Maximal Mitochondrial Respiratory Capacity Research Articles

Aerobic capacity and mitochondrial function in bipolar disorder: a longitudinal study during acute phases and after clinical remission.

Aerobic capacity has shown to predict physical and mental health-related quality of life in bipolar disorder (BD). However, the correlation between exercise respiratory capacity and mitochondrial function remains understudied. We aimed to assess longitudinally intra-individual differences in these factors during mood episodes and remission in BD. This study included eight BD patients admitted to an acute psychiatric unit. Incremental cardiopulmonary exercise test (CPET) was conducted during acute episodes (T0), followed by constant work rate cycle ergometry (CWRCE) to evaluate endurance time, oxygen uptake at peak exercise (VO2peak) and at the anaerobic threshold. The second test was repeated during remission (T1). Mitochondrial respiration rates were assessed at T0 and T1 in peripheral blood mononuclear cells. Endurance time, VO2peak, and anaerobic threshold oxygen consumption showed no significant variations between T0 and T1. Basal oxygen consumption at T1 tended to inversely correlate with maximal mitochondrial respiratory capacity (r=-0.690, p=0.058), and VO2peak during exercise at T1 inversely correlated with basal and minimum mitochondrial respiration (r=-0.810, p=0.015; r=-0.786, p=0.021, respectively). Our preliminary data showed that lower basal oxygen consumption may be linked to greater mitochondrial respiratory capacity, and maximum oxygen uptake during the exercise task was associated with lower basal mitochondrial respiration, suggesting that lower oxygen requirements could be associated with greater mitochondrial capacity. These findings should be replicated in larger samples stratified for manic and depressive states.

Insights into the mechanism of action of the chlorophyll derivative 13-2-hydroxypheophytine a on reducing neutral lipid reserves in zebrafish larvae and mice adipocytes

Obesity is a worldwide epidemic and natural products may hold promise in its treatment. The chlorophyll derivative 13-2-hydroxypheophytine (hpa) was isolated in a screen with zebrafish larvae to identify lipid reducing molecules from cyanobacteria. However, the mechanisms underlying the lipid-reducing effects of hpa in zebrafish larvae remain poorly understood. Thus, investigating the mechanism of action of hpa and validation in other model organisms such as mice represents important initial steps.In this study, we identified 14 protein targets of hpa in zebrafish larvae by thermal proteome profiling, and selected two targets (malate dehydrogenase and pyruvate kinase) involved in cellular metabolism for further validation by enzymatic measurements. Our findings revealed a dose-dependent inhibition of pyruvate kinase by hpa exposure using protein extracts of zebrafish larvae in vitro, and in exposure experiments from 3 to 5 days post fertilization in vivo. Analysis of untargeted metabolomics of zebrafish larvae detected 940 mass peaks (66 increased, 129 decreased) and revealed that hpa induced the formation of various phospholipid species (phosphoinositol, phosphoethanolamine, phosphatidic acid). Inter-species validation showed that brown adipocytes exposed to hpa significantly reduced the size of lipid droplets, increased maximal mitochondrial respiratory capacity, and the expression of PPARy during adipocyte differentiation.In line with our data, previous work described that reduced pyruvate kinase activity lowered hepatic lipid content via reduced pyruvate and citrate, and improved mitochondrial function via phospholipids. Thus, our data provide new insights into the molecular mechanism underlying the lipid reducing activities of hpa in zebrafish larvae, and species overlapping functions in reduction of lipids.

The antioxidant l-Ergothioneine prevents cystine lithiasis in the Slc7a9−/− mouse model of cystinuria

The high recurrence rate of cystine lithiasis observed in cystinuria patients highlights the need for new therapeutic options to address this chronic disease. There is growing evidence of an antioxidant defect in cystinuria, which has led to test antioxidant molecules as new therapeutic approaches. In this study, the antioxidant l-Ergothioneine was evaluated, at two different doses, as a preventive and long-term treatment for cystinuria in the Slc7a9−/− mouse model. l-Ergothioneine treatments decreased the rate of stone formation by more than 60% and delayed its onset in those mice that still developed calculi. Although there were no differences in metabolic parameters or urinary cystine concentration between control and treated mice, cystine solubility was increased by 50% in the urines of treated mice. We also demonstrate that l-Ergothioneine needs to be internalized by its transporter OCTN1 (Slc22a4) to be effective, as when administrated to the double mutant Slc7a9−/−Slc22a4−/− mouse model, no effect on the lithiasis phenotype was observed. In kidneys, we detected a decrease in GSH levels and an impairment of maximal mitochondrial respiratory capacity in cystinuric mice that l-Ergothioneine treatment was able to restore. Thus, l-Ergothioneine administration prevented cystine lithiasis in the Slc7a9−/− mouse model by increasing urinary cystine solubility and recovered renal GSH metabolism and mitochondrial function. These results support the need for clinical trials to test l-Ergothioneine as a new treatment for cystinuria.

Gitelman‐like syndrome caused by pathogenic variants in mitochondrial DNA

Gitelman syndrome is the most frequent hereditary salt‐losing tubulopathy characterized by hypokalemic alkalosis and hypomagnesemia. Gitelman syndrome is caused by biallelic pathogenic variants in SLC12A3, encoding the Na+‐Cl‐ cotransporter (NCC) expressed in the distal convoluted tubule. Pathogenic variants in CLCNKB, HNF1B, FXYD2 or KCNJ10 may result in renal phenocopies of Gitelman syndrome, as they can lead to reduced NCC activity. Nevertheless, ±10% of patients with a Gitelman syndrome phenotype remain genetically unsolved.After identification of mitochondrial DNA (mtDNA) variants in three families with Gitelman syndrome‐like electrolyte abnormalities, 156 families were investigated for variants in MT‐TI and MT‐TF, encoding the transfer RNAs for phenylalanine and isoleucine. Mitochondrial respiratory chain function was assessed in patient fibroblasts. In NCC‐expressing HEK293 cells, mitochondrial dysfunction was induced to assess the effect on thiazide‐sensitive 22Na+ transport.Genetic investigations revealed four mtDNA variants in 13 families: m.591C>T (n=7), m.616T>C (n=1), m.643A>G (n=1) (all in MT‐TF) and m.4291T>C (n=4, in MT‐TI). Variants were near homoplasmic in affected individuals. Importantly, affected members of six families with an MT‐TF variant additionally suffered from progressive chronic kidney disease. Maximal mitochondrial respiratory capacity was reduced in patient fibroblasts, caused by dysfunction of oxidative phosphorylation complex IV. In vitro pharmacological inhibition of complex IV, mimicking the effect of the mtDNA variants, demonstrated an inhibitory effect on NCC phosphorylation and NCC‐mediated sodium uptake.Pathogenic mtDNA variants in MT‐TF and MT‐TI can cause a Gitelman syndrome‐like syndrome. Genetic investigation of mtDNA should be considered in patients with unexplained Gitelman syndrome‐like tubulopathies.

Fatty acid oxidation enzyme Δ3, Δ2-enoyl-CoA isomerase 1 (ECI1) drives aggressive tumor phenotype and predicts poor clinical outcome in prostate cancer patients.

Prostate cancer (PCa) metastases are highly enriched with genomic alterations including a gain at the 16p13.3 locus, recently shown to be associated with disease progression and poor clinical outcome. ECI1, residing at the 16p13.3 gain region, encodes Δ3, Δ2-Enoyl-CoA Delta Isomerase 1 (ECI1), a key mitochondrial fatty acid β-oxidation enzyme. Although deregulated mitochondrial fatty acid β-oxidation is known to drive PCa pathogenesis, the role of ECI1 in PCa is still unknown. We investigated the impacts of ECI1 on PCa phenotype in vitro and in vivo by modulating its expression in cell lines and assessed the clinical implications of its expression in human prostate tissue samples. In vitro, ECI1 overexpression increased PCa cell growth while ECI1 deficiency reduced its growth. ECI1 also enhanced colony formation, cell motility, and maximal mitochondrial respiratory capacity. In vivo, PCa cells stably overexpressing ECI1 injected orthotopically in nude mice formed larger prostate tumors with higher number of metastases. Immunohistochemistry analysis of the human tissue microarray representing 332 radical prostatectomy cases revealed a stronger ECI1 staining in prostate tumors compared to corresponding benign tissues. ECI1 expression varied amongst tumors and was higher in cases with 16p13.3 gain, high Gleason grade, and advanced tumor stage. ECI1 overexpression was a strong independent predictor of biochemical recurrence after adjusting for known clinicopathologic parameters (hazard ratio: 3.65, P < 0.001) or the established CAPRA-S score (hazard ratio: 3.95, P < 0.001). ECI1 overexpression was also associated with significant increased risk of distant metastasis and reduced overall survival. Overall, this study demonstrates the functional capacity of ECI1 in PCa progression and highlights the clinical implication of ECI1 as a potential target for the management of PCa.

Mitochondrial Dysfunction and Alloimmunity in Accelerated Bronchiolitis Obliterans After Lung Transplantation

<h3>Purpose</h3> Bronchiolitis obliterans (BOS) remains a major cause of death after lung transplantation limiting the life of our lung transplant patients. Unfortunately, the mechanisms by which BOS develops remain poorly understood. We previously observed that human lung recipient carriers of a mutation in ATG16L1 (rs2241880), an autophagy related protein, developed earlier BOS relative to non-carriers. Prior work has shown that this mutation leads to protein instability resulting in deficiency of ATG16L1 in monocyte-derived (Mo-) macrophages. Although it is known that genetically encoded deficiencies in mitophagy, a specialized form of autophagy that targets the removal of damaged mitochondria promote clinical disease, it is not known if they play a role in BOS. To study this, we analyzed the effects of ATG16L1 deficiency in Mo-CD11c⁺ cells in a mouse orthotopic lung transplant model of BOS. <h3>Methods</h3> CD11cCreATG16L1^flox/flox (ATG16L1^Δ/Δ) and control CD11cCre recipients received major MHC-mismatched FVB lungs, with immunosuppression to induce acceptance. Following induced epithelial injury, allografts were assessed for obliterative airway lesions (OB) for up to 28d. Intragraft Mo-CD11c⁺ cells were analyzed for bulk RNA sequencing and by FACS for mitochondrial mass and ROS production. Mitophagic flux was visualized by confocal microscopy using Mt-kiema mitophagy reporter mice. Mo-CD11c⁺ cells were characterized metabolically using a Seahorse analyzer. Alloimmunity was examined via T cell proliferation assays and ELISA for IL1-β. <h3>Results</h3> Indeed, our mouse ATG16L1^Δ/Δ recipients also showed accelerated OB compared to controls. ATG16L1^Δ/Δ Mo-CD11c⁺ cells showed higher mitochondrial mass, elevated ROS production as well as loss of transcripts encoding subunits of mitochondria electron complexes. Confocal analysis revealed attenuated mitophagic flux. In line with these, were decreased maximal mitochondrial respiratory capacity, lower mitochondrial ATP production and increased utilization of glycolysis, as well as increased T cell proliferation and higher IL1-β suggesting metabolic adaptation of an M1 inflammatory phenotype. <h3>Conclusion</h3> These data reveal a new role for ATG16L1 as a regulator of mitochondrial quality control and alloimmunity with implication for lung transplant recipients that carry the ATG16L1 rs2241880 mutation.

Gitelman-Like Syndrome Caused by Pathogenic Variants in mtDNA.

Gitelman syndrome is the most frequent hereditary salt-losing tubulopathy characterized by hypokalemic alkalosis and hypomagnesemia. Gitelman syndrome is caused by biallelic pathogenic variants in SLC12A3, encoding the Na+-Cl- cotransporter (NCC) expressed in the distal convoluted tubule. Pathogenic variants of CLCNKB, HNF1B, FXYD2, or KCNJ10 may result in the same renal phenotype of Gitelman syndrome, as they can lead to reduced NCC activity. For approximately 10 percent of patients with a Gitelman syndrome phenotype, the genotype is unknown. We identified mitochondrial DNA (mtDNA) variants in three families with Gitelman-like electrolyte abnormalities, then investigated 156 families for variants in MT-TI and MT-TF, which encode the transfer RNAs for phenylalanine and isoleucine. Mitochondrial respiratory chain function was assessed in patient fibroblasts. Mitochondrial dysfunction was induced in NCC-expressing HEK293 cells to assess the effect on thiazide-sensitive 22Na+ transport. Genetic investigations revealed four mtDNA variants in 13 families: m.591C>T (n=7), m.616T>C (n=1), m.643A>G (n=1) (all in MT-TF), and m.4291T>C (n=4, in MT-TI). Variants were near homoplasmic in affected individuals. All variants were classified as pathogenic, except for m.643A>G, which was classified as a variant of uncertain significance. Importantly, affected members of six families with an MT-TF variant additionally suffered from progressive chronic kidney disease. Dysfunction of oxidative phosphorylation complex IV and reduced maximal mitochondrial respiratory capacity were found in patient fibroblasts. In vitro pharmacological inhibition of complex IV, mimicking the effect of the mtDNA variants, inhibited NCC phosphorylation and NCC-mediated sodium uptake. Pathogenic mtDNA variants in MT-TF and MT-TI can cause a Gitelman-like syndrome. Genetic investigation of mtDNA should be considered in patients with unexplained Gitelman syndrome-like tubulopathies.

Transcriptional and Metabolic Profiling of Nicotinamide-Enhanced Natural Killer (NAM-NK) Cells (GDA-201)

Adoptive transfer of NK cells is a promising immunotherapeutic modality, however limited NK cell persistence and proliferation in vivo have historically been barriers to clinical success. Nicotinamide (NAM), an allosteric inhibitor of NAD-dependent enzymes, has been shown to preserve cell function and prevent differentiation in ex vivo culture of NK (NAM-NK) and other cells. Clinical responses were observed in a Phase 1 trial of NAM-NK (GDA-201) in patients with refractory non-Hodgkin lymphoma (Bachanova, et. al., Blood 134:777, 2019). We now use transcriptional and metabolic profiling to characterize the mechanisms underlying the activity of NAM-NK.CD3 negative lymphocytes obtained from healthy donors were cultured for 14 days with IL-15 in the presence or absence of NAM (7 mM). Next generation sequencing (NGS), liquid chromatography-mass spectrometry (LC-MS)-based metabolite quantification, and glycolytic/mitochondrial respiration measurements were performed. Transcriptome and pathway enrichment analyses were performed with Ingenuity Pathway Analysis software. Extracted cellular and medium metabolites were analyzed on a Thermo Q-Exactive Plus mass spectrometer coupled with a Vanquish UHPLC system. Extracellular acidification (ECAR) and oxygen consumption rates (OCR) were quantified using a Seahorse Extracellular Flux Analyzer. Glycolysis/citric acid cycle (TCA) rates were measured using isotope-labelled glucose incorporation assays.Transcriptome analyses defined 1,204 differentially expressed (DE) genes in NAM-NK vs. control NK. Biological/functional enrichment and pathway analyses of DE-genes predicted upregulation of cell cycle, DNA replication (CDK4/CDKN2D, CyclinD/E, MAD2L), RNA transcription, translation (SMN1/2, ABCF1, EIF4B, RPL13, RPS6), protein synthesis (EIF2, PABPC1, SOS, 60S complex) mitochondrial energy metabolism (NDUFB8, ATP5G2/E, COX7B/C) migration, homing (CD62L, CD44, DNAM1), and cytokine/chemokine response (IL18R, CXCR3, CCR5, XCL1, SOCS3, LFA1) pathways, with concomitant downregulation of cell exhaustion, senescence (BATF1, FOXP1, STAT1, CD86, LGALS9, LAG3), apoptosis, necrosis (CASP1, MDM2, IKK3), stress response (CALR, HSP90, HSPH1), and lymphoid cellular maturation (IL-2Ra, CD40L, GATA3) pathways in NAM-NK. Metabolomic analyses showed a significant increase of intracellular NAD, NADH, NADP, NADPH, high-energy triphosphates (ATP, UTP, GTP) and overall energy charge ([ATP+0.5*ADP]/[ATP+ADP+AMP]) in NAM-NK. Cellular metabolic fitness analyses revealed increased basal and ATP-linked respiration, mitochondrial maximal respiratory capacity, and glycolytic capacity in NAM-NK compared to control NK. In addition, NAM increased the rate of glucose incorporation into TCA cycle intermediates (acetyl-CoA, succinyl-CoA), consistent with a more rapid glycolysis rate, increased TCA cycling, and improved glucose consumption efficiency.Taken together, results of transcriptome, metabolomic, mitochondrial respiration, and glycolytic rate analyses suggest that NAM pleiotropically modulates key cellular metabolic functions in ex vivo-expanded NK cells, resulting in increased response to cytokine stimulation and enhanced potency. NAM inhibits differentiation, cellular stress, and exhaustion pathways that are typically activated in culture. Moreover, NAM increases cellular metabolic fitness, energy charge, and efficiency of glucose consumption, potentially imparting a protective effect against oxidative stress in the tumor microenvironment. These data offer insight into the mechanism of improved persistence, proliferation, and cytotoxicity observed in in vivo and clinical studies of GDA-201. DisclosuresYackoubov: Gamida Cell: Current Employment. Pato: Gamida Cell: Current Employment. Rifman: Gamida Cell: Current Employment. Cohen: Gamida Cell: Current Employment. Hailu: Gamida Cell: Current Employment. Persi: Gamida Cell: Current Employment. Berhani-Zipori: Gamida Cell: Current Employment. Edri: Gamida Cell: Current Employment. Peled: Biokine Therapeutics Ltd: Current Employment; Gamida Cell: Research Funding. Cichocki: Gamida Cell: Research Funding; Fate Therapeutics, Inc: Patents & Royalties, Research Funding. Rabinowitz: Gamida Cell: Research Funding. Lodie: Gamida Cell: Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Adams: Gamida Cell: Current Employment. Simantov: Gamida Cell: Current Employment. Geffen: Gamida Cell: Current Employment.

Accelerated Bronchiolitis Obliterans Development after Lung Transplant Promoted by the ATG16l1 rs2241880 Mutation is Coupled to Mitochondrial Damage and Metabolic Alterations in Monocyte-Derived Alveolar Macrophages

Bronchiolitis obliterans (BOS) remains a major cause of death for lung transplant recipients, and mechanisms that drive BOS remain poorly understood. Genetically encoded deficiencies in mitophagy, a specialized autophagy that targets the removal of damaged mitochondria, have been shown to promote Parkinson's disease, but it is unclear if they play a role in other chronic diseases. Recent work has shown that the rs2241880 mutation in the autophagy regulator ATG16L1 leads to protein instability resulting in deficiency of ATG16L1 in monocyte-derived macrophages. We previously observed that human lung recipients that are carriers of rs2241880 had faster BOS development. Here, we analyzed the effects of ATG16L1 deficiency in monocyte-derived alveolar macrophages (Mo-AM) in a mouse orthotopic lung transplant model of BOS. CD11cCre ATG16L1flox/flox (ATG16L1Δ/Δ) and control CD11cCre recipients received major MHC-mismatched FVB lungs and were immunosuppressed to induce acceptance. Following induced epithelial injury, allografts were assessed for obliterative airway lesions (OB) for up to 28 days. Intragraft Mo-AM were analyzed for bulk RNA sequencing and by flow cytometry (FACS) for mitochondrial mass and ROS production. Mitophagic flux was visualized by confocal microscopy using Mt-kiema mitophagy reporter mice. Mo-AM were characterized metabolically using a Seahorse XF analyzer. When compared to controls, CD11cCre ATG16L1flox/flox recipients showed severe and accelerated OB development. RNAseq analysis of ATG16L1Δ/Δ Mo-AM demonstrated a loss of transcripts that encode several subunits of mitochondria electron complex I, II and V. Confocal and FACS analysis revealed attenuated mitophagic flux, high mitochondrial mass and elevated ROS production in ATG16L1Δ/Δ Mo-AM. In line with these observations were decreased maximal mitochondrial respiratory capacity, lower mitochondrial ATP production and increased utilization of glycolysis- consistent with the metabolic adaptation of an M1 inflammatory phenotype. Collectively, these data reveal a new role for ATG16L1 as a regulator of mitochondrial quality control with implications for lung recipients that carry the ATG16L1 rs2241880 mutation.

Powerhouses in the cold: mitochondrial function during thermal acclimation in montane mayflies.

Mitochondria provide the vast majority of cellular energy available to eukaryotes. Therefore, adjustments in mitochondrial function through genetic changes in mitochondrial or nuclear-encoded genes might underlie environmental adaptation. Environmentally induced plasticity in mitochondrial function is also common, especially in response to thermal acclimation in aquatic systems. Here, we examined mitochondrial function in mayfly larvae (Baetis and Drunella spp.) from high and low elevation mountain streams during thermal acclimation to ecologically relevant temperatures. A multi-substrate titration protocol was used to evaluate different respiratory states in isolated mitochondria, along with cytochrome oxidase and citrate synthase activities. In general, maximal mitochondrial respiratory capacity and oxidative phosphorylation coupling efficiency decreased during acclimation to higher temperatures, suggesting montane insects may be especially vulnerable to rapid climate change. Consistent with predictions of the climate variability hypothesis, mitochondria from Baetis collected at a low elevation site with highly variable daily and seasonal temperatures exhibited greater thermal tolerance than Baetis from a high elevation site with comparatively stable temperatures. However, mitochondrial phenotypes were more resilient than whole-organism phenotypes in the face of thermal stress. These results highlight the complex relationships between mitochondrial and organismal genotypes, phenotypes and environmental adaptation. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.

Skeletal muscle mitochondrial function and exercise capacity are not impaired in mice with knockout of STAT3.

Signal transducer and activator of transcription 3 (STAT3) was recently found to be localized to mitochondria in a number of tissues and cell types, where it modulates oxidative phosphorylation via interactions with the electron transport proteins, complex I and complex II. Skeletal muscle is densely populated with mitochondria although whether STAT3 contributes to skeletal muscle oxidative capacity is unknown. In the present study, we sought to elucidate the contribution of STAT3 to mitochondrial and skeletal muscle function by studying mice with muscle-specific knockout of STAT3 (mKO). First, we developed a novel flow cytometry-based approach to confirm that STAT3 is present in skeletal muscle mitochondria. However, contrary to findings in other tissue types, complex I and complex II activity and maximal mitochondrial respiratory capacity in skeletal muscle were comparable between mKO mice and floxed/wild-type littermates. Moreover, there were no genotype differences in endurance exercise performance, skeletal muscle force-generating capacity, or the adaptive response of skeletal muscle to voluntary wheel running. Collectively, although we confirm the presence of STAT3 in skeletal muscle mitochondria, our data establish that STAT3 is dispensable for mitochondrial and physiological function in skeletal muscle.NEW & NOTEWORTHY Whether signal transducer and activator of transcription 3 (STAT3) can regulate the activity of complex I and II of the electron transport chain and mitochondrial oxidative capacity in skeletal muscle, as it can in other tissues, is unknown. By using a mouse model lacking STAT3 in muscle, we demonstrate that skeletal muscle mitochondrial and physiological function, both in vivo and ex vivo, is not impacted by the loss of STAT3.

Mechanisms of Energy Metabolism in Skeletal Muscle Mitochondria Following Radiation Exposure.

An understanding of cellular processes that determine the response to ionizing radiation exposure is essential for improving radiotherapy and assessing risks to human health after accidental radiation exposure. Radiation exposure leads to many biological effects, but the mechanisms underlying the metabolic effects of radiation are not well known. Here, we investigated the effects of radiation exposure on the metabolic rate and mitochondrial bioenergetics in skeletal muscle. We show that ionizing radiation increased mitochondrial protein and mass and enhanced proton leak and mitochondrial maximal respiratory capacity, causing an increase in the fraction of mitochondrial respiration devoted to uncoupling reactions. Thus, mice and cells treated with radiation became energetically efficient and displayed increased fatty acid and amino acid oxidation metabolism through the citric acid cycle. Finally, we demonstrate that radiation-induced alterations in mitochondrial energy metabolism involved adenosine monophosphate-activated kinase signaling in skeletal muscle. Together, these results demonstrate that alterations in mitochondrial mass and function are important adaptive responses of skeletal muscle to radiation.

Novel Pilot Study Reveals that Heat Therapy Increases Muscle Mitochondrial Quality Control and Respiratory Efficiency in Healthy Human Subjects

Immense focus has been placed on novel treatment strategies to prevent or reverse insulin resistance and type 2 diabetes (T2D). We have shown that heat therapy (HT) in rodents protects against high‐fat diet induced skeletal muscle insulin resistance and impaired glucose homeostasis. However, it remains unknown whether this effect can be translated to humans. The purpose of this pilot study was to determine if HT via hot water immersion was well tolerated in humans and altered mitochondrial metabolism in skeletal muscle. Healthy male and female participants (n=5/sex) underwent one bout of HT increasing core temperature to 38.5°C for 1h. Muscle biopsies were taken one week prior and 24h post HT to determine whether HT impacted mitochondrial respiratory capacity and mitochondrial quality control via autophagy. HT was well tolerated in all subjects and resulted in acute increases in heart rate and reductions in systolic and diastolic blood pressure. HT did not significantly alter maximal mitochondrial respiratory capacity at basal, state3, state3S, or uncoupled states with either palmitoyl‐carnitine + palmitoyl‐CoA (palmitate) or pyruvate + glutamate + malate (PGM) as substrates. However, HT tended to improve respiratory efficiency as indicated by a reduced coupling control ratio (basal/state3) for PGM. Of interest, this improved efficiency and maintenance of mitochondrial respiratory capacity occurred despite HT‐induced reductions in mitochondrial content as inferred by citrate synthase activity. We believe this reduction in muscle mitochondrial content occured as a result of mitophagy ‐ the targeted degradation of damaged mitochondria. Our hypothesis is supported by the observed reductions (or degradation) of the autophagy/mitophagy related protein, microtubule‐associated Proteins 1A/1B light chain 3B‐ phosphatidylethanolamine (LC3II). For the first time, our data suggest that HT increases mitochondrial quality control (via enhanced autophagy/mitophagy) and enhances respiratory efficiency in human skeletal muscle. These data provide promising results for further analysis in metabolically impaired obese subjects.Support or Funding InformationNIH P30GM122731; Madison and Lila Self Graduate Fellowship (The University of Kansas)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

KU-596 decreases mitochondrial superoxide and improves bioenergetics following downregulation of manganese superoxide dismutase in diabetic sensory neurons

Neuronal mitochondrial dysfunction and oxidative stress are key pathophysiologic mechanisms of diabetic peripheral neuropathy (DPN). KU-596 is a small molecule modulator of heat shock protein 90 (Hsp90) that can reverse clinically relevant measures of DPN in diabetic animal models. Mechanistically, drug efficacy requires Hsp70 and correlates with improving mitochondrial maximal respiratory capacity (MRC) and decreasing oxidative stress in diabetic sensory neurons. The goal of this study was to determine if ex vivo treatment of diabetic neurons with KU-596 improves MRC by decreasing glucose-induced oxidative stress in an Hsp70-dependent manner. Sensory neurons were isolated from non-diabetic or diabetic mice wild type (WT) or Hsp70 knockout (Hsp70 KO) mice and treated with KU-596 in the presence of low or high glucose concentrations. In diabetic WT and Hsp70 KO neurons, hyperglycemia significantly increased superoxide levels, but KU-596 only decreased superoxide in WT neurons. Similarly, KU-596 significantly improved MRC in diabetic WT neurons maintained in high glucose but did not improve MRC in diabetic Hsp70 KO neurons under the same conditions. Since manganese superoxide dismutase (MnSOD) is the main mechanism to detoxify mitochondrial superoxide radicals, the cause and effect relationship between improved respiration and decreased oxidative stress was examined after knocking down MnSOD. Downregulating MnSOD in diabetic WT neurons increased hyperglycemia-induced superoxide levels, which was still significantly decreased by KU-596. However, KU-596 did not improve MRC following MnSOD knockdown. These data suggest that the ability of KU-596 to improve MRC is not necessarily dependent on decreasing mitochondrial superoxide in a MnSOD-dependent manner.

Intact Cell Lipidomics Reveal Changes to the Ratio of Cardiolipins to Phosphatidylinositols in Response to Kanamycin in HeLa and Primary Cells.

Antimicrobial resistance is a major threat the world is currently facing. Development of new antibiotics and the assessment of their toxicity represent important challenges. Current methods for addressing antibiotic toxicity rely on measuring mitochondrial damage using ATP and/or membrane potential as a readout. In this study, we propose an alternative readout looking at changes in the lipidome on intact and unprocessed cells by matrix-assisted laser desorption ionization mass spectrometry. As a proof of principle, we evaluated the impact of known antibiotics (levofloxacin, ethambutol, and kanamycin) on the lipidome of HeLa cells and mouse bone marrow-derived macrophages. Our methodology revealed that clinically relevant concentrations of kanamycin alter the ratio of cardiolipins to phosphatidylinositols. Unexpectedly, only kanamycin had this effect even though all antibiotics used in this study led to a decrease in the maximal mitochondrial respiratory capacity. Altogether, we report that intact cell-targeted lipidomics can be used as a qualitative method to rapidly assess the toxicity of aminoglycosides in HeLa and primary cells. Moreover, these results demonstrate there is no direct correlation between the ratio of cardiolipins to phosphatidylinositols and the maximal mitochondrial respiratory capacity.

Reduction of Mitochondrial Membrane Potential Leads to Enhancement of Type-II CD20-Antibody Cytotoxicity in Diffuse Large B-Cell Lymphoma

Diffuse Large B Cell Lymphoma (DLBCL) is the most prevalent non-Hodgkin lymphoma (NHL) in adults. Since the addition of the Type I anti-CD20 antibody Rituximab to chemotherapy, the overall survival of NHL patients has improved dramatically compared to the pre-Rituximab era. DLBCL however, has the worst survival rates out of all NHLs with an average 5-year survival of 55%. Unfortunately 40% of all DLBCL patients relapse within 2 years, and those that relapse or have refractory disease tend not to respond well to antibody-based salvage therapies. Since the discovery and utilisation of Rituximab, many have tried to enhance the efficacy of anti-CD20 antibodies in order to improve first-line treatment of DLBCL, leading to the evolution of Type II humanised anti-CD20 antibodies.The complete biological role of CD20 remains unclear, however it has been shown to act as part of an ion channel complex that is a component of the store operated calcium (Ca2+) system. This complex has the ability to facilitate mitochondrial membrane permeabilisation, resulting in reduced mitochondrial function.In order to investigate the effect of Type I- and Type II- anti-CD20 antibodies on mitochondrial function, we established a panel of 4 DLBCL cell lines. We used the XF Seahorse Mito Stress Test to reveal bioenergetic profiles of the cell lines before and after treatment with a panel of Type I and Type II anti-CD20 antibodies (2 Type-I and 2 Type-II anti-CD20 antibodies for each cell line). Basal oxidative phosphorylation (OxPhos), ATP production, and maximal and spare respiratory capacity of each sample were calculated as a measure of mitochondrial function. Next we used Metformin, a well-established inhibitor of oxidative phosphorylation to reduce the mitochondrial membrane potential (MMP) across our panel of cell lines. We confirmed MMP reduction by staining cells with JC-1, a chameleon dye used as an indicator of MMP and analysed samples using flow cytometry. We then used the XF Seahorse Mito Stress Test, this time to assess how combining each CD20-antibody with an OxPhos inhibitor effects mitochondrial function (10 conditions for each cell line). Finally, we used the same conditions to conduct clonogenic survival assays to see whether cytotoxicity of Type-I or Type-II anti-CD20 antibodies could be enhanced.We have observed that treatment with anti-CD20 antibodies results in a significant increase in the maximal respiratory capacity of our panel of cell lines. Conversely, pharmacological inhibition of oxidative phosphorylation causes a significant reduction in basal oxidative phosphorylation as well as a reduction in the maximal respiratory capacity of the cell lines in our panel. We also show that treatment combining an OxPhos inhibitor with either Type-I or Type-II CD20-antibodies prevents the increase in maximal respiratory capacity observed with CD20-antibody treatment alone. When analysing the clonogenic survival of cell lines we have found that only the cytotoxicity of Type-II anti-CD20 antibodies is enhanced by simultaneously treating cell lines with Metformin. We also used Annexin V/PI staining to assess cell death and show that inhibiting oxidative phosphorylation in conjunction with CD20-antibody treatment does not result in a significant increase in cell death across our panel of cell lines.Our data indicate for the first time that when cells are treated with CD20-antibodies they increase their maximal mitochondrial respiratory capacity to compensate for reduced basal mitochondrial function. We also show that inhibition of oxidative phosphorylation disables the cells from being able to compensate for the reduced mitochondrial function that is caused by CD20-antibody treatment. Importantly our data show that the reduction of mitochondrial function caused by combining Metformin with Type-II CD20 antibodies leads to a significant reduction in clonogenicity. We believe that understanding the mechanism of the inhibition of mitochondrial function will allow us to establish effective treatment combinations to significantly improve the efficacy of anti-CD20 antibody therapy in DLBCL. DisclosuresNo relevant conflicts of interest to declare.

The Extracellular Redox State Modulates Mitochondrial Function, Gluconeogenesis, and Glycogen Synthesis in Murine Hepatocytes

Circulating redox state changes, determined by the ratio of reduced/oxidized pairs of different metabolites, have been associated with metabolic diseases. However, the pathogenic contribution of these changes and whether they modulate normal tissue function is unclear. As alterations in hepatic gluconeogenesis and glycogen metabolism are hallmarks that characterize insulin resistance and type 2 diabetes, we tested whether imposed changes in the extracellular redox state could modulate these processes. Thus, primary hepatocytes were treated with different ratios of the following physiological extracellular redox couples: β-hydroxybutyrate (βOHB)/acetoacetate (Acoc), reduced glutathione (GSH)/oxidized glutathione (GSSG), and cysteine/cystine. Exposure to a more oxidized ratio via extracellular βOHB/Acoc, GSH/GSSG, and cysteine/cystine in hepatocytes from fed mice increased intracellular hydrogen peroxide without causing oxidative damage. On the other hand, addition of more reduced ratios of extracellular βOHB/Acoc led to increased NAD(P)H and maximal mitochondrial respiratory capacity in hepatocytes. Greater βOHB/Acoc ratios were also associated with decreased β-oxidation, as expected with enhanced lipogenesis. In hepatocytes from fasted mice, a more extracellular reduced state of βOHB/Acoc led to increased alanine-stimulated gluconeogenesis and enhanced glycogen synthesis capacity from added glucose. Thus, we demonstrated for the first time that the extracellular redox state regulates the major metabolic functions of the liver and involves changes in intracellular NADH, hydrogen peroxide, and mitochondrial respiration. Because redox state in the blood can be communicated to all metabolically sensitive tissues, this work confirms the hypothesis that circulating redox state may be an important regulator of whole body metabolism and contribute to alterations associated with metabolic diseases.

Diphenyl diselenide protects endothelial cells against oxidized low density lipoprotein-induced injury: Involvement of mitochondrial function.

Elevated levels of oxidized low density lipoprotein (oxLDL) are considered to be one of the major risk factors for atherosclerosis and cardiovascular morbidity. The early stages of atherosclerosis are initiated by the accumulation of oxLDL and the induction of toxic effects on endothelial cells, resulting in endothelial dysfunction. The aim of this study was to investigate how diphenyl diselenide (DD), an organoselenium compound, protect vascular endothelial cells against the toxic effects of oxLDL invitro. Our data showed that the treatment of bovine endothelial aortic cells (BAEC) with DD (0.1-1μM) for 24h protected from oxLDL-induced reactive species (RS) production and reduced glutathione (GSH) depletion. Moreover, DD (1μM) per se improved the maximal mitochondrial respiratory capacity and prevented oxLDL-induced mitochondrial damage. In addition, DD could prevent apoptosis induced by oxLDL in BAEC. Results from this study may provide insight into a possible molecular mechanism underlying DD suppression of oxLDL-mediated vascular endothelial dysfunction.

Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells

BackgroundCardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) have great potential as a cell source for therapeutic applications such as regenerative medicine, disease modeling, drug screening, and toxicity testing. This potential is limited, however, by the immature state of the cardiomyocytes acquired using current protocols. Tri-iodo-l-thyronine (T3) is a growth hormone that is essential for optimal heart growth. In this study, we investigated the effect of T3 on hiPSC-CM maturation. Methods and resultsA one-week treatment with T3 increased cardiomyocyte size, anisotropy, and sarcomere length. T3 treatment was associated with reduced cell cycle activity, manifest as reduced DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor p21. Contractile force analyses were performed on individual cardiomyocytes using arrays of microposts, revealing an almost two-fold higher force per-beat after T3 treatment and also an enhancement in contractile kinetics. This improvement in force generation was accompanied by an increase in rates of calcium release and reuptake, along with a significant increase in sarcoendoplasmic reticulum ATPase expression. Finally, although mitochondrial genomes were not numerically increased, extracellular flux analysis showed a significant increase in maximal mitochondrial respiratory capacity and respiratory reserve capability after T3 treatment. ConclusionsUsing a broad spectrum of morphological, molecular, and functional parameters, we conclude that T3 is a driver for hiPSC-CM maturation. T3 treatment may enhance the utility of hiPSC-CMs for therapy, disease modeling, or drug/toxicity screens.

P03 Effect of S-adenosyl-l-methionine (SAM), an allosteric activator of cystathionine-β-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro

Several series of in vitro and in vivo data presented at the current Meeting support the conclusion that colon cancer cells selectively overexpress CBS, which produces H2S, to maintain cellular bioenergetics, thereby supporting tumor growth and to promote angiogenesis and vasorelaxation. These studies, taken together, identify CBS-derived H2S as a tumor promoting factor and a potential future anticancer drug target. The purpose of the current study was to investigate the effect of S-adenosyl-l-methionine (SAM) on the proliferation and bioenergetics of the colon cancer cell line HCT116 in vitro. The non-transformed, the non-tumorigenic colon epithelial cell line NCM356, derived from the normal margin of a rectal cancer specimen, was used as a control. HCT116 cells were cultured in McCoy’s 5A medium, while NCM356 cells were cultured in DMEM supplemented with 10% fetal bovine serum. For assessment of cell proliferation, the xCELLigence system (Roche) was used. For the measurement of bioenergetic function, the XF24 Extracellular Flux Analyzer (Seahorse) was used. Oxygen consumption rate (OCR) after oligomycin (1.5 μg/ml) was used to assess ATP production rate and OCR after FCCP (0.5 μM) to assess maximal mitochondrial respiratory capacity. 2-deoxyglucose (100 mM) was used to estimate cellular glycolytic dependency and antimycin A (2 μg/m) and rotenone (2 μM) were used to inhibit the flux of electrons through complex III and I, to detect residual non-mitochondrial activity. Addition of SAM (0.1–1 mM) to HCT116 cells (which show a high expression of CBS) induced a concentration-dependent increase in cell proliferation between 0–50 h, while higher concentrations of SAM (3 mM) inhibited cell proliferation. However, at time periods between 50–100 h, the cells treated with intermediary concentrations of SAM (0.3–1 mM) showed a suppression of cell proliferation compared to the vehicle control; the proliferation-enhancing effect of SAM was only maintained throughout the entire experimental period with the 0.1 mM concentration of SAM. In contrast to HCT116 cells, NCM356 cells (which have only a low-level expression of CBS) were proliferating at a substantially slower rate, and SAM failed to stimulate their proliferation. Extracellular Flux Analysis of the effect of acute exposure of HCT116 cells to SAM (0.1–1 mM) induced a concentration-dependent increase in their oxygen consumption and bioenergetic function, while in NCM356 cells SAM failed to enhance oxygen consumption and cellular bioenergetics. Longer-term exposure of HCT116 cells to both low concentrations of SAM (0.1 mM) and high concentrations of SAM (1 mM) suppressed cellular bioenergetic responses. Studies presented at the current meeting demonstrate that the H2S-producing enzyme CBS is selectively overexpressed in human colorectal cancer cells, and significantly contributes to their proliferation, migration and invasion in vitro. The present data, demonstrating that short-term exposure of SAM to HCT116 cells exerts proliferative and positive bioenergetic effects that are in line with the effects of exogenously administered H2S in the same experimental system further support the view that CBS in colon cancer cells produces H2S in a regulated fashion, and that this H2S serves as an endogenous cancer cell proliferation and bioenergetic factor. Higher local concentrations of H2S are known to be antiproliferative, at least in part due to their inhibitory on mitochondrial function. Therefore, the longer-term inhibition of cell proliferation and bioenergetic function noted with SAM in the present experiments may either be attributed to the adverse autocrine effects of high concentrations of H2S, produced when CBS is ‘over-activated’ by SAM, or, alternatively, may be related to additional, CBS-independent pharmacological effects of SAM.