Effect of thyroid hormone on mitochondrial properties and oxidative stress in cells from patients with mtDNA defects

2009 ◽  
Vol 296 (2) ◽  
pp. C355-C362 ◽  
Author(s):  
Keir J. Menzies ◽  
Brian H. Robinson ◽  
David A. Hood
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Thyroid Hormone ◽  
Protein Expression ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Biogenesis ◽  
Mitochondrial Membrane ◽  
Ros Production ◽  
Mitochondrial Mass ◽  
Mtdna Mutations

Mitochondrial (mt)DNA mutations contribute to various disease states characterized by low ATP production. In contrast, thyroid hormone [3,3′,5-triiodothyronine (T3)] induces mitochondrial biogenesis and enhances ATP generation within cells. To evaluate the role of T3-mediated mitochondrial biogenesis in patients with mtDNA mutations, three fibroblast cell lines with mtDNA mutations were evaluated, including two patients with Leigh's syndrome and one with hypertrophic cardiomyopathy. Compared with control cells, patient fibroblasts displayed similar levels of mitochondrial mass, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), mitochondrial transcription factor A (Tfam), and uncoupling protein 2 (UCP2) protein expression. However, patient cells exhibited a 1.6-fold elevation in ROS production, a 1.7-fold elevation in cytoplasmic Ca2+ levels, a 1.2-fold elevation in mitochondrial membrane potential, and 30% less complex V activity compared with control cells. Patient cells also displayed 20–25% reductions in both cytochrome c oxidase (COX) activity and MnSOD protein levels compared with control cells. After T3 treatment of patient cells, ROS production was decreased by 40%, cytoplasmic Ca2+ was reduced by 20%, COX activity was increased by 1.3-fold, and ATP levels were elevated by 1.6-fold, despite the absence of a change in mitochondrial mass. There were no significant alterations in the protein expression of PGC-1α, Tfam, or UCP2 in either T3-treated patient or control cells. However, T3 restored the mitochondrial membrane potential, complex V activity, and levels of MnSOD to normal values in patient cells and elevated MnSOD levels by 21% in control cells. These results suggest that T3 acts to reduce cellular oxidative stress, which may help attenuate ROS-mediated damage, along with improving mitochondrial function and energy status in cells with mtDNA defects.

scholarly journals Artemisinin attenuated oxidative stress and apoptosis by inhibiting autophagy in MPP+-treated SH-SY5Y cells

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
Junqiang Yan ◽  
Hongxia Ma ◽  
Xiaoyi Lai ◽  
Jiannan Wu ◽  
Anran Liu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Protein Expression ◽  
Mitochondrial Membrane Potential ◽  
Western Blotting ◽  
Cell Apoptosis ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Neuroprotective Effect ◽  
Neuroprotective Effects

Abstract Background Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. The oxidative stress is an important component of the pathogenesis of PD. Artemisinin (ART) has antioxidant and neuroprotective effects. The purpose of this study is to explore the neuroprotective effect of ART on 1-methyl-4-phenyliodine iodide (MPP +)-treated SH-SY5Y cells and underlying mechanism. Methods We used MPP+-treated SH-SY5Y cells to study the neuroprotective effect of ART. Cell viability was measured by MTT assay after incubating the cells with MPP+ and/or ART for 24 h. DCFH-DA was used to detect the level of intracellular reactive oxygen species (ROS), and WST-8 was used to detect the level of superoxide dismutase (SOD). The level of intracellular reduced glutathione (GSH) was detected with 5,5΄-dithiobis-(2-nitrobenzoic acid), and the level of malondialdehyde (MDA) was assessed based on the reaction of MDA and thiobarbituric acid. A mitochondrial membrane potential detection kit (JC-1) was used to detect changes in the mitochondrial membrane potential (MMP), and an Annexin V-FITC cell apoptosis kit was used to detect cell apoptosis. The expression levels of caspase-3, cleaved caspase-3 and the autophagy-related proteins LC3, beclin-1, and p62 were detected by Western blotting. In addition, to verify the change in autophagy, we used immunofluorescence to detect the expression of LC3 and p62. Results No significant cytotoxicity was observed at ART concentrations up to 40 μM. ART could significantly increase the viability of SH-SY5Y cells treated with MPP+ and reduce oxidative stress damage and apoptosis. In addition, the Western blotting and immunofluorescence results showed that MPP+ treatment could increase the protein expression of beclin1 and LC3II/LC3I and decrease the protein expression of p62, indicating that MPP+ treatment could induce autophagy. Simultaneous treatment with ART and MPP+ could decrease the protein expression of beclin1 and LC3II/LC3I and increase the protein expression of p62, indicating that ART could decrease the level of autophagy induced by MPP+. Conclusion Our results indicate that ART has a protective effect on MPP+-treated SH-SY5Y cells by the antioxidant, antiapoptotic activities and inhibition of autophagy. Our findings may provide new hope for the prevention and treatment of PD.

scholarly journals Mir-141-3p Regulates Apoptosis and Mitochondrial Membrane Potential via Targeting Sirtuin1 in a 1-Methyl-4-Phenylpyridinium in vitro Model of Parkinson’s Disease

2020 ◽  
Vol 2020 ◽  
pp. 1-14
Author(s):  
Yumin Zheng ◽  
Li Dong ◽  
Na Liu ◽  
Xiaoguang Luo ◽  
Zhiyi He
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Western Blot ◽  
Pc12 Cells ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Luciferase Reporter ◽  
Ros Production ◽  
Reporter Assay ◽  
Induced Apoptosis

Objectives. Parkinson’s disease (PD) is a common neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons in the substantia nigra. The present study investigated miR-141-3p/sirtuin1 (SIRT1) activity in a 1-methyl-4-phenylpyridinium- (MPP+-) induced PC12-cell model of PD. Methods. PC12 cells were exposed to MMP+ following induction of differentiation by nerve growth factor (NGF). miR-141-3p and SIRT1 expressions were examined using RT-qPCR and western blot. Cell viability was evaluated using the MTT assay. Apoptosis percentage, reactive oxygen species (ROS) production, and mitochondrial membrane potential (Δψm) were evaluated using flow cytometry. Expression of Nuclear factor-kappa B- (NF-κB-) related proteins was determined by western blot. Bioinformatic analysis, RT-qPCR, and luciferase reporter assay were used to confirm the interaction between miR-141-3p and SIRT1. Results. miR-141-3p was upregulated, and SIRT1 was downregulated in MPP+-treated PC12 cells. MPP+ treatment also upregulated nitric oxide synthase 1 (Nos1) and α-synuclein. miR-141-3p induced apoptosis, oxidative stress, mitochondrial dysfunction, and downregulated the SIRT1 mRNA expression. The luciferase reporter assay showed that SIRT1 was the target of miR-141-3p. SIRT1 transfection attenuated apoptosis, ROS production and maintained Δψm. SIRT1 also downregulated Nos1, tumor necrosis factor-α (TNF-α), interleukin 1 beta (IL-1β), interleukin 6(IL-6) and upregulated B cell lymphoma 2 (Bcl-2) protein. In addition, SIRT1 activator resveratrol blocked the effects of miR-141-3p mimic on Nos1, α-synuclein, and mitochondrial membrane potential. SIRT1 inhibitor sirtinol reversed the biological effects of miR-141-3p. Conclusion. Increased miR-141-3p induced apoptosis, oxidative stress, and mitochondrial dysfunction in MPP+-treated PC12 cells by directly targeting the SIRT1 expression. Our study provided a potential therapeutic strategy for PD.

scholarly journals Thrombopoietin Protects Cardiomyocytes from Iron-Overload Induced Oxidative Stress and Mitochondrial Injury

2015 ◽  
Vol 36 (5) ◽  
pp. 2063-2071 ◽  
Author(s):  
Shing Chan ◽  
Godfrey Chifung Chan ◽  
Jieyu Ye ◽  
Qizhou Lian ◽  
Jianliang Chen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Iron Overload ◽  
Protective Effect ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Lipid Hydroperoxides ◽  
Ros Production ◽  
H9c2 Cells ◽  
Induced Apoptosis

Background/Aims: Thalassaemia accompanied with iron-overload is common in Hong Kong. Iron-overload induced cardiomyopathy is the commonest cause of morbidity and mortality in patients with β-thalassaemia. Chronic iron-overload due to blood transfusion can cause cardiac failure. Decreased antioxidant defence and increased ROS production may lead to oxidative stress and cell injury. Iron-overload may lead to heart tissue damage through lipid peroxidation in response to oxidative stress, and a great diversity of toxic aldehydes are formed when lipid hydroperoxides break down in heart and plasma. Methods: Iron entry into embryonic heart H9C2 cells was determined by calcein assay using a fluorometer. Reactive oxygen species (ROS) production in cells treated with FeCl3 or thrombopoietin (TPO) was monitored by using the fluorescent probe H2DCFDA. Changes in mitochondrial membrane potential of H9C2 cells were quantified by using flow cytometry. Results: We demonstrated that iron induced oxidative stress and apoptosis in cardiomyocytes, and that iron increased ROS production and reduced cell viability in a dose-dependent manner. Iron treatment increased the proportion of cells with JC-1 monomers, indicating a trend of drop in the mitochondrial membrane potential. TPO exerted a cardio-protective effect on iron-induced apoptosis. Conclusions: These findings suggest that iron-overload leads to the generation of ROS and further induces apoptosis in cardiomyocytes via mitochondrial pathways. TPO might exert a protective effect on iron-overload induced apoptosis via inhibiting oxidative stress and suppressing the mitochondrial pathways in cardiomyocytes.

scholarly journals Artemisinin attenuated oxidative stress and apoptosis by inhibiting autophagy in MPP+-treated SH-SY5Y cell

2021 ◽  
Author(s):  
Junqiang Yan ◽  
Hongxia Ma ◽  
Xiaoyi Lai ◽  
Jiannan Wu ◽  
Anran Liu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Protein Expression ◽  
Mitochondrial Membrane Potential ◽  
Western Blotting ◽  
Cell Apoptosis ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Neuroprotective Effect ◽  
Neuroprotective Effects

Abstract Background Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. The oxidative stress is an important component of the pathogenesis of PD. Artemisinin (ART) have antioxidant and neuroprotective effects. The purpose of this study was to explore the neuroprotective effect of ART on 1-methyl-4-phenyliodine iodide (MPP+)-treated SH-SY5Y cells and underlying mechanism .Methods We used MPP+-treated SH-SY5Y cells to study the neuroprotective effect of ART. Cell viability was measured by MTT assay after incubating the cells with MPP+ and/or ART for 24 h. DCFH-DA was used to detect the level of intracellular reactive oxygen species (ROS), and WST-8 was used to detect the level of superoxide dismutase (SOD). The level of intracellular reduced glutathione (GSH) was detected using 5,5'-dithiobis-(2-nitrobenzoic acid), and the level of malondialdehyde (MDA) was assessed by measuring the reaction of MDA and thiobarbituric acid. A mitochondrial membrane potential detection kit (JC-1) was used to detect changes in the mitochondrial membrane potential (MMP), and an Annexin V-FITC cell apoptosis kit was used to detect cell apoptosis. The expression levels of caspase-3, cleaved caspase-3 and the autophagy-related proteins LC3, beclin-1, and p62 were detected by Western blotting. In addition, to verify the change in autophagy, we used immunofluorescence to detect the expression of LC3 and p62.Results No significant cytotoxicity was observed at ART concentrations up to 40 μM. ART could significantly increase the viability of SH-SY5Y cells treated with MPP+ and reduce oxidative stress damage and apoptosis. In addition, the Western blotting and immunofluorescence results showed that MPP+ treatment could increase the protein expression of beclin1 and LC3II/LC3I and decrease the protein expression of P62, indicating that MPP+ treatment could induce autophagy. Simultaneous treatment with ART and MPP+ could decrease the protein expression of beclin1 and LC3II/LC3I and increase the protein expression of p62, indicating that ART could decrease the level of autophagy induced by MPP+.Conclusion Our results indicate that ART has a protective effect on MPP+-treated SH-SY5Y cells by the antioxidant, antiapoptotic activities and inhibition of autophagy. Our findings may provide new hope for the prevention and treatment of PD.

scholarly journals Two Subpopulations of Human Monocytes That Differ by Mitochondrial Membrane Potential

Biomedicines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 153
Author(s):  
Nikita G. Nikiforov ◽  
Anastasia Ryabova ◽  
Marina V. Kubekina ◽  
Igor D. Romanishkin ◽  
Kirill A. Trofimov ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Primary Culture ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Function ◽  
Mitochondrial Membrane ◽  
Aminolevulinic Acid ◽  
Protoporphyrin Ix ◽  
Intima Media Thickness ◽  
Mtdna Mutations ◽  
Proinflammatory Activity

Atherosclerosis is associated with a chronic local inflammatory process in the arterial wall. Our previous studies have demonstrated the altered proinflammatory activity of circulating monocytes in patients with atherosclerosis. Moreover, atherosclerosis progression and monocyte proinflammatory activity were associated with mitochondrial DNA (mtDNA) mutations in circulating monocytes. The role of mitochondria in the immune system cells is currently well recognized. They can act as immunomodulators by releasing molecules associated with bacterial infection. We hypothesized that atherosclerosis can be associated with changes in the mitochondrial function of circulating monocytes. To test this hypothesis, we performed live staining of the mitochondria of CD14+ monocytes from healthy donors and atherosclerosis patients with MitoTracker Orange CMTMRos dye, which is sensitive to mitochondrial membrane potential. The intensity of such staining reflects mitochondrial functional activity. We found that parts of monocytes in the primary culture were characterized by low MitoTracker staining (MitoTracker-low monocytes). Such cells were morphologically similar to cells with normal staining and able to metabolize 5-aminolevulinic acid and accumulate the heme precursor protoporphyrin IX (PplX), indicative of partially preserved mitochondrial function. We assessed the proportion of MitoTracker-low monocytes in the primary culture for each study subject and compared the results with other parameters, such as monocyte ability to lipopolysaccharide (LPS)-induced proinflammatory activation and the intima-media thickness of carotid arteries. We found that the proportion of MitoTracker-low monocytes was associated with the presence of atherosclerotic plaques. An increased number of such monocytes in the primary culture was associated with a reduced proinflammatory activation ability of cells. The obtained results indicate the presence of circulating monocytes with mitochondrial dysfunction and the association of such cells with chronic inflammation and atherosclerosis development.

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.

Abstract 371: Mitochondrial Antiviral Signaling (MAVS) Protects Cardiomyocytes Under Oxidative Stress by Interfering with Bax-Mediated Cell Death

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Toshitaka Yajima ◽  
Stanley Park ◽  
Hanbing Zhou ◽  
Michinari Nakamura ◽  
Mitsuyo Machida ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Membrane Potential ◽  
Cytochrome C ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Cytochrome C Release ◽  
Antiviral Signaling ◽  
Cell Death Pathway ◽  
The Impact

MAVS is a mitochondrial outer membrane protein that activates innate antiviral signaling by recognizing cytosolic viral RNAs and DNAs. While the discovery of MAVS is the first molecular evidence that links mitochondria to innate immune mechanisms, it is still unclear whether MAVS affects mitochondrial cell death as a member of caspase activation and recruitment domain (CARD)-containing proteins. We found that MAVS interacts with Bax through CARD by Yeast two-hybrid and a series of immunoprecipitation (IP) assay, which led us to hypothesize that MAVS functions not only in the innate antiviral mechanisms but also in the mitochondrial cell death pathway. Methods: 1) We examined molecular interaction between MAVS and Bax under oxidative stress by IP using isolated myocytes with H2O2 stimulation and the heart post ischemia-reperfusion (I/R). 2) We evaluated the effect of MAVS on mitochondrial membrane potential and apoptosis under H2O2 stimulation using isolated myocytes with adenoviral MAVS knockdown. 3) We investigated the impact of MAVS on %myocardial infarction (%MI) post I/R using cardiac-specific MAVS knockout (cKO) and transgenic (cTg) mice which we have originally generated. 4) We examined the effect of MAVS on recombinant Bax (rBax)-mediated cytochrome c release using isolated mitochondria from wild type (WT) and MAVS KO mice. Results: 1) The amount of Bax pulled down with MAVS was significantly increased in isolated myocytes with 0.2 mM H2O2 compared to those without stimulation (mean±SD; 1.808±0.14, n=5, p<0.001) and in the heart post I/R compared to sham (2.2±1.19, n=3, p=0.0081). 2) Myocytes with MAVS knockdown showed clear abnormalities in mitochondrial membrane potential and caspace-3 cleavage with 0.2 mM H2O2 compared to control cardiomyocytes. 3) MAVS cKO had significantly larger %MI than WT (81.9 ± 5.8% vs. 42.6 ± 13.6%, n=8, p=0.0008). In contrast, MAVS cTg had significantly smaller %MI that WT (30.0 ± 4.8% vs. 49.2 ± 4.8%, n=10, p=0.0113). 4) Mitochondria from MAVS KO exhibited cytochrome c release after incubation with 2.5 μ g of rBax while those from WT required 10 μ g of rBax. Conclusion: These results demonstrate that MAVS protects cardiomyocyte under oxidative stress by interfering with Bax-mediated cytochrome c release from mitochondria.

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