Electron Transport Chain Complex II Sustains High Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells

The role of mitochondria in the fate determination of hematopoietic stem and progenitor cells (HSPCs) is not solely limited to the switch from glycolysis to oxidative phosphorylation, but also involves alterations in mitochondrial features and properties, including mitochondrial membrane potential (ΔΨmt). Several research groups have used mitochondrial dyes and have showed that long term multi-lineage reconstitution is enriched in low ΔΨmt fraction. However, hematopoietic stem cells (HSCs) exhibit higher pump activity than mature populations, and this causes the enhanced extrusion of mitochondrial dyes used for measuring ΔΨmt, such as tetramethylrhodamine methyl ester (TMRM), which in turn can lead to biased results (Bonora M. et al, 2018). In this study, while considering the activity of xenobiotic efflux pumps in HSCs, we have assessed the equilibrium between electron transport chain (ETC) complexes and ATP production in order to elucidate the mechanism that sustain mitochondrial membrane potential in HSPCs. We first used flow analysis of HSCs and other bone marrow populations, stained by TMRM in presence of Verapamil, an efflux pump inhibitor, to show a downward trend in ΔΨmt along with hematopoietic differentiation. To validate high ΔΨmt as a key feature of HSCs, we measured Ki67 positivity to assess whether ΔΨmt is associated with cell cycle quiescence in HSPCs. When Lin-Sca-1+c-Kit+ (LSK) cells were separated into two fractions, based on their TMRM intensity, we found that the percentage of Ki67+ cells in LSK-High was lower than the one in LSK-Low, and were comparable to CD150+CD48- HSC-enriched fraction. Consistently, phenotypic HSCs preferentially reside in the TMRM high population, with less Ki67 positivity. Since ΔΨmt levels in the cells is determined by the balance between proton pumping (by ETC) and proton flow (by ATP synthase/complex V), we next assessed ETC complexes. The expression of ATP5A, a key subunit of complex V, and of NDUFV1, a subunit of complex I, were particularly weak in HSPCs and drastically increased following differentiation process, while no differences were detected in complex II subunit SDHA expression between HSCs and mature populations. Likewise, the activity of complex I increased following differentiation process, while the activity of complex II remained stable among HSC, LSK, and Lin− fractions. Interestingly, when the respective ratios of complex I and II to complex V were calculated, compared to complex I, a significantly higher ratio of complex II: complex V was found in HSPCs. Collectively, these data support the hypothesis that HSPCs have low proton flow comparing mature populations, but similar proton pumping activity, especially due to complex II, which finally results in a higher ΔΨmt. In order to deeply investigate the contribution of complex I, II and III to sustain ΔΨmt, the reduction of TMRM intensity after the administration of low dosages of their specific inhibitors (Rotenone, TTFA and Antimycin A, respectively) was analyzed. The reduction of TMRM intensity by Rotenone was observed in the committed cells, and the addition of Antimycin A led to a drop in TMRM intensity in all hematopoietic lineages. Critically, complex II inhibition by TTFA caused a substantial decrease of ΔΨmt, particularly in HSPCs. Finally, we investigated the functional importance of each ETC complex in HSCs, founding that TTFA, but not Rotenone, caused a reduction in in vitro colony-replating capacity, and a similar effect was observed after administration of Antimycin A. Altogether this study highlights complex II as a key regulator of ΔΨmt in HSPCs and suggests the distinct roles of complex I and complex II in hematopoiesis. Further characterization of the precise mechanisms regulating mitochondrial controls in HSCs will contribute to a better understanding of an active role of mitochondria in HSC homeostasis. Disclosures No relevant conflicts of interest to declare.

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Electron transport chain complex II sustains high mitochondrial membrane potential in hematopoietic stem and progenitor cells

Stem Cell Research ◽

10.1016/j.scr.2019.101573 ◽

2019 ◽

Vol 40 ◽

pp. 101573 ◽

Cited By ~ 6

Author(s):

Claudia Morganti ◽

Massimo Bonora ◽

Kyoko Ito ◽

Keisuke Ito

Keyword(s):

Membrane Potential ◽

Electron Transport ◽

Progenitor Cells ◽

Mitochondrial Membrane Potential ◽

Electron Transport Chain ◽

Mitochondrial Membrane ◽

Chain Complex ◽

Hematopoietic Stem ◽

Transport Chain ◽

Stem And Progenitor Cells

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Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

Journal of Visualized Experiments ◽

10.3791/60057 ◽

2019 ◽

Cited By ~ 1

Author(s):

Claudia Morganti ◽

Massimo Bonora ◽

Keisuke Ito

Keyword(s):

Membrane Potential ◽

Progenitor Cells ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Efflux Pumps ◽

Hematopoietic Stem ◽

Flow Cytometric ◽

Stem And Progenitor Cells

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Anaerobic respiration sustains mitochondrial membrane potential in a prolyl hydroxylase pathway-activated cancer cell line in a hypoxic microenvironment

AJP Cell Physiology ◽

10.1152/ajpcell.00255.2013 ◽

2014 ◽

Vol 306 (4) ◽

pp. C334-C342 ◽

Cited By ~ 10

Author(s):

Eiji Takahashi ◽

Michihiko Sato

Keyword(s):

Membrane Potential ◽

Electron Transport ◽

Cell Line ◽

Mitochondrial Membrane Potential ◽

Cancer Cell ◽

Mitochondrial Membrane ◽

Cancer Cell Line ◽

Prolyl Hydroxylase ◽

Complex Ii ◽

Pathway Activation

To elucidate how tumor cells produce energy in oxygen-depleted microenvironments, we studied the possibility of mitochondrial electron transport without oxygen. We produced well-controlled oxygen gradients (ΔO2) in monolayer-cultured cells. We then visualized oxygen levels and mitochondrial membrane potential (ΔΦm) in individual cells by using the red shift of green fluorescent protein (GFP) fluorescence and a cationic fluorescent dye, respectively. In this two-dimensional tissue model, ΔΦm was abolished in cells >500 μm from the oxygen source [the anoxic front (AF)], indicating limitations in diffusional oxygen delivery. This result perfectly matched GFP-determined ΔO2. In cells pretreated with dimethyloxaloylglycine (DMOG), a prolyl hydroxylase domain-containing protein (PHD) inhibitor, the AF was expanded to 1,500–2,000 μm from the source. In these cells, tissue ΔO2 was substantially decreased, indicating that PHD pathway activation suppressed mitochondrial respiration. The expansion of the AF and the reduction of ΔO2 were much more prominent in a cancer cell line (Hep3B) than in the equivalent fibroblast-like cell line (COS-7). Hence, the results indicate that PHD pathway-activated cells can sustain ΔΦm, despite significantly decreased electron flux to complex IV. Complex II inhibition abolished the effect of DMOG in expanding the AF, although tissue ΔO2 remained shallow. Separate experiments demonstrated that complex II plays a substantial role in sustaining ΔΦm in DMOG-pretreated Hep3B cells with complex III inhibition. From these results, we conclude that PHD pathway activation can sustain ΔΦm in an otherwise anoxic microenvironment by decreasing tissue ΔO2 while activating oxygen-independent electron transport in mitochondria.

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Palmitic Acid, but Not Lauric Acid, Induces Metabolic Inflammation, Mitochondrial Fragmentation, and a Drop in Mitochondrial Membrane Potential in Human Primary Myotubes

Frontiers in Nutrition ◽

10.3389/fnut.2021.663838 ◽

2021 ◽

Vol 8 ◽

Author(s):

Domenico Sergi ◽

Natalie Luscombe-Marsh ◽

Nenad Naumovski ◽

Mahinda Abeywardena ◽

Nathan O'Callaghan

Keyword(s):

Insulin Resistance ◽

Membrane Potential ◽

Electron Transport ◽

Mitochondrial Membrane Potential ◽

Electron Transport Chain ◽

Mitochondrial Membrane ◽

Chain Complex ◽

Mitochondrial Fragmentation ◽

Metabolic Inflammation ◽

Transport Chain

The chain length of saturated fatty acids may dictate their impact on inflammation and mitochondrial dysfunction, two pivotal players in the pathogenesis of insulin resistance. However, these paradigms have only been investigated in animal models and cell lines so far. Thus, the aim of this study was to compare the effect of palmitic (PA) (16:0) and lauric (LA) (12:0) acid on human primary myotubes mitochondrial health and metabolic inflammation. Human primary myotubes were challenged with either PA or LA (500 μM). After 24 h, the expression of interleukin 6 (IL-6) was assessed by quantitative polymerase chain reaction (PCR), whereas Western blot was used to quantify the abundance of the inhibitor of nuclear factor κB (IκBα), electron transport chain complex proteins and mitofusin-2 (MFN-2). Mitochondrial membrane potential and dynamics were evaluated using tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and immunocytochemistry, respectively. PA, contrarily to LA, triggered an inflammatory response marked by the upregulation of IL-6 mRNA (11-fold; P < 0.01) and a decrease in IκBα (32%; P < 0.05). Furthermore, whereas PA and LA did not differently modulate the levels of mitochondrial electron transport chain complex proteins, PA induced mitochondrial fragmentation (37%; P < 0.001), decreased MFN-2 (38%; P < 0.05), and caused a drop in mitochondrial membrane potential (11%; P < 0.01) compared to control, with this effect being absent in LA-treated cells. Thus, LA, as opposed to PA, did not trigger pathogenetic mechanisms proposed to be linked with insulin resistance and therefore represents a healthier saturated fatty acid choice to potentially preserve skeletal muscle metabolic health.

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Depolarized Mitochondrial Membrane Potential and Protection with Duroquinone in Isolated Perfused Lungs from Rats Exposed to Hyperoxia

Journal of Applied Physiology ◽

10.1152/japplphysiol.00565.2021 ◽

2021 ◽

Author(s):

Said H. Audi ◽

Swetha Ganesh ◽

Pardis Taheri ◽

Xiao Zhang ◽

Ranjan K. Dash ◽

...

Keyword(s):

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Complex I ◽

Mitochondrial Membrane ◽

Rhodamine 6G ◽

Computational Approach ◽

Experimental Protocol ◽

Perfusion System ◽

The Impact ◽

Mitochondrial Uncoupler

Dissipation of mitochondrial membrane potential (Δψm) is a hallmark of mitochondrial dysfunction. our objective was to use a previously developed experimental-computational approach to estimate tissue Δψm in intact lungs of rats exposed to hyperoxia, and to evaluate the ability of duroquinone (DQ) to reverse any hyperoxia-induced depolarization of lung Δψm. Rats were exposed to hyperoxia (>95% O2) or normoxia (room air) for 48 hrs, after which lungs were excised and connected to a ventilation-perfusion system. The experimental protocol consisted of measuring the concentration of the fluorescent dye rhodamine 6G (R6G) during three single-pass phases: loading, washing, and uncoupling, in which the lungs were perfused with and without R6G, and with the mitochondrial uncoupler FCCP, respectively. For normoxic lungs, the protocol was repeated with 1) rotenone (complex I inhibitor), 2) rotenone and either DQ or its vehicle (DMSO), and 3) rotenone, glutathione (GSH), and either DQ or DMSO added to the perfusate. Hyperoxic lungs were studied with and without DQ and GSH added to the perfusate. Computational modeling was used to estimate lung Δψm from R6G data. Rat exposure to hyperoxia resulted in partial depolarization (-33 mV) of lung Δψm, and complex I inhibition depolarized lung Δψm by -83 mV. Results also demonstrate the efficacy of DQ to fully reverse both rotenone-induced and hyperoxia-induced lung Δψm depolarization. This study demonstrates hyperoxia-induced Δψm depolarization in intact lungs, and the utility of this approach for assessing the impact of potential therapies such as exogenous quinones that target mitochondria in intact lungs.

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Abstract P352: Prostaglandin E2 Reduces Mitochondrial Function in Adult Mouse Cardiomyocytes

Hypertension ◽

10.1161/hyp.70.suppl_1.p352 ◽

2017 ◽

Vol 70 (suppl_1) ◽

Author(s):

Pamela Harding ◽

Timothy D Bryson ◽

Indrani Datta ◽

Yun Wang ◽

Albert Levin

Keyword(s):

Membrane Potential ◽

Ejection Fraction ◽

Prostaglandin E2 ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Function ◽

Complex I ◽

Mitochondrial Membrane ◽

Receptor Subtypes ◽

Atp Levels ◽

Complex I Activity

Hypertension is a leading cause of heart failure and both conditions are characterized by increased prostaglandin E2 (PGE2) which signals through 4 receptor subtypes (EP1-EP4) to elicit diverse physiologic effects. We previously reported that cardiomyocyte-specific deletion of the EP4 receptor results in a phenotype of dilated cardiomyopathy in male mice that is characterized by reduced ejection fraction. Subsequent gene array on left ventricles from these mice, coupled with Ingenuity Pathway Analysis (IPA) demonstrated that genes differentiating WT mice and EP4 KO mice with low ejection fraction were significantly overrepresented in mitochondrial (p=2.51x10 -28 ) and oxidative phosphorylation (p=3.16 x10 -30 ) pathways. We therefore hypothesized that PGE2 could reduce mitochondrial function. To test this hypothesis, we used isolated mouse cardiomyocytes (AVM) from 16-18 week old male C57Bl/6 mice and treated them with 1 μM PGE2 for various times. Mitochondrial gene expression was examined using a RT-profiler kit for mitochondrial energy metabolism, complex I activity with a spectrophotometric assay, ATP levels with a bioluminescence assay and mitochondrial membrane potential using JC-1 staining. Treatment of AVM with PGE2 for 4 hrs reduced expression of multiple genes from mitochondrial pathways including sub units of mitochondrial NADH dehydrogenase ubiquinone flavoprotein (Nduf), a component of complex I. In accord with the mRNA data, Complex I activity was reduced by 50% (p < 0.05) by 4 hr treatment with PGE2, from 1.32 ± 0.36 to 0.66 ± 0.08 mOD/min. Cytochrome c oxidase subunit 8 (Cox8c) mRNA was also reduced from a control value of 1.00 to -1.75 ± 0.20 (p < 0.005) after PGE2 treatment. Immuno-fluorescence showed that JC-1 aggregates were reduced after 1 or 3 hr treatment with either 1 μM PGE2 or the EP3 agonist, sulprostone, suggesting reduced mitochondrial membrane potential. Subsequent experiments also showed that ATP levels were reduced 16% from 11.18 ± 0.71 nmol to 9.39 ± 0.83 nmol after treatment with sulprostone for only 1 hr. Taken together, these results suggest that increased PGE2 in hypertension may contribute to impaired mitochondrial function and provide yet another link between inflammation and cardiac dysfunction.

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Olfactomedin 4 Is Essential for Superoxide Production and Sensitizes Oxidative Stress-Induced Apoptosis in Neutrophils.

Blood ◽

10.1182/blood.v114.22.1356.1356 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1356-1356

Author(s):

Wenli Liu ◽

Yueqin Liu ◽

Ruihong Wang ◽

Cuiling Li ◽

Chuxia Deng ◽

...

Keyword(s):

Oxidative Stress ◽

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Respiratory Chain ◽

Complex I ◽

Mitochondrial Membrane ◽

Mitochondrial Respiratory Chain ◽

H2o2 Production ◽

Induced Apoptosis ◽

Mitochondrial Respiratory

Abstract Abstract 1356 Poster Board I-378 Introduction Olfactomedin 4 (OLFM4), also called hGC-1, GW112 and pDP4, was first identified and specifically expressed in hematopoietic myeloid cells. OLFM4 expression in myeloid cells is regulated by transcription factors, PU1 and NF-κB. It has significant homology in its C-terminal domain with other olfactomedin-related proteins. OLFM4 encodes a 510 amino acid N-linked glycoprotein. The exact biological function of OLFM4, especially in neutrophils, is currently undefined. To characterize the in vivo function of OLFM4, we generated OLFM4 deficient mice (OLFM4-/-) and investigated its potential role in neutrophil functioins. Results 1) In this study, we showed that OLFM4 is a secreted glycoprotein and is also localized in the mitochondria, cytoplasm and cell membrane fractions of neutrophils. We demonstrated that OLFM4 interacts with GRIM-19 (Genes associated with Retinoid-IFN-induced Mortality-19), an apoptosis related protein, in the neutrophil mitochondria using co-immuoprecipitation assay. GRIM-19 is a subunit of complex I of mitochondrial respiratory chain and is essential for maintenance of mitochondrial membrane potential. Our result suggests that OLFM4 appears to be a novel component of complex I of mitochondrial respiratory chain and may be involved in regulation of mitochondrial membrane potential. 2) Mice heterozygous (OLFM4+/-) and homozygous (OLFM4-/-) for the null mutation in OLFM4 appeared to have normal development, fertility, and viability relative to wild-type (WT) mice. Whole blood analysis, differential leukocyte counts, blood chemistry and bone marrow smears were normal in OLFM4-/- mice, suggesting that OLFM4 is not essential for normal development and hematopoiesis in mice. 3) In response to LPS, fMLP and E.coli bacteria challenge, neutrophils from OLFM4-/- mice showed significantly reduced superoxide (O2−) and hydrogen peroxide (H2O2) production compared with WT mice. These results suggest that OLFM4 is an essential component to mediate O2− and H2O2 production in the neutrophil mitochondria under inflammation stimuli. 4) Exogenous H2O2 induced neutrophil apoptosis in a time and dose dependent manner in WT mice, but this induction of apoptosis was significantly reduced in OLFM4-/- mice. This result suggests that OLFM4 sensitizes and mediates H2O2-induced apoptosis in neutrophils. 5) Furthermore, we demonstrated that H2O2-stimulated mitochondrial membrane permeability reduction and caspase-3 and caspase-9 activation were inhibited in the neutrophils of OLFM4-/- mice. This result confirmed our hypothesis that OLFM4 may be involved in maintenance of mitochondrial membrane potential and suggests that OLFM4 may have opposite role as GRIM-19. 6) Moreover, Bax association with mitochondria and the cytoplasmic translocation of Omi/HtrA2 and Smac/DIABLO in response to H2O2 were inhibited in the neutrophils of OLFM4-/- mice. Conclusion Our results suggest: 1) OLFM4 has multiple subcellular localizations including mitochondria, cytoplasm, and cell membrane in neutrophils. The interaction of OLFM4 with GRIM-19 in the mitochondria suggests that OLFM4 is novel component of complex I of mitochondrial respiratory chain in the mitochondria of neutrophils, 2) OLFM4 is a novel mitochondrial molecule that is essential for O2− and H2O2 production in the neutrophils in the presence of inflammation stimuli, 3) Loss of OLFM4 in neutrophils does not trigger spontaneous apoptosis. However, OLFM4 sensitizes oxidative stress-induced apoptosis in mouse neutrophils. OLFM4 is involved in the regulation of mitochondria membrane potential and sensitizes cytoplasmic translocation of Omi/HtrA2 and Smac/DIABLO and caspases-3 and caspase-9 mediated apoptosis in the presence of oxidative stress. Disclosures No relevant conflicts of interest to declare.

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Time-dynamics of mitochondrial membrane potential reveal an inhibition of ATP synthesis in mitosis

10.1101/772244 ◽

2019 ◽

Cited By ~ 2

Author(s):

Joon Ho Kang ◽

Georgios Katsikis ◽

Max A. Stockslager ◽

Daniel Lim ◽

Michael B. Yaffe ◽

...

Keyword(s):

Cell Division ◽

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Atp Synthesis ◽

Electrical Circuit ◽

Time Dynamics ◽

Delay Cell ◽

Transport Chain ◽

A Cell

AbstractThe energetic demands of a cell are believed to increase during mitosis 1–7. As cells transit from G2 into mitosis, mitochondrial electron transport chain (ETC) activity increases 4,8,9, and cellular ATP levels progressively decrease until the metaphase-anaphase transition 3,7,10, consistent with elevated consumption. The rates of ATP synthesis during mitosis, however, have not been quantified. Here, we monitor mitochondrial membrane potential of single lymphocytes and demonstrate that cyclin-dependent kinase 1 (CDK1) activity causes mitochondrial hyperpolarization from G2/M until the metaphase-anaphase transition. By using an electrical circuit model of mitochondria, we quantify the time-dynamics of mitochondrial membrane potential under normal and perturbed conditions to extract mitochondrial ATP synthesis rates in mitosis. We found that mitochondrial ATP synthesis decreases by approximately 50 % during early mitosis, when CDK1 is active, and increases back to G2 levels during cytokinesis. Consistently, acute inhibition of mitochondrial ATP synthesis failed to delay cell division. Our results provide a quantitative understanding of mitochondrial bioenergetics in mitosis and challenge the traditional dogma that cell division is a highly energy demanding process.

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