Abstract 406: Oxidative Stress Mediated Myocardial Lipid Dysfunction

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Manikandan Panchatcharam ◽  
Mini Chandra ◽  
Jonathan Fox ◽  
Shenuarin Bhuiyan ◽  
Wayne Orr ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Oxygen Consumption ◽  
Oxidative Phosphorylation ◽  
Ischemia Reperfusion ◽  
Cardiac Injury ◽  
Negative Regulator ◽  
Membrane Pore ◽  
Mitochondrial Superoxide ◽  
Rate Of Oxygen Consumption

Lipid phosphate phosphatases-3 control the conversion of bioactive lipid phosphates to their dephosphorylated counterparts. Oxidative stress transactivates microRNA-92a, which is a negative regulator of LPP3. We found that LPP3 expression was markedly downregulated in ischemic regions after ischemia/reperfusion (I/R) injury. We observed a similar trend in the myocardium from patients samples with acute MI at 24h. Our in vitro studies indicate that overexpression of LPP3 protects the cardiomyocyte against reactive oxygen species (ROS)-induced cardiac injury and knockout of LPP3 gene in the myocardium increases cardiac dysfunction and mortality. Using XF24 Seahorse analyzer we determined the effect of ROS on respiration in pluripotent stem cell-derived cardiomyocytes (iPSC-CM). Adding Phorbol 12-myristate 13-acetate (PMA) to these cells immediately increased oxygen consumption as compared to LPP3 overexpressed cells. This apparent increase in respiration was reversible by oligomycin, which blocks ATP synthase. The rate of oxygen consumption per cell was significantly lower in stimulated compared to LPP3 overexpressed iPSC-CM. The most noticeable difference in the O 2 consumption was found in the presence of carbonilcyanide p-trifluromethoxyphenylhydrazone (FCCP). FCCP is an inner membrane pore opener which resets the proton gradient between mitochondrial matrix and interspace, resulting in continuous transport of protons and consuming O 2 at the maximum potential. Remarkably, while the FCCP treatment increased O 2 consumption in LPP3 overexpressed cells (P<0.05), the treatment showed no effect on the O 2 consumption in the PMA stimulated alone. The result indicated that the low basal oxidative phosphorylation activity in stimulated cells was due to unusually low oxidative phosphorylation potential. To explore the free radical regulation of LPP3 overexpressed cells, superoxide anion was measured using dihydroethidium, a fluorescent cholesterol analog. The levels of superoxide radicals in PMA treated cells were consistently and significantly higher than the levels in LPP3 overexpressed cells (P<0.05). In turn, the radicals can be removed by adding MitoTEMPO (a specific scavenger of mitochondrial superoxide).

Abstract 203: Regulation of Lipid Phosphate Phosphatase 3 in Cardiac Ischemia/Reperfusion Injury and Hypertrophy

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Mini Chandra ◽  
Jonathan Fox ◽  
Wayne Orr ◽  
Christopher Kevil ◽  
Sumitra Miriyala ◽  
...  
Keyword(s):  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Cardiac Injury ◽  
Negative Regulator ◽  
Cardiomyocyte Hypertrophy ◽  
Acute Mi ◽  
Lipid Phosphate ◽  
Lipid Phosphate Phosphatases ◽  
Old Mice

Generation of reactive oxygen species (ROS) has been implicated in myocardial infarction (MI), stroke and sudden cardiac death. Mitochondrial respiration is a major source of ROS production and lipids regulate mitochondrial oxidative metabolism and homeostasis through effects on mitochondrial fusion and fission and on the activity of mitochondrial membrane proteins. Lipid phosphate phosphatases (LPPs) control the conversion of bioactive lipid phosphates to their dephosphorylated counterparts. These include phosphatidic acid (PA), and lysophosphatidic acid (LPA). Oxidative stress was identified to transactivate microRNA-92a, which is a negative regulator of LPP3. We found that LPP3 expression was markedly down regulated in ischemic regions after ischemia/reperfusion (I/R) injury. We observed a similar trend in the myocardium from patients with acute MI at 24h. Our in vitro studies indicate that overexpression of LPP3 protects the cardiomyocyte against ROS-induced cardiac injury and reduction of LPP3 by conditional specific cardiac knockout of the LPP3 gene in mice increases cardiac dysfunction and mortality. These mice are viable and fertile but showed increased mortality ~8 months (Fig1). Blood pressure was similar in LPP3 fl/fl (96 ± 9 mmHg; n = 19) and Myh6- LPP3 Δ mice (92 ± 7 mmHg; n = 19), although heart rates were significantly higher in Myh6- LPP3 Δ 3 month old mice (642 ± 21 bpm, compared to LPP3 fl/fl with 600± 17 bpm; P<0.001). Knockdown of LPP3 enhanced cardiomyocyte hypertrophy induced by LPA based on analysis of sarcomere organization, cell surface area, levels of fetal genes ANP and BNP, and ANF release from nuclei, which are hallmarks of cardiomyocyte hypertrophy, indicating that LPP3 negatively regulates cardiomyocyte hypertrophy induced by LPA.

scholarly journals The Long Noncoding RNA Hotair Regulates Oxidative Stress and Cardiac Myocyte Apoptosis during Ischemia-Reperfusion Injury

2020 ◽  
Vol 2020 ◽  
pp. 1-19 ◽  
Author(s):  
Kai Meng ◽  
Jiao Jiao ◽  
Rui-Rui Zhu ◽  
Bo-Yuan Wang ◽  
Xiao-Bo Mao ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Calcium Binding ◽  
Cardiac Myocyte ◽  
Heart Diseases ◽  
Ischemia Reperfusion ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Myocyte Apoptosis ◽  
Cardiac Myocyte Apoptosis

Oxidative stress and subsequent cardiac myocyte apoptosis play central roles in the initiation and progression of myocardial ischemia-reperfusion (I/R) injury. Homeobox transcript antisense intergenic RNA (Hotair) was previously implicated in various heart diseases, yet its role in myocardial I/R injury has not been clearly demonstrated. Mice with cardiac-restricted knockdown or overexpression of Hotair were exposed to I/R surgery. H9c2 cells were cultured and subjected to hypoxia/reoxygenation (H/R) stimulation to further verify the role and underlying mechanisms of Hotair in vitro. Histological examination, molecular detection, and functional parameters were determined in vivo and in vitro. In response to I/R or H/R treatment, Hotair expression was increased in a bromodomain-containing protein 4-dependent manner. Cardiac-restricted knockdown of Hotair exacerbated, whereas Hotair overexpression prevented I/R-induced oxidative stress, cardiac myocyte apoptosis, and cardiac dysfunction. Mechanistically, we observed that Hotair exerted its beneficial effects via activating AMP-activated protein kinase alpha (AMPKα). Further detection revealed that Hotair activated AMPKα through regulating the enhancer of zeste homolog 2/microRNA-451/calcium-binding protein 39 (EZH2/miR-451/Cab39) axis. We provide the evidence that endogenous lncRNA Hotair is an essential negative regulator for oxidative stress and cardiac myocyte apoptosis in myocardial I/R injury, which is dependent on AMPKα activation via the EZH2/miR-451/Cab39 axis.

MiR-485-5p Promotes Neuron Survival through Mediating Rac1/Notch2 Signaling Pathway after Cerebral Ischemia/Reperfusion

2020 ◽  
Vol 17 (3) ◽  
pp. 259-266 ◽  
Author(s):  
Xuan Chen ◽  
Sumei Zhang ◽  
Peipei Shi ◽  
Yangli Su ◽  
Dong Zhang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Viability ◽  
Therapeutic Option ◽  
Ischemia Reperfusion ◽  
Glucose Deprivation ◽  
Small Gtpase ◽  
Luciferase Reporter ◽  
Pathological Feature ◽  
In Cells

Objective: Ischemia-reperfusion (I/R) injury is a pathological feature of ischemic stroke. This study investigated the regulatory role of miR-485-5p in I/R injury. Methods: SH-SY5Y cells were induced with oxygen and glucose deprivation/reoxygenation (OGD/R) to mimic I/R injury in vitro. Cells were transfected with designated constructs (miR-485- 5p mimics, miR-485-5p inhibitor, lentiviral vectors overexpressing Rac1 or their corresponding controls). Cell viability was evaluated using the MTT assay. The concentrations of lactate dehydrogenase, malondialdehyde, and reactive oxygen species were detected to indicate the degree of oxidative stress. Flow cytometry and caspase-3 activity assay were used for apoptosis assessment. Dual-luciferase reporter assay was performed to confirm that Rac family small GTPase 1 (Rac1) was a downstream gene of miR-485-5p. Results: OGD/R resulted in decreased cell viability, elevated oxidative stress, increased apoptosis, and downregulated miR-485-5p expression in SH-SY5Y cells. MiR-485-5p upregulation alleviated I/R injury, evidenced by improved cell viability, decreased oxidative markers, and reduced apoptotic rate. OGD/R increased the levels of Rac1 and neurogenic locus notch homolog protein 2 (Notch2) signaling-related proteins in cells with normal miR-485-5p expression, whereas miR- 485-5p overexpression successfully suppressed OGD/R-induced upregulation of these proteins. Furthermore, the delivery of vectors overexpressing Rac1 in miR-485-5p mimics-transfected cells reversed the protective effect of miR-485-5p in cells with OGD/R-induced injury. Conclusion: This study showed that miR-485-5p protected cells following I/R injury via targeting Rac1/Notch2 signaling suggest that targeted upregulation of miR-485-5p might be a promising therapeutic option for the protection against I/R injury.

scholarly journals Calycosin-7-O-β-D-glucoside Attenuates OGD/R-Induced Damage by Preventing Oxidative Stress and Neuronal Apoptosis via the SIRT1/FOXO1/PGC-1α Pathway in HT22 Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Xiangli Yan ◽  
Aiming Yu ◽  
Haozhen Zheng ◽  
Shengxin Wang ◽  
Yingying He ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Neuronal Apoptosis ◽  
Ischemia Reperfusion ◽  
Glucose Deprivation ◽  
Pathological Process ◽  
Neuroprotective Effects ◽  
Ht22 Cells ◽  
Positive Effects ◽  
Antioxidant Stress

Neuronal apoptosis induced by oxidative stress is a major pathological process that occurs after cerebral ischemia-reperfusion. Calycosin-7-O-β-D-glucoside (CG) is a representative component of isoflavones in Radix Astragali (RA). Previous studies have shown that CG has potential neuroprotective effects. However, whether CG alleviates neuronal apoptosis through antioxidant stress after ischemia-reperfusion remains unknown. To investigate the positive effects of CG on oxidative stress and apoptosis of neurons, we simulated the ischemia-reperfusion process in vitro using an immortalized hippocampal neuron cell line (HT22) and oxygen-glucose deprivation/reperfusion (OGD/R) model. CG significantly improved cell viability and reduced oxidative stress and neuronal apoptosis. In addition, CG treatment upregulated the expression of SIRT1, FOXO1, PGC-1α, and Bcl-2 and downregulated the expression of Bax. In summary, our findings indicate that CG alleviates OGD/R-induced damage via the SIRT1/FOXO1/PGC-1α signaling pathway. Thus, CG maybe a promising therapeutic candidate for brain injury associated with ischemic stroke.

Proximal tubule dysfunction in cystine-loaded tubules: effect of phosphate and metabolic substrates

1996 ◽  
Vol 271 (3) ◽  
pp. F717-F722
Author(s):  
G. Bajaj ◽  
M. Baum
Keyword(s):  
Oxygen Consumption ◽  
Proximal Tubule ◽  
Tricarboxylic Acid Cycle ◽  
Dimethyl Ester ◽  
Proximal Tubules ◽  
Intracellular Atp ◽  
Metabolic Substrates ◽  
Rate Of Oxygen Consumption ◽  
Intracellular Phosphate

Intracellular cystine loading by use of cystine dimethyl ester (CDME) results in a generalized inhibition in proximal tubule transport due, in part, to a decrease in intracellular ATP. The present study examined the importance of phosphate and metabolic substrates in the proximal tubule dysfunction produced by cystine loading. Proximal tubule intracellular phosphorus was 1.8 +/- 0.1 in control tubules and 1.1 +/- 0.1 nmol/mg protein in proximal tubules incubated in vitro with CDME P < 0.001). Infusion of sodium phosphate in rabbits and subsequent incubation of proximal tubules with a high-phosphate medium attenuated the decrease in proximal tubule respiration and prevented the decrease in intracellular ATP with cystine loading. Tricarboxylic acid cycle intermediates have been shown to preserve oxidative metabolism in phosphate-depleted proximal tubules. In proximal tubules incubated with either 1 mM valerate or butyrate, there was a 42 and 34% reduction (both P < 0.05) in the rate of oxygen consumption with cystine loading. However, tubules incubated with 1 mM succinate or citrate had only a 13 and 14% P = NS) reduction in the rate of oxygen consumption, respectively. These data are consistent with a limitation of intracellular phosphate in the pathogenesis of the proximal tubule dysfunction with cystine loading.

scholarly journals Oxidative Stress Induction of DJ-1 Protein in Reactive Astrocytes Scavenges Free Radicals and Reduces Cell Injury

2009 ◽  
Vol 2 (1) ◽  
pp. 36-42 ◽  
Author(s):  
Takashi Yanagida ◽  
Jun Tsushima ◽  
Yoshihisa Kitamura ◽  
Daijiro Yanagisawa ◽  
Kazuyuki Takata ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Injury ◽  
Ischemia Reperfusion ◽  
Primary Cultures ◽  
Artery Occlusion ◽  
Reactive Astrocytes ◽  
Glutathione S Transferase ◽  
Triphenyltetrazolium Chloride ◽  
Ischemic Region

Astrocytes, one of the predominant types of glial cells, function as both supportive and metabolic cells for the brain. Under cerebral ischemia/reperfusion-induced oxidative conditions, astrocytes accumulate and activate in the ischemic region. DJ-1 has recently been shown to be a sensor of oxidative stress in living cells. However, the function of astrocytic DJ-1 is still unknown. In the present study, to clarify the effect of astrocytic DJ-1 protein under massive oxidative insult, we used a focal ischemic rat model that had been subjected to middle cerebral artery occlusion (MCAO) and reperfusion. We then investigated changes in the distribution of DJ-1 in astrocytes, DJ-1 release from cultured astrocytes, and the effects of recombinant DJ-1 protein on hydrogen peroxide (H2O2)-induced death in normal and DJ-1-knockdown SH-SY5Y cells and on in vitro scavenging of hydroxyl radicals (•OH) by electron spin resonance spectrometry. At 24 h after 2-h MCAO and reperfusion, an infarct lesion was markedly observed using magnetic resonance imaging and 2,3,5-triphenyltetrazolium chloride staining. In addition, reactive astrocytes enhanced DJ-1 expression in the penumbral zone of the ischemic core and that DJ-1 protein was extracellularly released from astrocytes by H2O2 in in vitro primary cultures. Although DJ-1-knockdown SH-SY5Y cells were markedly vulnerable to oxidative stress, treatment with glutathione S-transferase-tagged recombinant human DJ-1 protein (GST-DJ-1) significantly inhibited H2O2-induced cell death. In addition, GST-DJ-1 protein directly scavenged•OH. These results suggest that oxidative stress induces the release of astrocytic DJ-1 protein, which may contribute to astrocyte-mediated neuroprotection.

scholarly journals Mitochondrial Spare Reserve Capacity : A New Predictive Metabolic Biomarker for Aggressiveness of Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 7-7
Author(s):  
Quentin Fovez ◽  
Bruno Quesnel ◽  
William Laine ◽  
Raeeka Khamari ◽  
Celine Berthon ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Oxygen Consumption ◽  
Oxidative Phosphorylation ◽  
Mutation Rate ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Reserve Capacity ◽  
Glycolytic Activity ◽  
Acute Myeloid

Introduction The persistence of leukemic cells after treatment limits the effectiveness of anticancer drugs and is the cause of relapse in patients with acute myeloid leukemia (AML). After exposure to chemotherapeutic drugs, the survival of leukemic cells is mainly supported by mitochondrial energy metabolism. Several preclinical studies have shown that the combination of mitochondrial oxidative phosphorylation inhibitors with various anticancer treatments constitutes an effective therapeutic combination in vitro to eradicate the surviving leukemic cells. Evaluating the mitochondrial bioenergetic activity of blasts from AML patients could therefore provide predictive information on treatment response. The basal oxygen consumption of cells varies according to hematopoietic differentiation and depends on the energy needs in the in vitro condition of measurement. But it is necessary to treat the cells with uncoupling agents (eg FCCP) to assess the maximum activity that the respiratory chain could reach to respond to energy stress. Then, the switch from a basal level of oxygen consumption to a maximum level defines the mitochondrial spare reserve capacity (SRC). In this study, we propose to determine whether spare reserve capacity of blasts is a potential biomarker of AML aggressiveness in patients and to characterize the biochemical processes involved in the control of SRC in leukemic cells. Results Using the XFe24 Seahorse fluorometric oximeter, we first determined the mitochondrial oxygen consumption and glycolytic activity in hematopoietic cells (monocytes, lymphocytes, dendritic cells) of healthy donors, in AML patient blasts at diagnosis or at relapse and in AML cell lines (HL-60, MOLM-13, THP-1, KG1, OCI-AML3, MV-4-11, U-937). All measures have been assessed from freshly collected samples of peripheral blood and of bone marrow. As expected, AMLs are characterized by low oxidative phosphorylation activity compared to normal hematopoietic cells. From all the OXPHOS values obtained we defined a SRC threshold above which the SRC is considered high. This threshold has been set at a capacity to increase basal respiration by 250%. From patients blasts, we have therefore defined two groups characterized by high (n=14) or low (n=21) mitochondrial spare reserve capacity. Blasts with high SRC exhibit high glycolytic activity suggesting a link between spare reserve capacity and glucose metabolism. Using U-13C6 glucose and pharmacological inhibitors, we have demonstrated that the utilization of the mitochondrial spare reserve capacity of leukemic cells is supported through glycolysis and that mitochondrial oxidation of pyruvate is a key element for SRC recruitment. Mitochondrial pyruvate carrier inhibitors (as UK-5099) or gene silencing of BRP44 abolish the SRC of leukemic cells highlighting the importance of pyruvate oxidation to increase oxygen consumption. Since high mutation rate is recognized as an unfavorable prognostic factor in AML, we have also sequenced 45 commonly genes mutated in AMLs characterized by high or low SRC blasts. Interestingly, DNA sequencing analysis showed that AML with low SRC blasts have a higher mutation rate than high SRC blasts and also exhibited exclusive mutations such as ASXL1 (25%), IDH2 (25%), NPM1 (25%), IDH1 (13%), JAK2 (13%) and SF3B1 (13%). Conclusion Currently, most of the clinical biomarkers used to predict AML aggressiveness are based on DNA analysis, but the emergence of mutations is not always associated with phenotypic changes. This study shows that the mitochondrial spare reserve capacity of blasts represents a new functional biomarker based on the assessment of the energetic phenotype and could help the clinicians to determine the prognosis of AML. Moreover we have showed that altering pyruvate metabolism highly decrease spare reserve capacity of blasts and then could be evaluated as metabolic strategies to improve the therapeutic response in patients with AML. Disclosures Kluza: Daiichi-Sankyo: Research Funding.

scholarly journals In vivo and In vitro Antioxidant Activities of Aqueous and Ethanol Stem Bark Extracts of Vitex doniana on Doxorubicin-induced Oxidative Stress in Rats

2021 ◽  
pp. 44-55
Author(s):  
Mohammed Aliyu Sulaiman ◽  
Daniel Dahiru ◽  
Mohammed Auwal Ibrahim ◽  
Ahmed Ibrahim Hayatu
Keyword(s):  
Oxidative Stress ◽  
Antioxidant Activities ◽  
Ischemia Reperfusion ◽  
Oral Treatment ◽  
Stem Bark ◽  
Albino Rats ◽  
Associated Diseases ◽  
Vitex Doniana

Background: Oxidative stress is involved in the pathogenesis of hypertension, myocardial ischemia-reperfusion injury, atherosclerosis, muscular dystrophy, aging and other associated diseases. Vitex doniana is used in Adamawa, northern Nigeria to treat oxidative stress associated diseases. However, the antioxidative effects of the plant have not been scientifically examined in oxidative stress experimental animal models. The aim of this study is to investigate the in vitro and in vivo antioxidant activities of aqueous and ethanol stem bark extracts of Vitex doniana in oxidative stress model of rats. Methods: The study used 35 adult albino rats weighing 175 ± 25 g, of which 30 were induced with oxidative stress by intraperitoneal injection of doxorubicin (10 mg/kg) for three consecutive days. Animals were treated by oral administration of silymarin (100 mg/kg) and Vitex doniana aqueous or ethanol extract (100 mg/kg and 200 mg/kg) for 14 consecutive days before they were sacrificed on the 15th day and blood was analyzed for biochemical indices of oxidative stress. Results: The results of the phytochemistry showed the presence of alkaloids, tannins, flavonoids, steroids, phenols, saponins, terpenoids, glycosides: and total flavonoids (52.70 ± 1.60 mg/ml and 75.40 ± 0.80 mg/ml), total phenols (21.45 ± 1.54 mg/ml and 26.50 ± 1.22 mg/ml) for aqueous and ethanol stem bark extracts respectively. The extracts scavenged DPPH radical, reduced Fe3+ and inhibited lipid peroxidation. Doxorubicin significantly (p<0.05) lowered the levels of SOD, CAT, GR and TAS and significantly (p<0.05) but, increased the level of LPO. Oral treatment with Vitex doniana extracts significantly (p<0.05) increased the activities of CAT, GR, SOD and TAS while LPO was significantly (p<0.05) lowered. Vitex doniana stem bark extracts significantly (p<0.05) improved the biochemical derangements observed in the induced untreated animals in comparable manner to that of Silymarin. Conclusion: The present study provides the scientific rationale for the use of Vitex doniana stem bark in traditional medicine and has a viable antioxidative capacity both in vitro and in vivo.

Abstract 357: Manganese Superoxide Dismutase: a Novel Mediator of Heart Failure Development and Progression

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Sumitra Miriyala ◽  
Mini Chandra ◽  
Benjamin Maxey ◽  
Daret K St. Clair ◽  
Manikandan Panchatcharam
Keyword(s):  
Superoxide Dismutase ◽  
Oxygen Consumption ◽  
Manganese Superoxide Dismutase ◽  
Consumption Rate ◽  
Primary Source ◽  
Wild Type ◽  
Membrane Pore ◽  
Manganese Superoxide ◽  
Rate Of Oxygen Consumption ◽  
In Cells

Manganese Superoxide Dismutase (MnSOD), an antioxidant enzyme that catalyzes the conversion of superoxide radicals (O 2 •-) in mitochondria. Constitutive activation mitochondrial reactive oxygen species (ROS) has been implicated in both the pathogenesis and the progression of cardiovascular disease. Absence of SOD2 (gene that encodes MnSOD) is found to be embryonic lethal in animal models due to impairment of mitochondrial function, most noticeably in the heart. In our earlier investigation, we have shown that the MnSOD mimetic, MnTnBuOE-2-PyP 5+ distributes 3-fold more in mitochondria than in cytosol. The exceptional ability of MnTnBuOE-2-PyP 5+ to dismute O 2 •- parallels its ability to reduce ONOO– and CO3–. Based on our earlier reports, we have generated mice that specifically lack MnSOD in cardiomyocytes (Mhy6-SOD2 Δ ). These mice showed early mortality ~4 months due to cardiac mitochondrial dysfunction. Oxidative phosphorylation (OXPHOS) in mitochondria is the predominant mode for O 2 consumption in cells, and the mitochondria are the primary source of ROS in cells due to leaked electrons. FACS analyses using Mito-Tracker Green indicated that the mass of mitochondria per cell was slightly decreased in the Mhy6-SOD2 Δ to the wild type. We then examined OXPHOS levels in Mhy6-SOD2 Δ v.s. wild type using a Seahorse XF analyzer. The rate of oxygen consumption per cells was signi[[Unable to Display Character: &#64257;]]cantly lower in Mhy6-SOD2 Δ cardiomyocytes than that in wild type. The most noticeable difference in the O 2 consumption was found in the presence of FCCP (H+ ionophore / uncoupler). FCCP is an inner membrane pore opener which resets the proton gradient between the mitochondrial matrix and the interspace, resulting in continuous transport of protons and consuming O 2 at the maximum potential. Remarkably, while the FCCP treatment increased O 2 consumption in wild type, the treatment showed no effect on the O 2 consumption in the Mhy6-SOD2 Δ cardiomyocytes. The result indicated that the low basal OXPHOS activity in Mhy6-SOD2 Δ was due to unusually low OXPHOS potential. We examined glycolysis in these cells by measuring extracellular acidi[[Unable to Display Character: &#64257;]]cation (ECAR) and the pattern exactly opposite to that of oxygen consumption rate (OCR) was observed for glycolysis rates between Mhy6-SOD2 Δ and wild type.

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