Inhibition Of Mitochondrial Respiratory Chain Research Articles

Mitochondrial Esterase Activity Measured at the Single Organelle Level by Nano-flow Cytometry.

Monitoring mitochondrial esterase activity is crucial not only for investigating mitochondrial metabolism but also for assessing the effectiveness of mitochondrial-targeting prodrugs. However, accurately detecting esterase activity within mitochondria poses challenges due to its ubiquitous presence in cells and the uncontrolled localization of fluorogenic probes. To overcome this hurdle and reveal variations among different mitochondria, we isolated mitochondria and preserved their activity and functionality in a buffered environment. Subsequently, we utilized a laboratory-built nano-flow cytometer in conjunction with an esterase-responsive calcein-AM fluorescent probe to measure the esterase activity of individual mitochondria. This approach enabled us to investigate the influence of temperature, pH, metal ions, and various compounds on the mitochondrial esterase activity without any interference from other cellular constituents. Interestingly, we observed a decline in the mitochondrial esterase activity following the administration of mitochondrial respiratory chain inhibitors. Furthermore, we found that mitochondrial esterase activity was notably higher in the presence of a high concentration of ATP compared to that of ADP and AMP. Additionally, we noticed a correlation between elevated levels of complex IV and increased mitochondrial esterase activity. These findings suggest a functional connection between the mitochondrial respiratory chain and mitochondrial esterase activity. Moreover, we detected an upsurge in mitochondrial esterase activity during the early stages of apoptosis, while cellular esterase activity decreased. This highlights the significance of analyzing enzyme activity within specific organelle subregions. In summary, the integration of a nano-flow cytometer and fluorescent dyes introduces a novel method for quantifying mitochondrial enzyme activity with the potential to uncover the alterations and unique functions of other mitochondrial enzymes.

Inhibition of the mitochondrial respiratory chain reduces catecholamine‑stimulated lipolysis via increasing lactate production in 3T3‑L1 adipocytes.

Adipose tissue serves a significant role in the regulation of energy metabolism in the body. The re‑esterification of the fatty acids generated during lipolysis is critical for efficient lipolysis. However, the effect of the intracellular energy state on lipolytic activity and fatty acid re‑esterification during lipolysis is not yet fully understood. The present study aimed to assess the effect of the intracellular energy state on lipolytic activity and fatty acid re‑esterification during lipolysis. 3T3‑L1 adipocytes were incubated with mitochondrial respiratory chain inhibitors, oligomycin A or rotenone, during isoproterenol stimulation; and glycerol, glucose and lactate concentrations in the medium were measured. Western blot analysis was performed to examine the phosphorylation levels of cAMP‑dependent protein kinase A (PKA). The results showed that inhibition of mitochondrial ATP synthesis decreased catecholamine‑stimulated lipolysis without affecting PKA signaling. The inhibition of mitochondrial respiration increased glucose uptake and lactate production, indicating that a large amount of glucose taken up into the cell was preferentially used for ATP production rather than for re‑esterification. In conclusion, the results of the present study suggested that the energy state during lipolysis may influence lipolytic activity by suppressing fatty acid re‑esterification.

Commentary: Inhibitors of mitochondrial respiratory chain in the treatment of type 2 diabetes

Commentary: Inhibitors of mitochondrial respiratory chain in the treatment of type 2 diabetes

Ginkgo Biloba and Long COVID: In Vivo and In Vitro Models for the Evaluation of Nanotherapeutic Efficacy.

Coronavirus infections are neuroinvasive and can provoke injury to the central nervous system (CNS) and long-term illness consequences. They may be associated with inflammatory processes due to cellular oxidative stress and an imbalanced antioxidant system. The ability of phytochemicals with antioxidant and anti-inflammatory activities, such as Ginkgo biloba, to alleviate neurological complications and brain tissue damage has attracted strong ongoing interest in the neurotherapeutic management of long COVID. Ginkgo biloba leaf extract (EGb) contains several bioactive ingredients, e.g., bilobalide, quercetin, ginkgolides A-C, kaempferol, isorhamnetin, and luteolin. They have various pharmacological and medicinal effects, including memory and cognitive improvement. Ginkgo biloba, through its anti-apoptotic, antioxidant, and anti-inflammatory activities, impacts cognitive function and other illness conditions like those in long COVID. While preclinical research on the antioxidant therapies for neuroprotection has shown promising results, clinical translation remains slow due to several challenges (e.g., low drug bioavailability, limited half-life, instability, restricted delivery to target tissues, and poor antioxidant capacity). This review emphasizes the advantages of nanotherapies using nanoparticle drug delivery approaches to overcome these challenges. Various experimental techniques shed light on the molecular mechanisms underlying the oxidative stress response in the nervous system and help comprehend the pathophysiology of the neurological sequelae of SARS-CoV-2 infection. To develop novel therapeutic agents and drug delivery systems, several methods for mimicking oxidative stress conditions have been used (e.g., lipid peroxidation products, mitochondrial respiratory chain inhibitors, and models of ischemic brain damage). We hypothesize the beneficial effects of EGb in the neurotherapeutic management of long-term COVID-19 symptoms, evaluated using either in vitro cellular or in vivo animal models of oxidative stress.

β-lapachone-mediated WST1 Reduction as Indicator for the Cytosolic Redox Metabolism of Cultured Primary Astrocytes

Electron cycler-mediated extracellular reduction of the water-soluble tetrazolium salt 1 (WST1) is frequently used as tool for the determination of cell viability. We have adapted this method to monitor by determining the extracellular WST1 formazan accumulation the cellular redox metabolism of cultured primary astrocytes via the NAD(P)H-dependent reduction of the electron cycler β-lapachone by cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1). Cultured astrocytes that had been exposed to β-lapachone in concentrations of up to 3 µM remained viable and showed an almost linear extracellular accumulation of WST1 formazan for the first 60 min, while higher concentrations of β-lapachone caused oxidative stress and impaired cell metabolism. β-lapachone-mediated WST1 reduction was inhibited by the NQO1 inhibitors ES936 and dicoumarol in a concentration-dependent manner, with half-maximal inhibition observed at inhibitor concentrations of about 0.3 µM. β-lapachone-mediated WST1 reduction depended strongly on glucose availability, while mitochondrial substrates such as lactate, pyruvate or ketone bodies allowed only residual β-lapachone-mediated WST1 reduction. Accordingly, the mitochondrial respiratory chain inhibitors antimycin A and rotenone hardly affected astrocytic WST1 reduction. Both NADH and NADPH are known to supply electrons for reactions catalysed by cytosolic NQO1. Around 60% of the glucose-dependent β-lapachone-mediated WST1 reduction was prevented by the presence of the glucose-6-phosphate dehydrogenase inhibitor G6PDi-1, while the glyceraldehyde-3-phosphate dehydrogenase inhibitor iodoacetate had only little inhibitory potential. These data suggest that pentose phosphate pathway-generated NADPH, and not glycolysis-derived NADH, is the preferred electron source for cytosolic NQO1-catalysed reductions in cultured astrocytes.

Total Synthesis of Caldorazole, a Potent Mitochondrial Respiratory Chain Inhibitor without Chiral Centers.

Caldorazole (1) is a novel polyketide that was isolated from a marine cyanobacterium in 2022. It is a unique natural product that exhibits potent inhibitory activity against mitochondrial respiratory chain complex I despite having no chiral centers. To establish a method for obtaining caldorazole without relying on biological resources and for constructing a useful synthetic route for studies of its structure-activity relationship, we achieved the first total synthesis of caldorazole using a convergent synthetic route.

Small molecule-mediated rapid maturation of human induced pluripotent stem cell-derived cardiomyocytes

BackgroundHuman induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) do not display all hallmarks of mature primary cardiomyocytes, especially the ability to use fatty acids (FA) as an energy source, containing high mitochondrial mass, presenting binucleation and increased DNA content per nuclei (polyploidism), and synchronized electrical conduction. This immaturity represents a bottleneck to their application in (1) disease modelling—as most cardiac (genetic) diseases have a middle-age onset—and (2) clinically relevant models, where integration and functional coupling are key. So far, several methods have been reported to enhance iPSC-CM maturation; however, these protocols are laborious, costly, and not easily scalable. Therefore, we developed a simple, low-cost, and rapid protocol to promote cardiomyocyte maturation using two small molecule activators of the peroxisome proliferator-activated receptor β/δ and gamma coactivator 1-alpha (PPAR/PGC-1α) pathway: asiatic acid (AA) and GW501516 (GW).Methods and ResultsMonolayers of iPSC-CMs were incubated with AA or GW every other day for ten days resulting in increased expression of FA metabolism-related genes and markers for mitochondrial activity. AA-treated iPSC-CMs responsiveness to the mitochondrial respiratory chain inhibitors increased and exhibited higher flexibility in substrate utilization. Additionally, structural maturity improved after treatment as demonstrated by an increase in mRNA expression of sarcomeric-related genes and higher nuclear polyploidy in AA-treated samples. Furthermore, treatment led to increased ion channel gene expression and protein levels.ConclusionsCollectively, we developed a fast, easy, and economical method to induce iPSC-CMs maturation via PPAR/PGC-1α activation. Treatment with AA or GW led to increased metabolic, structural, functional, and electrophysiological maturation, evaluated using a multiparametric quality assessment.

Effects of chronic exposure of naturally weathered microplastics on oxidative stress level, behaviour, and mitochondrial function of adult zebrafish (Danio rerio)

Microplastics (MPs) are a big and growing environmental concern, with studies showing sublethal to acute biological impacts on typical aquatic organisms. However, little is known about the biological effects of naturally weathered MPs, particularly focusing on mitochondria dysfunction as the key trigger of the biological effects. Therefore, in this study, naturally weathered MPs were produced from day-to-day life products, characterized, and chronically exposed (21 days) to adult zebrafish at the concentration of 0.1 and 1 mg/L. Locomotion and unconditioned anxiety-like behaviour was assessed. Mitochondrial respiration, membrane potential, mitochondrial complex activity and oxidative-related parameters were evaluated in the brain and liver. The results revealed the weathered MPs as a copolymer of propylene and ethylene that induced anxiety-like behaviour. There was an increase in brain catalase activity while the brain lactate dehydrogenase activity was inhibited after exposure to 1 mg/L. Brain glutathione levels were increased while their ratio was not affected. Mitochondrial respiratory chain complex Ⅱ and IV were also significantly decreased in the brain, although not compromising mitochondrial function. On the other hand, exposure to 1 mg/L caused a deficiency in liver mitochondrial respiration and decreased mitochondrial membrane potential, which were associated with the mitochondrial respiratory chain inhibition. An increase in hepatic superoxide dismutase and catalase activity was noticed, supporting the occurrence of ROS-induced ROS release as the potential trigger for the mitochondrial dysfunction. Overall, these findings highlight the potential indirect and cumulative environmental effects these particles may pose to aquatic ecosystems.

P16-16 Mitochondrial respiratory chain inhibitor pesticides alter expression of dopa decarboxylase and growth differentiation factor 15 in SH-SY5Y neurons

P16-16 Mitochondrial respiratory chain inhibitor pesticides alter expression of dopa decarboxylase and growth differentiation factor 15 in SH-SY5Y neurons

Diphenyleneiodonium Triggers Cell Death of Acute Myeloid Leukemia Cells by Blocking the Mitochondrial Respiratory Chain, and Synergizes with Cytarabine.

Simple SummaryAcute myeloid leukemia (AML) is an aggressive heterogeneous cancer of the blood, of which 70% of cases develop relapse. Relapse is mainly due to chemoresistant leukemic cells (LCs) that are characterized by high mitochondrial oxidative phosphorylation (OxPhos) status, i.e., cells that are dependent on the mitochondrial respiratory chain (MRC) function. The aim of our study was to determine whether diphenyleneiodonium (DPI)—known as a potent inhibitor of flavoproteins—could be used to target AML cells. Herein, we demonstrate that DPI disrupts the mitochondrial function of AML cell lines. Interestingly, we found that cells with high OxPhos are more sensitive to the apoptotic effects of DPI. Moreover, we showed that DPI sensitizes AML cell lines to cytarabine (Ara-C) treatment, suggesting that MRC inhibitors could be employed to target LCs that are resistant to this chemotherapeutic agent.Acute myeloid leukemia (AML) is characterized by the accumulation of undifferentiated blast cells in the bone marrow and blood. In most cases of AML, relapse frequently occurs due to resistance to chemotherapy. Compelling research results indicate that drug resistance in cancer cells is highly dependent on the intracellular levels of reactive oxygen species (ROS). Modulating ROS levels is therefore a valuable strategy to overcome the chemotherapy resistance of leukemic cells. In this study, we evaluated the efficiency of diphenyleneiodonium (DPI)—a well-known inhibitor of ROS production—in targeting AML cells. Results showed that although inhibiting cytoplasmic ROS production, DPI also triggered an increase in the mitochondrial ROS levels, caused by the disruption of the mitochondrial respiratory chain. We also demonstrated that DPI blocks mitochondrial oxidative phosphorylation (OxPhos) in a dose-dependent manner, and that AML cells with high OxPhos status are highly sensitive to treatment with DPI, which synergizes with the chemotherapeutic agent cytarabine (Ara-C). Thus, our results suggest that targeting mitochondrial function with DPI might be exploited to target AML cells with high OxPhos status.

Mitochondrial respiratory chain dysfunction alters ER sterol sensing and mevalonate pathway activity

Mitochondrial dysfunction induces a strong adaptive retrograde signaling response; however, many of the downstream effectors of this response remain to be discovered. Here, we studied the shared transcriptional responses to three different mitochondrial respiratory chain inhibitors in human primary skin fibroblasts using QuantSeq 3′-RNA-sequencing. We found that genes involved in the mevalonate pathway were concurrently downregulated, irrespective of the respiratory chain complex affected. Targeted metabolomics demonstrated that impaired mitochondrial respiration at any of the three affected complexes also had functional consequences on the mevalonate pathway, reducing levels of cholesterol precursor metabolites. A deeper study of complex I inhibition showed a reduced activity of endoplasmic reticulum–bound sterol-sensing enzymes through impaired processing of the transcription factor Sterol Regulatory Element-Binding Protein 2 and accelerated degradation of the endoplasmic reticulum cholesterol-sensors squalene epoxidase and HMG-CoA reductase. These adaptations of mevalonate pathway activity affected neither total intracellular cholesterol levels nor the cellular free (nonesterified) cholesterol pool. Finally, measurement of intracellular cholesterol using the fluorescent cholesterol binding dye filipin revealed that complex I inhibition elevated cholesterol on intracellular compartments. Taken together, our study shows that mitochondrial respiratory chain dysfunction elevates intracellular free cholesterol levels and therefore attenuates the expression of mevalonate pathway enzymes, which lowers endogenous cholesterol biosynthesis, disrupting the metabolic output of the mevalonate pathway. We conclude that intracellular disturbances in cholesterol homeostasis may alter systemic cholesterol management in diseases associated with declining mitochondrial function.

Mitochondrial dysfunction triggers the pathogenesis of Parkinson's disease in neuronal C/EBPβ transgenic mice.

Respiratory chain complex I deficiency elicits mitochondrial dysfunction and reactive oxidative species (ROS), which plays a crucial role in Parkinson's disease (PD) pathogenesis. However, it remains unclear whether the impairment in other complexes in the mitochondrial oxidative phosphorylation chain is also sufficient to trigger PD onset. Here we show that inhibition of Complex II or III in the electron transport chain (ETC) induces the motor disorder and PD pathologies in neuronal Thy1-C/EBPβ transgenic mice. Through a cell-based screening of mitochondrial respiratory chain inhibitors, we identified TTFA (complex II inhibitor) and Atovaquone (complex III inhibitor), which robustly block the oxidative phosphorylation functions, strongly escalate ROS, and activate C/EBPβ/AEP pathway that triggers dopaminergic neuronal cell death. Oral administration of these inhibitors to Thy1-C/EBPβ mice elicits constipation and motor defects, associated with Lewy body-like inclusions. Deletion of SDHD (Succinate dehydrogenase) gene from the complex II in the Substantia Nigra of Thy1-C/EBPβ mice triggers ROS and PD pathologies, resulting in motor disorders. Hence, our findings demonstrate that mitochondrial ETC inactivation triggers PD pathogenesis via activating C/EBPβ/AEP pathway.

ING2 tumor suppressive protein translocates into mitochondria and is involved in cellular metabolism homeostasis.

ING2 (Inhibitor of Growth 2) is a tumor suppressor gene that has been implicated in critical biological functions (cell-cycle regulation, replicative senescence, DNA repair and DNA replication), most of which are recognized hallmarks of tumorigenesis occurring in the cell nucleus. As its close homolog ING1 has been recently observed in the mitochondrial compartment, we hypothesized that ING2 could also translocate into the mitochondria and be involved in new biological functions. In the present study, we demonstrate that ING2 is imported in the inner mitochondrial fraction in a redox-sensitive manner in human cells and that this mechanism is modulated by 14-3-3η protein expression. Remarkably, ING2 is necessary to maintain mitochondrial ultrastructure integrity without interfering with mitochondrial networks or polarization. We observed an interaction between ING2 and mtDNA under basal conditions. This interaction appears to be mediated by TFAM, a critical regulator of mtDNA integrity. The loss of mitochondrial ING2 does not impair mtDNA repair, replication or transcription but leads to a decrease in mitochondrial ROS production, suggesting a detrimental impact on OXPHOS activity. We finally show using multiple models that ING2 is involved in mitochondrial respiration and that its loss confers a protection against mitochondrial respiratory chain inhibition in vitro. Consequently, we propose a new tumor suppressor role for ING2 protein in the mitochondria as a metabolic shift gatekeeper during tumorigenesis.

Boosting mitochondria activity by silencing MCJ overcomes cholestasis-induced liver injury

Background & AimsMitochondria are the major organelles for the formation of reactive oxygen species (ROS) in the cell, and mitochondrial dysfunction has been described as a key factor in the pathogenesis of cholestatic liver disease. The methylation-controlled J-protein (MCJ) is a mitochondrial protein that interacts with and represses the function of complex I of the electron transport chain. The relevance of MCJ in the pathology of cholestasis has not yet been explored.MethodsWe studied the relationship between MCJ and cholestasis-induced liver injury in liver biopsies from patients with chronic cholestatic liver diseases, and in livers and primary hepatocytes obtained from WT and MCJ-KO mice. Bile duct ligation (BDL) was used as an animal model of cholestasis, and primary hepatocytes were treated with toxic doses of bile acids. We evaluated the effect of MCJ silencing for the treatment of cholestasis-induced liver injury.ResultsElevated levels of MCJ were detected in the liver tissue of patients with chronic cholestatic liver disease when compared with normal liver tissue. Likewise, in mouse models, the hepatic levels of MCJ were increased. After BDL, MCJ-KO animals showed significantly decreased inflammation and apoptosis. In an in vitro model of bile-acid induced toxicity, we observed that the loss of MCJ protected mouse primary hepatocytes from bile acid-induced mitochondrial ROS overproduction and ATP depletion, enabling higher cell viability. Finally, the in vivo inhibition of the MCJ expression, following BDL, showed reduced liver injury and a mitigation of the main cholestatic characteristics.ConclusionsWe demonstrated that MCJ is involved in the progression of cholestatic liver injury, and our results identified MCJ as a potential therapeutic target to mitigate the liver injury caused by cholestasis.Lay summaryIn this study, we examine the effect of mitochondrial respiratory chain inhibition by MCJ on bile acid-induced liver toxicity. The loss of MCJ protects hepatocytes against apoptosis, mitochondrial ROS overproduction, and ATP depletion as a result of bile acid toxicity. Our results identify MCJ as a potential therapeutic target to mitigate liver injury in cholestatic liver diseases.

Neurotoxicity and underlying cellular changes of 21 mitochondrial respiratory chain inhibitors

Inhibition of complex I of the mitochondrial respiratory chain (cI) by rotenone and methyl-phenylpyridinium (MPP +) leads to the degeneration of dopaminergic neurons in man and rodents. To formally describe this mechanism of toxicity, an adverse outcome pathway (AOP:3) has been developed that implies that any inhibitor of cI, or possibly of other parts of the respiratory chain, would have the potential to trigger parkinsonian motor deficits. We used here 21 pesticides, all of which are described in the literature as mitochondrial inhibitors, to study the general applicability of AOP:3 or of in vitro assays that are assessing its activation. Five cI, three complex II (cII), and five complex III (cIII) inhibitors were characterized in detail in human dopaminergic neuronal cell cultures. The NeuriTox assay, examining neurite damage in LUHMES cells, was used as in vitro proxy of the adverse outcome (AO), i.e., of dopaminergic neurodegeneration. This test provided data on whether test compounds were unspecific cytotoxicants or specifically neurotoxic, and it yielded potency data with respect to neurite degeneration. The pesticide panel was also examined in assays for the sequential key events (KE) leading to the AO, i.e., mitochondrial respiratory chain inhibition, mitochondrial dysfunction, and disturbed proteostasis. Data from KE assays were compared to the NeuriTox data (AO). The cII-inhibitory pesticides tested here did not appear to trigger the AOP:3 at all. Some of the cI/cIII inhibitors showed a consistent AOP activation response in all assays, while others did not. In general, there was a clear hierarchy of assay sensitivity: changes of gene expression (biomarker of neuronal stress) correlated well with NeuriTox data; mitochondrial failure (measured both by a mitochondrial membrane potential-sensitive dye and a respirometric assay) was about 10–260 times more sensitive than neurite damage (AO); cI/cIII activity was sometimes affected at > 1000 times lower concentrations than the neurites. These data suggest that the use of AOP:3 for hazard assessment has a number of caveats: (i) specific parkinsonian neurodegeneration cannot be easily predicted from assays of mitochondrial dysfunction; (ii) deriving a point-of-departure for risk assessment from early KE assays may overestimate toxicant potency.

A Fast, Simple, and Affordable Technique to Measure Oxygen Consumption in Living Zebrafish Embryos.

In all animal species, oxygen consumption is a key process that is partially impaired in a large number of pathological situations and thus provides informative details on the physiopathology of the disease. In this study, we describe a simple and affordable method to precisely measure oxygen consumption in living zebrafish larvae using a spectrofluorometer and the MitoXpress Xtra Oxygen Consumption Assay. In addition, we used zebrafish larvae treated with mitochondrial respiratory chain inhibitors, antimycin A or rotenone, to verify that our method enables precise and reliable measurements of oxygen consumption.

Unmasking the Amphotericin B Resistance Mechanisms in Candida haemulonii Species Complex

The polyene amphotericin B (AMB) exerts a powerful and broad antifungal activity. AMB acts by (i) binding to ergosterol, leading to pore formation at the fungal plasma membrane with subsequent ion leakage, and (ii) inducing the intracellular accumulation of reactive oxygen species (ROS). Herein, we have deciphered the AMB resistance mechanisms in clinical isolates of Candida haemulonii complex (C. haemulonii, C. duobushaemulonii, C. haemulonii var. vulnera) in comparison to other clinically relevant non-albicans Candida species. Membrane gas chromatography-mass spectrometry analysis revealed that the vast majority of sterols were composed of ergosterol pathway intermediates, evidencing the absence of AMB target. Supporting this data, C. haemulonii species complex demonstrated poor membrane permeability after AMB treatment. Regarding the oxidative burst, AMB induced the formation of ROS in all species tested; however, this phenomenon was slightly seen in C. haemulonii complex isolates. Our results indicated that these isolates displayed altered respiratory status, as revealed by their poor growth in nonfermented carbon sources, low consumption of oxygen, and derisive mitochondrial membrane potential. The use of specific inhibitors of mitochondrial respiratory chain (complex I-IV) revealed no effects on the yeast growth, highlighting the metabolic shift to fermentative pathway in C. haemulonii strains. Also, C. haemulonii complex proved to be highly resistant to oxidative burst agents, which can be correlated with a high activity of antioxidant enzymes. Our data demonstrated primary evidence suggesting that ergosterol content, mitochondrial function, and fungal redox homeostasis are involved in AMB fungicidal effects and might explain the resistance presented in this multidrug-resistant, emergent, and opportunistic fungal complex.

Optimizing oxygen delivery in sepsis: A review

Multiple organ failure syndrome (MOFS) is a hallmark of sepsis. The continued dysfunction of microvascular perfusion has been implicated as the inciting factor for this. Multiple etiopathological factors are involved in producing this disequilibrium in the demand, supply, and extraction of oxygen as a result of derecruitment of microcirculation. This is further complicated by mitochondrial dysfunction in the form of the inhibition of mitochondrial respiratory chain of enzymes, leading to difficulty in extraction of the oxygen at the cellular level. Eventually, although hemodynamic stability of systemic/macrovascular circulation may have been achieved, the process of deficient and defective delivery of oxygen to the tissues goes on relentlessly. The indicators and monitoring of this process of impairment of oxygen delivery (DO2) have been discussed in this review. In addition, the review also encompasses various therapeutic modalities and their efficacy, based on the evidence. The latest guidelines regarding optimizing the DO2 in sepsis are also included here.

Loss of ZBTB7A Enhances Glycolysis and Beta Oxidation in Myeloid Leukemia

ZBTB7A is a transcription factor with function in hematopoietic lineage fate decisions (reviewed in Lunardi et al., 2013, Blood). Moreover, ZBTB7A has been shown to also be involved in regulation of glycolysis in solid tumors (Liu et al., 2014, Genes Dev.). Recently, we found ZBTB7A frequently mutated in acute myeloid leukemia (AML) with t(8;21) translocation. What is more, high expression of ZBTB7A correlated with better clinical outcome in cytogenetically normal AML (Hartmann et al., 2016, Nat Commun). The functional role of ZBTB7A and its alterations in myeloid malignancies, however, remains unclear, especially concerning the regulation of leukemia metabolism. To investigate the effect of ZBTB7A mutations in leukemia, we generated K562 ZBTB7A knockout (KO) cells using CRISPR/Cas9, followed by RNA-Seq in KO and control cells. Thereby, we confirmed de-repression of the previously reported ZBTB7A target gene SLC2A3 (glucose transporter 3) as well as upregulation of several other glycolysis related genes (PGM2, PGM3, SLC2A1 and ENO2). ZBTB7A binding to all of these candidate target genes was validated using publicly available ChIP-Seq data from K562 cells (ENCSR000BME). Interestingly, Gene Set Enrichment Analysis revealed deregulation of distinct pathways related to fatty acid metabolism in this model (Figure 1A). KO cells overexpress genes involved in the fatty acid beta oxidation pathway (ACAA2, ACOX1, ACSL1, ACADVL, CPT1A and CPT1B) as well as genes related to other fatty acid metabolism (EPHX2, FADS2 and others) (Figure 1B). We could further validate ACAA2, ACOX1, ACADVL and CPT1A as direct ZBTB7A targets using ChIP-Seq data. Of special interest are CPT1A and CPT1B, which are targetable through Etomoxir treatment. KO cells showed an increased sensitivity to this drug compared to control (IC50= 120.6 and 125.5 µM in KOs vs 228.4 µM in control, p<0.0001). Moreover, analysis of RNA-Seq data from patients with AML t(8;21) revealed a significantly higher expression of EPHX2 (p=0.049), FADS2 (p=0.003) and FASD1 (p=0.021) in patients harboring ZBTB7A mutations (Figure 1C) using a two-tailed unpaired Student's t-test. In order to evaluate our findings on a functional level, we performed metabolic flux assays in K562 ZBTB7A KO vs control cells. Using Seahorse technology (Agilent), we found that KO cells show a modest increase in extracellular acidification rate (ECAR), indicating a higher glycolysis. This effect becomes more obvious after mitochondrial respiratory chain inhibition: 41.20 and 51.66 mpH/min in KO clones vs 34.33 mpH/min in control (p=0.002 and p<0.001, respectively) (Figure 1D). This result suggests that loss of ZBTB7A may confer an advantage to cells in specific microenvironment with low oxygen availability, such as the bone marrow. Moreover, metabolic flux assays also revealed a nearly 50% increase in oxygen consumption rate (OCR) in KO cells after 1h glucose starvation: 140.53 and 148.53 pmol/min in KO clones vs 96.46 pmol/min in control (p<0.001 in both comparisons) (Figure 1E). Since the cells were deprived from glucose, the observed oxygen consumption may arise mainly from glutamate metabolism or fatty acid oxidation. Deprivation from glutamate reduced overall OCR but KO cells still showed increased oxygen consumption compared to control. These results therefore suggest that an increased beta oxidation of fatty acids leads to the higher OCR observed in KO cells. In summary, we have demonstrated that the previously described role of ZBTB7A as a regulator of glycolysis in solid tumors is also relevant in myeloid malignancies. In addition, we identified the beta oxidative pathway and fatty acid synthesis as novel mechanisms underlying the perturbed function of ZBTB7A in tumor metabolism. ZBTB7A downregulation or mutation may lead to an increased energy production providing an advantage to leukemia cells. These findings likely have therapeutic implications, as metabolic inhibitors such as 2-deoxy-d-glucose and Etomoxir may specifically target ZBTB7A deficient malignancies. Figure Disclosures Hiddemann: Celgene: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria; Bayer: Research Funding; Vector Therapeutics: Consultancy, Honoraria.

Induction of mitochondrial dysfunction in patients under cardiopulmonary by-pass: preliminary results.

Cardiac surgery is often performed by cardiopulmonary by-pass (CPB), generally associated with organ dysfunction. The aim of this work was to determine if and how this phenomenon is related to mitochondrial damage. To this purpose, the effect of the addition of serum from CPB patients to human fibroblasts cultures on mitochondrial respiratory chain and oxidative phosphorylation (OXPHOS) activities was investigated. Serum samples of five patients were obtained before (pre-CPB) and after 6 h CPB weaning (CPB). Mitochondrial OXPHOS activities were examined by polarographic and spectrophotometric assays, and reactive oxygen species (ROS) production was measured by a spectrofluorimeter. Addition of CPB serum to fibroblasts determined a decrease of mitochondrial oxygen consumption due to an inhibition of mitochondrial respiratory chain and some OXPHOS enzymes activities. This inhibition seems to be mainly related to a reduced activity of complex I. Our data represent the first translational research evidence showing that CPB determines mitochondrial dysfunction which leads to impairment of OXPHOX activities and to an increase in ROS production, compromising tissue bioenergetic efficiency.