation, the cells were incubated with rotenone (20 μM) and sesamol at different concentrations (10-100 μM). Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. The reactive oxygen species, mitochondrial membrane potential and nuclear morphology were determined by dichlorofluorescein diacetate, rhodamine 123 and 4’, 6-diamidino-2-phenylindole, respectively. Thiobarbituric acid reactive substances, activities of catalase, superoxide dismutase, and glutathione peroxidase and glutathione level were determined by standard assays.</p><p><strong>Results: </strong>Sesamol significantly increased the cell viability and decreased the rotenone-induced cell death in SH-SY5Y cells. Sesamol antagonized rotenone-induced reactive oxygen species generation, loss of mitochondrial membrane potential and nuclear damage. Sesamol also decreased thiobarbituric acid reactive substances level, increased the activities of catalase, superoxide dismutase, glutathione peroxidase and increased the level of glutathione in rotenone-induced cells.</p><p><strong>Conclusion: </strong>The results obtained strongly indicate the promising neuroprotective role of sesamol against rotenone-induced death in SH-SY5Y cells.</p>

scholarly journals Rat liver GTP-binding proteins mediate changes in mitochondrial membrane potential and organelle fusion

1999 ◽  
Vol 276 (3) ◽  
pp. C611-C620 ◽  
Author(s):  
Jorge Daniel Cortese
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Rat Liver ◽  
Mitochondrial Membrane ◽  
Binding Proteins ◽  
Mitochondrial Fusion ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Morphology ◽  
Gtp Binding Proteins ◽  
Gtp Binding

The variety of mitochondrial morphology in healthy and diseased cells can be explained by regulated mitochondrial fusion. Previously, a mitochondrial outer membrane fraction containing fusogenic, aluminum fluoride (AlF4)-sensitive GTP-binding proteins (mtg) was separated from rat liver (J. D. Cortese, Exp. Cell Res. 240: 122–133, 1998). Quantitative confocal microscopy now reveals that mtg transiently increases mitochondrial membrane potential (ΔΨ) when added to permeabilized rat hepatocytes (15%), rat fibroblasts (19%), and rabbit myocytes (10%). This large mtg-induced ΔΨ increment is blocked by fusogenic GTPase-specific modulators such as guanosine 5′- O-(3-thiotriphosphate), excess GTP (>100 μM), and AlF4, suggesting a linkage between ΔΨ and mitochondrial fusion. Accordingly, stereometric analysis shows that decreasing ΔΨ or ATP synthesis with respiratory inhibitors limits mtg- and AlF4-induced mitochondrial fusion. Also, a specific G protein inhibitor ( Bordetella pertussis toxin) hyperpolarizes mitochondria and leads to a loss of AlF4-dependent mitochondrial fusion. These results place mtg-induced ΔΨ changes upstream of AlF4-induced mitochondrial fusion, suggesting that GTPases exert ΔΨ-dependent control of the fusion process. Mammalian mitochondrial morphology thus can be modulated by cellular energetics.

The Protective Effect of Stichopus japonicus Fucoidan on PC12 Cells with Hypoxia/Reoxygenation Injury

2014 ◽  
Vol 556-562 ◽  
pp. 610-614
Author(s):  
Shu Liang Song ◽  
Wei Wang ◽  
Xiao Chen Wang ◽  
Hao Liang ◽  
Yun Shan Wang ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Protective Effect ◽  
Pc12 Cells ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Apoptotic Pathway ◽  
Apoptosis Pathway ◽  
Stichopus Japonicus ◽  
Reoxygenation Injury ◽  
Hypoxia Reoxygenation

In this study the effect of fucoidan SJP-3 extracted from Stichopus Japonicus was used to evaluate the protective effect on PC12 cells with hypoxia/reoxygenation injury. SJP-3 can stabilize the mitochondrial membrane potential and resistant Cyt-C/mitochondrial apoptotic pathway via increasing the Bcl-2 expression and decreasing expression of the Bax, caspase-3, caspase-9 suggesting that SJP-3 may exert a protective effects on neural cells with hypoxia/reoxygenation injury through the protection of mitochondria, the stability of mitochondrial membrane potential and inhibition of mitochondrial apoptosis pathway.

Grape Seed Proanthocyanidins Protect N2a Cells against Ischemic Injury via Endoplasmic Reticulum Stress and Mitochondrial-associated Pathways

2019 ◽  
Vol 18 (4) ◽  
pp. 334-341 ◽  
Author(s):  
Kun Fu ◽  
Liqiang Chen ◽  
Lifeng Miao ◽  
Yan Guo ◽  
Wei Zhang ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Cell Viability ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Grape Seed ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Grape Seed Proanthocyanidins ◽  
N2a Cells ◽  
Caspase 12

Background/Objective: Grape seed proanthocyanidins (GSPs) are a group of polyphenolic bioflavonoids, which possess a variety of biological functions and pharmacological properties. We studied the neuroprotective effects of GSP against oxygen-glucose deprivation/reoxygenation (OGD/R) injury and the potential mechanisms in mouse neuroblastoma N2a cells. Methods: OGD/R was conducted in N2a cells. Cell viability was evaluated by CCK-8 and LDH release assay. Apoptosis was assessed by TUNEL staining and flow cytometry. Protein levels of cleaved caspase-3, Bax and Bcl-2 were detected by Western blotting. CHOP, GRP78 and caspase-12 mRNA levels were assessed by real-time PCR. JC-1 dying was used to detect mitochondrial membrane potential. ROS levels, activities of endogenous antioxidant enzymes and ATP production were examined to evaluate mitochondrial function. Results: GSP increased cell viability after OGD/R injury in a dose-dependent manner. Furthermore, GSP inhibited cell apoptosis, reduced the mRNA levels of CHOP, GRP78 and caspase-12 (ER stressassociated genes), restored mitochondrial membrane potential and ATP generation, improved activities of endogenous anti-oxidant ability (T-AOC, GXH-Px, and SOD), and decreased ROS level. Conclusion: Our findings suggest that GSP can protect N2a cells from OGD/R insult. The mechanism of anti-apoptotic effects of GSP may involve attenuating ER stress and mitochondrial dysfunction.

scholarly journals Analysis of mitochondrial morphology and function with novel fixable fluorescent stains.

1996 ◽  
Vol 44 (12) ◽  
pp. 1363-1372 ◽  
Author(s):  
M Poot ◽  
Y Z Zhang ◽  
J A Krämer ◽  
K S Wells ◽  
L J Jones ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Epifluorescence Microscopy ◽  
Mitochondrial Morphology ◽  
Microtubule Network ◽  
Multiple Features ◽  
Permeabilized Cells ◽  
Dye Staining ◽  
And Function

Investigation of mitochondrial morphology and function has been hampered because photostable, mitochondrion-specific stains that are retained in fixed, permeabilized cells have not been available. We found that in live cell preparations, the CMXRos and H2-CMXRos dyes were more photostable than rhodamine 123. In addition, fluorescence and morphology of mitochondria stained with the CMXRos and CMXRos-H2 dyes were preserved even after formaldehyde fixation and acetone permeabilization. Using epifluorescence microscopy, we showed that CMXRos and H2-CMXRos dye fluorescence fully co-localized with antibodies to subunit I of cytochrome c oxidase, indicating that the dyes specifically stain mitochondria. Confocal microscopy of these mitochondria yielded colored banding patterns, suggesting that these dyes and the mitochondrial enzyme localize to different suborganellar regions. Therefore, these stains provide powerful tools for detailed analysis of mitochondrial fine structure. We also used poisons that decrease mitochondrial membrane potential and an inhibitor of respiration complex II to show by flow cytometry that the fluorescence intensity of CMXRos and H2-CMXRos dye staining responds to changes in mitochondrial membrane potential and function. Hence, CMXRos has the potential to monitor changes in mitochondrial function. In addition, CMXRos staining was used in conjunction with spectrally distinct fluorescent probes for the cell nucleus and the microtubule network to concomitantly evaluate multiple features of cell morphology.

Metformin Collaborates With PINK1/Mfn2 Overexpression Prevented Isoproterenol-Induced Cardiomyocyte Injury By Improving Mitochondrial Function

2021 ◽  
Author(s):  
Zhuang Ma ◽  
Zuheng Liu ◽  
Yuting Xue ◽  
Hao Zhang ◽  
Wenjun Xiong ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Energy Metabolism ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Biogenesis ◽  
Mitochondrial Membrane ◽  
Mitochondrial Morphology ◽  
Dependent Manner ◽  
Combination Strategy ◽  
Mitochondrial Energy Metabolism ◽  
Atp Production

Abstract Background: Both mitochondrial quality control and energy metabolism are critical in maintaining the physiological function of cardiomyocytes. Previous studies indicated that PGC-1α is a transcription co-activator in promoting mitochondrial energy metabolism which would be beneficial for cardiomyocytes. However, PGC-1α overexpression in heart tissues could also result in the development of cardiomyopathy. This discrepancy in vivo and in vitro might be due to neglecting the elimination of damaged mitochondrial. Thus, an integration strategy of mitochondrial biogenesis and mitophagy might be beneficial.Methods: We studied the function of PINK1 in mitophagy in isoproterenol (Iso)-induced cardiomyocyte injury. Adenovirus was used to provoke an overexpression of the PINK1/Mfn2 protein. Mitochondrial morphology was examined via electron microscopy and confocal microscopy. Cardiomyocytes injury were measured by mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and apoptosis. Metformin was used to increase mitochondrial biogenesis, the level of which was detected via immunoblotting. Additionally, mitochondrial respiratory function was measured by ATP production and oxygen consumption rate (OCR). Results: Cardiomyocytes treated with Iso had high levels of PINK1 and low levels of Mfn2 in a time-dependent manner. PINK1 overexpression promoted mitophagy, alleviated Iso-induced reduction in MMP, reduced ROS production and the apoptotic rate. In addition to increasing mitophagy, metformin could promote mitochondrial biogenensis and the overexpression of Mfn2 induce mitochondrial fusion. Moreover, metformin treatment and PINK1/Mfn2 overexpression reduced the mitochondrial dysfunction by inhibiting the generation of ROS, and leading to an increase in both ATP production and mitochondrial membrane potential in Iso-induced cardiomyocytes injury. Conclusion: Our findings indicate that a combination strategy may help ameliorate myocardial injury through mitophagy and mitochondrial biogenesis.

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