Mitochondrial Oxygen Consumption Rate Research Articles

Electrical Pulse Stimulation Protects C2C12 Myotubes against Hydrogen Peroxide-Induced Cytotoxicity via Nrf2/Antioxidant Pathway.

Skeletal muscle contraction evokes numerous biochemical alterations that underpin exercise benefits. This present study aimed to elucidate the mechanism for electrical pulse stimulation (EPS)-induced antioxidant adaptation in C2C12 myotubes. We found that EPS significantly upregulated Nrf2 and a broad array of downstream antioxidant enzymes involved in multiple antioxidant systems. These effects were completely abolished by pretreatment with a ROS scavenger, N-acetylcysteine. MitoSOX-Red, CM-H2DCFDA, and EPR spectroscopy revealed a significantly higher ROS level in mitochondria and cytosol in EPS cells compared to non-stimulated cells. Seahorse and Oroboros revealed that EPS significantly increased the maximal mitochondrial oxygen consumption rate, along with an upregulated protein expression of mitochondrial complexes I/V, mitofusin-1, and mitochondrial fission factor. A post-stimulation time-course experiment demonstrated that upregulated NQO1 and GSTA2 last at least 24 h following the cessation of EPS, whereas elevated ROS declines immediately. These findings suggest an antioxidant preconditioning effect in the EPS cells. A cell viability study suggested that the EPS cells displayed 11- and 36-fold higher survival rates compared to the control cells in response to 2 and 4 mM H2O2 treatment, respectively. In summary, we found that EPS upregulated a large group of antioxidant enzymes in C2C12 myotubes via a contraction-mitochondrial-ROS-Nrf2 pathway. This antioxidant adaptation protects cells against oxidative stress-associated cytotoxicity.

Mitochondrial players as novel therapeutic options in arrhythmogenic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Joint Project Südtirol- FWF (grant number 23623; Italy-Austria) and the Department of Innovation, Research and University of the Autonomous Province of Bolzano-South Tyrol (Italy) Background Mitochondria are dynamic organelles that sense and respond to environmental, nutritional, and pathological changes. These organelles can smartly adapt to different stressful conditions, by changing the way they network or the energy sources they use. Over the past years, numerous disorders have been associated with mitochondrial dysfunction, including cardiomyopathies. Arrhythmogenic Cardiomyopathy (ACM) is a cardiac disease characterized by the progressive replacement of the myocardium with fibro-fatty tissue. The current treatment primarily focuses on managing the symptoms rather than addressing the underlying issue of tissue substitution. Purpose: Therefore, in this study, we explored the mitochondrial and metabolic phenotype in ACM and tested the efficacy of a mitochondria-targeting library to rescue the fibro-fatty phenotype in ACM Methods: Human primary cardiac stromal cells (CStCs) were cultured in adipogenic medium (AM), a condition used to model the ACM fibro-fatty phenotype. Mitochondria-related analyses of ultrastructure, membrane potential, network connection, respiratory capacity, ROS production and redox system were performed at day (d) d0, d3, d7, d15 of AM exposure. A mitochondria-targeting library (MedChem Express) was tested on ACM-CStCs exposed to AM for 7 days. The extent of lipid accumulation was determined using the neutral lipid staining dye BODIPY 493/503, cell numbers were measured using Hoechst counterstaining, and images acquired using a high-content imaging system Results: At d0, the mitochondrial network was more fragmented in ACM compared to healthy CStCs (CTR). This result was in line with the observation of a decrease in mitochondrial respiratory capacity and an increase in ROS production. In addition, AM exposure led to faster and higher lipid accumulation in ACM CStCs, which was associated with higher mitochondrial oxygen consumption rates observed in ACM CStCs after 7 days of AM exposure. When ACM CStCs were simultaneously exposed to AM and 407 mitochondrial-directed compounds, about 25% of the compounds demonstrated the ability to reduce lipid accumulation Conclusion: ACM CStCs demonstrate an increased risk for mitochondrial network impairment. Moreover, prolonged exposure of ACM CStCs cells to AM negatively affects lipid deposition as well as mitochondrial and metabolic parameters. This highlights the potential for using specific mitochondria-targeting compounds to reduce lipid accumulation.

Investigation of the Antarctic krill tripeptide Phe-Pro-Phe (FPF) for its antioxidant properties and mechanism

Oxidative stress causes damage to cells and brain injury-associated disorders. The study aimed to investigate how the tripeptide Phe-Pro-Phe (FPF), derived from an antioxidant-rich Antarctic krill peptide Ser-Ser-Asp-Ala-Phe-Phe-Pro-Phe-Arg (SSDAFFPFR), could protect against oxidative stress caused by scopolamine. Utilizing mouse and cell models to induce oxidative stress injury with scopolamine, coupled with Fourier transform infrared (FTIR) microscopy, immunofluorescence, and mitochondrial function assays, the study demonstrated that FPF significantly reduced malondialdehyde (MDA) and reactive oxygen species (ROS) levels, as well as decreasing the expression level of the apoptotic protein Caspase-3, while also increasing superoxide dismutase (SOD) activity and unsaturated lipid levels. Furthermore, FPF significantly improved mitochondrial aerobic respiration oxygen consumption rate (OCR) and anaerobic glycolytic metabolism extracellular acidification rate (ECAR) in mouse hippocampal neuron (HT22) cells. The results indicated that FPF protected against scopolamine-induced oxidative stress by regulating anti-oxidative activity, enhancing Caspase-3 expression, increasing unsaturated lipid levels, and improving cellular energy metabolism.

Sulforaphane Exposure Prevents Cadmium-Induced Toxicity and Mitochondrial Dysfunction in the Nematode Caenorhabditis elegans by Regulating the Insulin/Insulin-like Growth Factor Signaling (IIS) Pathway.

Cadmium (Cd) is a heavy metal that is highly toxic to humans and animals. Its adverse effects have been widely associated with mitochondrial alterations. However, there are not many treatments that target mitochondria. This study aimed to evaluate the impact of sulforaphane (SFN) pre-exposure against cadmium chloride (CdCl2)-induced toxicity and mitochondrial alterations in the nematode Caenorhabditis elegans (C. elegans), by exploring the role of the insulin/insulin-like growth factor signaling pathway (IIS). The results revealed that prior exposure to SFN protected against CdCl2-induced mortality and increased lifespan, body length, and mobility while reducing lipofuscin levels. Furthermore, SFN prevented mitochondrial alterations by increasing mitochondrial membrane potential (Δψm) and restoring mitochondrial oxygen consumption rate, thereby decreasing mitochondrial reactive oxygen species (ROS) production. The improvement in mitochondrial function was associated with increased mitochondrial mass and the involvement of the daf-16 and skn-1c genes of the IIS signaling pathway. In conclusion, exposure to SFN before exposure to CdCl2 mitigates toxic effects and mitochondrial alterations, possibly by increasing mitochondrial mass, which may be related to the regulation of the IIS pathway. These discoveries open new possibilities for developing therapies to reduce the damage caused by Cd toxicity and oxidative stress in biological systems, highlighting antioxidants with mitochondrial action as promising tools.

Decreased mitochondrial respiration associates with frailty in community-dwelling older adults.

Aging population has led to an increased prevalence of chronic and degenerative pathologies. A manifestation of unhealthy aging is frailty, a geriatric syndrome that implies a non-specific state of greater vulnerability. Currently, methods for frailty diagnosis are based exclusively on clinical observation. The aim of this study is to determine whether the bioenergetic capacity defined as mitochondrial oxygen consumption rate (OCR) of peripheral circulation mononuclear cells (PBMC) associates with the frailty phenotype in older adults and with their nutritional status. This is a cross-sectional analytic study of 58 participants 70years and older, 18 frail and 40 non-frail adults, from the ALEXANDROS cohort study, previously described. Participants were characterized through sociodemographic and anthropometric assessments. Frail individuals displayed a higher frequency of osteoporosis and depression. The mean age of the participants was 80.2 ± 5.2years, similar in both groups of men and women. Regarding the nutritional status defined as the body mass index, most non-frail individuals were normal or overweight, while frail participants were mostly overweight or obese. We observed that OCR was significantly decreased in frail men (p < 0.01). Age was also associated with significant differences in oxygen consumption in frail patients, with lower oxygen consumption being observed in those over 80years of age. Therefore, the use of PBMC can result in an accessible fingerprint that may identify initial stages of frailty in a minimally invasive way.

Altered Hepatic Mitochondrial Bioenergetics and Oxidant Emission with Ischemia and Reperfusion

Rationale: Ischemia-reperfusion injury (IRI) is inevitable in liver transplantation (LT) which can be mitigated by normothermic machine perfusion (NMP) before LT. The current NMP liver viability criteria for LT mainly rely on bile production and perfusate lactate levels, factors that can also be affected by external variables such as NMP conditions and perfusate additives. Incorporating mitochondrial bioenergetic data could improve the predictive value of current NMP viability criteria for LT. Thus, the goal of this study was to evaluate mitochondrial oxygen consumption rate (OCR), membrane potential (Δψ), and H2O2 oxidant emission in control livers and in livers with IRI and to explore underlying mechanisms responsible for any observed differences. Methods: Healthy control livers and livers exposed to 60 minutes warm ischemia time (WIT) through in situ clamping of the portal vein and hepatic artery followed by 60 minutes reperfusion (IRI) were harvested from adult male Sprague-Dawley rats. Mitochondria from control and IRI livers were isolated using established protocols and assessed for their bioenergetics responses. Three different substrate combinations, namely, pyruvate+malate (PM), glutamate+malate (GM), and succinate were used, followed by addition(s) of ADP and the uncoupler FCCP. Two different ADP addition protocols were designed to determine how different substrates influence mitochondrial OCR, Δψ, and H2O2 emission during oxidative phosphorylation (OxPhos) between control and IRI conditions. In one protocol, a single saturated dose of ADP addition, and in the other, sequentially increasing doses of ADP additions were made following substrate addition. The OCR and Δψ were measured simultaneously using a dual chamber Oroboros Oxygraph-2k Instrument coupled to a fluorometer using the TMRM dye. The H2O2 emission was measured spectrofluorometrically using the amplex red and horseradish peroxidase assay. Results: The measured data show that the kinetics and effciency of OxPhos defining mitochondrial OCR and Δψ responses in the liver are negatively affected by IRI, characterized by enhanced H2O2 emission, in a substrate-dependent manner. Interestingly, the respiratory rates are higher when GM and succinate are utilized as substrates, whereas the use of PM shows comparatively lower respiration in both conditions. Control mitochondria exhibited higher respiratory rates than IRI mitochondria irrespective of the substrate utilized. The ADP-induced state 3 OCR in IRI mitochondria was 50% lower than that in control mitochondria resulting in doubling the duration of state 3 OCR. Similar differences were observed in Δψ for both ADP addition protocols. Compromised mitochondrial bioenergetics during IRI was observed using both single and sequentially increasing doses of ADP and was concomitant with a higher rate of H2O2 emission. Conclusion: This study provided novel quantitative data demonstrating the substrate specific changes in mitochondrial bioenergetics in hepatic IRI, which can be used to improve the NMP viability criteria for LT. The obtained bioenergetics and H2O2 emission data indicate that, during IRI, mitochondrial function is highly affected which may critically impact energy dependent metabolism and lactate/pyruvate ratio. NIH R01-HL151587. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Electrical pulse stimulation protects C2C12 myotubes against H2O2-induced injury via activating Nrf2/antioxidant pathway

Background: In response to various physiological stimuli during exercise, skeletal muscle (SkM), a highly plastic tissue, is capable of functional and structural remodeling. The present study aimed to elucidate antioxidant adaptation of SkM to contraction by exposing C2C12 myotubes to electrical pulse stimulation (EPS). Methods: C2C12 myoblasts were differentiated to myotubes by changing the media from complete media (DMEM/High Glucose, 10% fetal bovine serum, and 1% antibiotics) to differentiation media (DMEM/High Glucose, 5% horse serum, and 1% antibiotics). The myotubes were seeded at 50,000 cells/well in a 6-well plate where they underwent EPS at 10 V, 10 ms, 50 Hz, 0.3 sec/3 sec for 1 hour/day for 4 days. Results: (1) By completing experiments for time-course (0 – 6 days) and dose-effect (0 – 10 voltage) tests, we found that NQO1 and GSTA2 proteins, two well-known targets of Nrf2, were maximally upregulated with 3.3 and 12.1 fold increase, respectively, by an EPS at 10 voltage for 4 days; therefore, these parameters were used in the following experiments. (2) MitoSOX-Red, CM-H2DCFDA, confocal microscopy, and EPR assay revealed significantly higher ROS levels in mitochondria, cytosol, and whole cell of EPS-cells than control-cells. Western blot data suggests that EPS-cells express significantly higher Nrf2 and a broad array of downstream antioxidant enzymes (SOD1, SOD2, Cat, GPX, NQO1, GSTA2, GSTA4, PRX1, and TRX). This phenomenon was completely abolished by pretreatment with N-acetylcysteine, a ROS scavenger. These data suggest that contraction enhances muscle antioxidant defense through ROS-dependent, Nrf2-activated multi-antioxidant mechanisms, including the first line of antioxidant components, glutathione system, thioredoxin system, and peroxiredoxin system. (3) We further found that EPS significantly upregulated expression of mitochondrial complexes I/V, mitofusin-1, and mitochondrial fission factor, and significantly increased mitochondrial basal oxygen consumption rate and ATP-linked respiration, suggesting an enhanced mitochondrial function and biogenesis. (4) A post-stimulation time-course experiment demonstrated that the upregulated NQO1 and GSTA2 protein expression lasts at least 24 hours after EPS, suggesting that EPS induces antioxidant preconditioning in the cells. (5) A cell viability study using CCK-8 assay demonstrated that EPS-cells have a significantly higher survival rate than control cells in response to H2O2 treatment. Conclusion: We found that EPS dramatically upregulated a large group of antioxidant enzymes in C2C12 myotubes via activating Nrf2. These cells display an increased mitochondrial function and enhanced tolerance to oxidative challenge. These data suggest that contraction can evoke antioxidant adaptation in muscle via mitochondrion-ROS-Nrf2 pathway, which provides myocytes with protection against oxidative stress-associated injury. This project is funded by NIH R01 HL 160820. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Unacylated Ghrelin Protects Against Muscle Wasting, Mitochondrial Dysfunction, and Neuromuscular Junction Disruption in Tumor Bearing Mice

Background: Cancer cachexia is a complex metabolic syndrome that severely impacts mobility, treatment strategies, and life quality of the patients. However, no treatments are available to mitigate the debilitating consequences of cancer cachexia. Ghrelin is a hormone released from the stomach that increases appetite upon acylation by binding to GHSR1a receptors in the brain. Because of its orexigenic effects and increases in body weight, ghrelin GHSR1a receptor analogs were tested as clinical trials to mitigate muscle wasting and loss of strength in cancer patients in several countries. The analogs (i.e., Anamorelin) increased body weight of the non-small lung cancer patients in European countries and Japan, but failed to increase strength, which is the critical component in cancer cachexia. Mainly due to the lack of improvement in strength and patients’ quality of life, Anamorelin was not approved for clinical use in Europe. While the lack of increases in strength is, at least in part, associated with the adipogenic effects of acylated ghrelin, unacylated ghrelin directly improves satellite cell renewal, muscle growth, and mitochondrial function independent of GHSR1a receptor activation and increases in appetite. Unacylated ghrelin is the predominant population in circulation comprising proximately 90-95% of total ghrelin in humans and rodents. Hypothesis: Unacylated ghrelin projects against muscle wasting, loss of strength, and metabolic dysfunction of tumor bearing mice. Methods and Results: Supplementing the cancer cell conditioned medium (CM) with unacylated ghrelin preserved myotube size. Unacylated ghrelin also diminished the cancer cell CM-induced upregulation of MuRF-1, muscle specific ubiquitin ligase. Interestingly, unacylated ghrelin normalized transcriptional upregulation of denervation markers (i.e., sarcolipin, Runx1, GADD45a) in myotubes treated with CM. To further investigate the therapeutic effects of unacylated ghrelin in vivo, we inoculated lung cancer cell line (LL2, ATCC®) in the flank of 4 months old male mice. Two weeks after the injection prior to development of cachexia, mice were provided with unacylated ghrelin containing drinking water or untreated water as control. Tumor bearing (TB) mice treated with unacylated ghrelin showed 30-40% increases in gastrocnemius and quadriceps weights, while soleus muscle mass was unchanged. In addition to the muscle quantity, unacylated ghrelin increased force generating capacity was also improved by unacylated ghrelin. Notably, unacylated ghrelin increased mitochondrial oxygen consumption rate and protected against neuromuscular junction disruption and denervation in TB control mice. Conclusion: Unacylated ghrelin protects against cancer cachexia by targeting multiple risk factors in skeletal muscle wasting, including protein balance, metabolic alterations, and neuromuscular junction disruption. Our findings show protective effects of unacylated ghrelin against cachexia and neuromuscular impairment in tumor bearing mice, indicating its therapeutic potential in cancer patients. NIA R00 064143 to Ahn B. Pilot funds from the Center for Redox Biology in Medicine, and Cancer Genetics and Metabolism program at the Wake Forest Baptist Comprehensive Cancer Center. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Impact of Sex Hormones on Renal Mitochondrial Bioenergetics in Aging

Introduction. Aging is accompanied by changes in multiple factors, such as accumulation of mitochondrial dysfunction and the decline of sex hormone levels throughout life. Females are generally protected from renal diseases until menopause, potentially as a result of the presence of female sex hormones. Sex hormones govern a plethora of crucial renal processes, including metabolism and mitochondrial bioenergetics. However, there remains a gap in knowledge regarding the role of sex hormones in mitochondrial bioenergetics in renal aging. We hypothesized in this study that removal of sex hormones will exacerbate the aging-induced decline in renal mitochondrial function. Methods. Male and female Sprague Dawley (SD) rats were randomly divided into groups, and gonadectomy or sham surgery was performed before 4 wks of age. At 6 and 12 months of age kidneys were perfused to remove blood, then cortical tissues were immediately collected for mitochondrial isolations using differential centrifugation or snap-frozen/fixed for biochemical assays. Spectrofluorimetry was utilized to measure mitochondrial membrane potential (ΔΨm) and H2O2 production using TMRM and Amplex Red. Seahorse assay was done to measure oxygen consumption rate (OCR). OriginPro was used for statistical analysis (two-way ANOVA with Holm-Sidak post hoc). Results. Male OCR parameters (basal, ADP-linked, maximal and spare capacity) were lower than OCR observed in females at both 6 and 12 months of age (p&lt;0.05). Orchiectomy resulted in an increase in male OCR parameters at both ages (p&lt;0.05); ovariectomy did not affect renal cortical OCR (p&gt;0.05 for both ages). Of note, gonadectomy in males increased the OCR up to the female level (p&gt;0.05 for both ages). Cortical mitochondrial ΔΨm displayed similar values at 6 months across all groups. However, at 12 months we report lower ΔΨm in males vs female shams (p&lt;0.001). Of note, the male gonadectomy increased ΔΨm up to the level of female values, while ovariectomy had no effect (p=0.3). Female cortical H2O2 production was higher than in males (p&lt;0.001), whereas ovariectomy lowered H2O2 at both time points (p&lt;0.001), indicative of ΔΨm-independent ROS sources. Interestingly, the effect of testosterone on H2O2 production was dependent on age. At 6 months old, orchiectomy did not alter H2O2 production compared to shams, while at 12 months, orchiectomy led to higher H2O2 production (p&lt;0.001). Conclusions. Renal mitochondrial OCR was found to be independent of age in either sex at 6 and 12 months of age. Gonadectomy data allowed us to speculate that testosterone suppresses renal OCR, and the decreased membrane potential in males is likely driving the OCR changes. The observed ΔΨm-independent sources of H2O2 remain to be explored in future experiments. These data established the impact of sex hormones on mitochondrial bioenergetics and will help elucidate sex-specific targets that can mitigate the effects of aging on kidney function. Funding for this project came from 1U54HL169191. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mitochondrial reactive oxygen species impact human fibroblast responses to protracted γ-ray exposures

Purpose: Continuous exposure to ionizing radiation at a low dose rate poses significant health risks to humans on deep space missions, prompting the need for mechanistic studies to identify countermeasures against its deleterious effects. Mitochondria are a major subcellular locus of radiogenic injury, and may trigger secondary cellular responses through the production of reactive oxygen species (mtROS) with broader biological implications. Methods and Materials: To determine the contribution of mtROS to radiation-induced cellular responses, we investigated the impacts of protracted γ-ray exposures (IR; 1.1 Gy delivered at 0.16 mGy/min continuously over 5 days) on mitochondrial function, gene expression, and the protein secretome of human HCA2-hTERT fibroblasts in the presence and absence of a mitochondria-specific antioxidant mitoTEMPO (MT; 5 µM). Results: IR increased fibroblast mitochondrial oxygen consumption (JO2) and H2O2 release rates (JH2O2) under energized conditions, which corresponded to higher protein expression of NADPH Oxidase (NOX) 1, NOX4, and nuclear DNA-encoded subunits of respiratory chain Complexes I and III, but depleted mtDNA transcripts encoding subunits of the same complexes. This was associated with activation of gene programs related to DNA repair, oxidative stress, and protein ubiquination, all of which were attenuated by MT treatment along with radiation-induced increases in JO2 and JH2O2. IR also increased secreted levels of interleukin-8 and Type I collagens, while decreasing Type VI collagens and enzymes that coordinate assembly and remodeling of the extracellular matrix. MT treatment attenuated many of these effects while augmenting others, revealing complex effects of mtROS in fibroblast responses to IR. Conclusion: These results implicate mtROS production in fibroblast responses to protracted radiation exposure, and suggest potentially protective effects of mitochondrial-targeted antioxidants against radiogenic tissue injury in vivo.

The troglitazone derivative EP13 disrupts energy metabolism through respiratory chain complex I inhibition in breast cancer cells and potentiates the antiproliferative effect of glycolysis inhibitors

BackgroundThe metabolism of cancer cells generally differs from that of normal cells. Indeed, most cancer cells have a high rate of glycolysis, even at normal oxygen concentrations. These metabolic properties can potentially be exploited for therapeutic intervention. In this context, we have developed troglitazone derivatives to treat hormone-sensitive and triple-negative breast cancers, which currently lack therapeutic targets, have an aggressive phenotype, and often have a worse prognosis than other subtypes. Here, we studied the metabolic impact of the EP13 compound, a desulfured derivative of Δ2-troglitazone that we synthetized and is more potent than its parent compounds.MethodsEP13 was tested on two triple-negative breast cancer cell lines, MDA-MB-231 and Hs578T, and on the luminal cell line MCF-7. The oxygen consumption rate (OCR) of the treated cell lines, Hs578T mammospheres and isolated mitochondria was measured using the XFe24 Seahorse analyser. ROS production was quantified using the MitoSOX fluorescent probe. Glycolytic activity was evaluated through measurement of the extracellular acidification rate (ECAR), glucose consumption and lactate production in extracellular medium. The synergistic effect of EP13 with glycolysis inhibitors (oxamate and 2-deoxyglucose) on cell cytotoxicity was established using the Chou-Talalay method.ResultsAfter exposure to EP13, we observed a decrease in the mitochondrial oxygen consumption rate in MCF7, MDA-MB-231 and Hs578T cells. EP13 also modified the maximal OCR of Hs578T spheroids. EP13 reduced the OCR through inhibition of respiratory chain complex I. After 24 h, ATP levels in EP13-treated cells were not altered compared with those in untreated cells, suggesting compensation by glycolysis activity, as shown by the increase in ECAR, the glucose consumption and lactate production. Finally, we performed co-treatments with EP13 and glycolysis inhibitors (oxamate and 2-DG) and observed that EP13 potentiated their cytotoxic effects.ConclusionThis study demonstrates that EP13 inhibits OXPHOS in breast cancer cells and potentiates the effect of glycolysis inhibitors.

Mitochondrial respiratory capacity in patients with acute episodes of bipolar disorder compared with clinical remission

IntroductionBipolar disorder (BD) is a chronic and recurrent disease characterized by acute mood episodes alternated with periods of euthymia. The available literature postulates that a biphasic dysregulation of mitochondrial bioenergetics might be observed in BD.ObjectivesWe aimed to explore differences in in vivo mitochondrial respiration (1) intra-individually: longitudinally within patients during an acute mood episode of BD and after clinical remission, and (2) inter-individually: between patients with BD on depressive or manic episodes and healthy controls (HC).MethodsPatients admitted to our acute psychiatric ward with a manic episode or bipolar depression were recruited. Different mitochondrial oxygen consumption rates (OCRs) were assessed during the acute episode (T0) and after clinical remission (T1) in one million of peripheral blood mononuclear cells (PBMC): Routine, Leak, ETC and Rox. They were measured as picomoles of oxygen per million cells (pmol O2/million). This experiment was also conducted in HC. High-resolution respirometry was performed at 37°C by polarographic oxygen sensors in a two-chamber Oxygraph-2k system. Manic and depressive symptoms were assessed using standardized psychometric scales. Oxygen consumption capacity was compared (1) intra-individually, during acute episodes and after clinical remission, and (2) inter-individually, during acute manic and depressive episodes, and in HC. Statistical analyses were performed with SPSS, GraphPad and R Statistics.Results20 patients with BD (15 manic, 5 depressed) and 10 HC were included. A significant increase in the maximal oxygen consumption capacity (ETC) was observed in clinical remission (27.4 ± 17.4) compared to the acute episodes (21.1 ± 11.7, p = 0.001), which remained significant after subtracting Rox from the other rates (p = 0.001). At T1, patients admitted with a manic episode tended to show higher mean ETC (31.2 ± 18.7) compared with T0 (24.1 ± 12.0, p = 0.074); the tendency persisted after Rox subtraction (p = 0.076). Patients admitted with a depressive episode also showed higher ETC means in T1 (16.3 ± 3.8) compared to T0 (12.1 ± 3.4), but there were not significant differences (p = 0.231). When HC, manic and depressive patients at T0 were compared between them, significant differences were observed in ETC (H =8.5; p =0.014) and Rox (H =13.8; p = 0.001). After Rox deduction, differences in ETC remained (H =11.7; p = 0.003). Individuals with bipolar depression showed lower ETC rates (12.1 ± 3.4) than those with a manic episode (24.1 ± 12.0; t = -3.5, p = 0.003), which was also found after Rox deduction (p = 0.001).ConclusionsIn both manic and depressive episodes in BD, mitochondrial respiration might be reduced and increase after clinical remission. Further studies with larger samples will allow to confirm these results and also to identify potential mitochondrial state-dependent biomarkers.Disclosure of InterestA. Giménez-Palomo Grant / Research support from: AGP is supported by a Rio Hortega 2021 grant (CM21/00094) from the Spanish Ministry of Health financed by ISCIII and cofinanced by Fondo Social Europeo Plus (FSE+)., M. Guitart-Mampel: None Declared, A. Meseguer: None Declared, M. Valentí: None Declared, L. Bracco: None Declared, H. Andreu: None Declared, E. Vieta: None Declared, G. Garrabou: None Declared, I. Pacchiarotti: None Declared

Abstract 7057: Validation of OXPHOS gene amplification as a driver of treatment resistance in non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer deaths in the U.S. and worldwide. Up to 90% of all lung cancers are non-small cell lung cancer (NSCLC). NSCLC is a complex and highly aggressive disease typical for poor prognosis and high rates of treatment resistance. Clinical management of NSCLC varies according to the stage. High-dose stereotactic body radiation therapy (SBRT) is the standard of care for localized non-resectable NSCLC. However, tumor hypoxia is a significant barrier to effective radiation therapy. In the last decade, first line NSCLC treatment has been substantially reinforced with the introduction of immunotherapy targeting immune checkpoints such as programmed cell death 1 (PD-1), yet long-term disease control occurs in less than 25% of NSCLC patients. Therefore, understanding the mechanisms of treatment resistance is essential to address the dire need of introducing novel synergistic therapies to enhance anti-cancer treatment response in refractory NSCLC tumors. The basis of tumor hypoxia has traditionally been attributed to the oxygen supply deficit as malformed tumor vasculature fails to meet the high demand of the rapidly proliferating tumor mass. However, our preliminary analysis of NSCLC patient datasets in the Cancer Genome Atlas (TCGA) PanCancer dataset revealed a significant correlation between high-level expression of nuclear genes encoding mitochondrial subunits essential for oxidative phosphorylation (OXPHOS), and high-level expression of hypoxia-regulated genes (Buffa hypoxia score). Furthermore, we have observed a direct correlation between copy number amplification (CNA) of several OXPHOS genes and hypoxia levels in patient samples. Because mitochondrial function consumes up to 90% of available cellular oxygen, its activity may be indirectly regulating oxygen availability in the tumor microenvironment by rapidly consuming oxygen upon its delivery to the tumor. Moreover, we have identified that almost 40% of NSCLC patients harbor CNA of these essential OXPHOS genes. To provide mechanistic insight into these clinical observations, we have generated mouse syngeneic NSCLC cell lines with modified mRNA expression levels of OXPHOS genes (representing deletion, diploid and amplified number of copies) identified in our screen and observed a direct effect on the rates of mitochondrial oxygen consumption in these cells. Our study investigates the previously unknown role of OXPHOS gene copy number in driving mitochondrial oxygen demand and thus contributing to tumor hypoxia and treatment resistance in NSCLC. Citation Format: Martin Benej, Katarina Benejova, Rebecca Hoyd, Caroline Wheeler, Daniel Spakowicz, Nicholas C. Denko. Validation of OXPHOS gene amplification as a driver of treatment resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7057.

Abstract 4064: hetIL-15 and Fenofibrate combination alters the metabolic fitness of tumor-infiltrating CD8+T cells leading to TNBC tumor eradication in mice

Abstract Introduction: Tumor-infiltrating cytotoxic CD8+T cells frequently undergo an altered state of differentiation referred to as "exhaustion" and, as a result, they fail to control tumor growth. IL-15 is a cytokine which stimulates the generation, proliferation and cytotoxic function of tumor CD8+T and NK cells. We have produced the native heterodimeric form of IL-15 (hetIL-15) which has advanced in clinical trials. The aim of this study was to overcome the exhaustion of the tumor-infiltrating CD8+T cells and to increase their cytotoxicity, using Fenofibrate (FF), a PPAR-α agonist, in combination with hetIL-15. Experimental Procedures: We have evaluated the therapeutic efficacy of hetIL-15 immunotherapy as a monotherapy and in combination with FF in the murine EO771 orthotopic breast cancer model. The effects of hetIL-15 and/or FF on immune cells and on tumor microenvironment were analyzed by flow cytometry, transcriptomics, metabolomics and proteomics. The metabolic profile of tumor-infiltrating T cells was performed with Seahorse analysis. Results and Conclusions: hetIL-15 monotherapy resulted in tumor eradication in 40% of treated mice and increased survival. Seahorse analysis of tumor-infiltrating CD8+T cells confirmed a rise in oxygen consumption rate (OCR) with substantial increase of spare respiratory capacity. Upon hetIL-15 treatment, tumor-infiltrating CD8+T cells showed elevated extracellular acidification rate (ECAR), resulting in a pronounced shift in the OCR/ECAR ratio in comparison to control, confirming their increased proliferating status. These tumor-infiltrating CD8+T cells also showed increased mitochondrial potential and/or mass and fatty acid (FA) uptake, as evidenced by increased MitoTracker and Bodipy staining, respectively. Transcriptomic analysis revealed increased expression of genes involved in several metabolic pathways such as oxidative phosphorylation, FA oxidation and glycolysis. Monotherapy with FF had no effect on tumor growth delay, whereas the FF- hetIL-15 combination resulted in complete eradication of the tumors in 85%. Combination therapy reshaped the tumor microenvironment, resulting in higher IFN-γ and in differentially expressed metabolites as shown by tumor proteomics and metabolomics, respectively. The combination treatment increased the mitochondrial function, FA uptake and OCR, revealing a more metabolically active phenotype. We conclude that hetIL-15 supports a favorable metabolic profile of intratumoral effector lymphocytes, important for their function. Our data suggest that metabolic reprogramming of tumor specific CD8+T cells using FF is a promising strategy to overcome T cell exhaustion and to promote survival in a metabolically hostile tumor microenvironment. Citation Format: Dimitris Stellas, Sevasti Karaliota, Vasiliki Stravokefalou, Katherine C. Goldfarbmuren, Breana Myers, George N. Pavlakis, Barbara K. Felber. hetIL-15 and Fenofibrate combination alters the metabolic fitness of tumor-infiltrating CD8+T cells leading to TNBC tumor eradication in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4064.

Impaired mitochondrial respiration in upper compared to lower body differentiated human adipocytes and adipose tissue.

Abdominal obesity is associated with increased cardiometabolic disease risk, while lower body fat seems to confer protection against obesity-related complications. The functional differences between upper and lower body adipose tissue (AT) remain poorly understood. We aimed to examine whether mitochondrial respiration is impaired in abdominal as compared to femoral differentiated human multipotent adipose-derived stem cells (hMADS; primary outcome) and AT in postmenopausal women. In this cross-sectional study, 23 postmenopausal women with normal weight or obesity were recruited at the University of Birmingham/Queen Elizabeth Hospital Birmingham (Birmingham, UK). We collected abdominal and femoral subcutaneous AT biopsies to determine mitochondrial oxygen consumption rates in differentiated abdominal and femoral hMADS. Furthermore, we assessed OXPHOS protein expression and mtDNA content in abdominal and femoral AT as well as hMADS. Finally, we explored in vivo fractional oxygen extraction and carbon dioxide release across abdominal and femoral subcutaneous AT in a subgroup of the same individuals with normal weight or obesity. We found lower basal and maximal uncoupled mitochondrial oxygen consumption rates in abdominal compared to femoral hMADS. In line, in vivo fractional oxygen extraction and carbon dioxide release were lower across abdominal than femoral AT. OXPHOS protein expression and mtDNA content did not significantly differ between abdominal and femoral differentiated hMADS and AT. The present findings demonstrate that in vitro mitochondrial respiration and in vivo oxygen fractional extraction are lower in upper compared to lower body differentiated hMADS and AT, respectively, in postmenopausal women.

Alcohol Promotes Lipogenesis in Sebocytes-Implications for Acne.

The oral consumption of alcohol (ethanol) has a long tradition in humans and is an integral part of many cultures. The causal relationship between ethanol consumption and numerous diseases is well known. In addition to the well-described harmful effects on the liver and pancreas, there is also evidence that ethanol abuse triggers pathological skin conditions, including acne. In the present study, we addressed this issue by investigating the effect of ethanol on the energy metabolism in human SZ95 sebocytes, with particular focus on qualitative and quantitative lipogenesis. It was found that ethanol is a strong trigger for lipogenesis, with moderate effects on cell proliferation and toxicity. We identified the non-oxidative metabolism of ethanol, which produced fatty acid ethyl esters (FAEEs), as relevant for the lipogenic effect-the oxidative metabolism of ethanol does not contribute to lipogenesis. Correspondingly, using the Seahorse extracellular flux analyzer, we found an inhibition of the mitochondrial oxygen consumption rate as a measure of mitochondrial ATP production by ethanol. The ATP production rate from glycolysis was not affected. These data corroborate that ethanol-induced lipogenesis is independent from oxygen. In sum, our results give a causal explanation for the prevalence of acne in heavy drinkers, confirming that alcoholism should be considered as a systemic disease. Moreover, the identification of key factors driving ethanol-dependent lipogenesis may also be relevant in the treatment of acne vulgaris.

Activation of Mitochondrial Oxygen Consumption Rate by Delivering Coenzyme Q10 to Mitochondria of Rat Skeletal Muscle Cell (L6)

Numerous mitochondria are present in skeletal muscle cells. Muscle disease and aging impair mitochondrial functioning in the skeletal muscle. However, there have been few reports of therapeutic intervention via drug delivery to mitochondria owing to methodological difficulties. We surmised that mitochondrial activation is associated with improved skeletal muscle function. In this study, we attempted to activate the mitochondrial respiratory capacity in rat skeletal muscle cells (L6 cells) by delivering Coenzyme Q10 (CoQ10), a mitochondrial functional activator, to mitochondria using MITO-Porter, a nanoparticle that facilitates mitochondria-targeted drug delivery. Cellular uptake was confirmed by measuring the amount of fluorescence-modified MITO-Porter taken up by cells using flow cytometry. Intracellular dynamics of MITO-Porter was observed using confocal laser scanning microscopy. Mitochondrial function was assessed by measuring the mitochondrial oxygen consumption rate using an extracellular flux analyzer. The results indicated MITO-Porter-assisted delivery of CoQ10 to the mitochondria activated mitochondrial respiratory capacity in L6 cells. We believe that our results indicate the possibility of skeletal muscle therapy using mitochondrial drug delivery.

Mitophagy regulates mitochondrial number following pharmacological induction of mitochondrial biogenesis in renal proximal tubule cells.

Background: Mitochondrial biogenesis (MB) induction through the activation of the 5-Hydroxytriptamine (5-HT) 1F receptor (HTR1F) is a promising mechanism for the treatment of diseases characterized by mitochondrial dysfunction, such as acute kidney injury (AKI). While several studies report pharmacological activation of MB in the proximal tubule, it is unclear how the proximal tubule regulates itself once the pharmacological activation is removed. Mitophagy is the process of selective mitochondria degradation. We hypothesize that mitophagy decreases mitochondrial number after pharmacological stimulation and restore mitochondrial homeostasis. Methods: Renal proximal tubules were treated at time 0hr with LY344864 or vehicle for 24h and then removed. LY344864, a selective HTR1F agonist, induces MB in renal proximal tubules as previously reported (Gibbs et al., Am J Physiol Renal Physiol, 2018, 314(2), F260-F268). Vehicle and pharmacological reagents were added at the 24h time point. Electron microscopy was used to assess mitochondrial morphology, number, and autolysosomes. Seahorse Bioscience XF-96 extracellular flux analyzer was used to measure maximal mitochondrial oxygen consumption rates (FCCP-OCR), a functional marker of MB. Results: LY344864 treatment increased FCCP-OCR, phosphorylation of protein kinase B (AKT), peroxisome proliferator-activated receptor γ coactivator-1alpha (PGC-1α), and mitochondrial number after 24h. These endpoints decreased to baseline 24h after LY344864 removal. Treatment with ROC-325, an autophagy inhibitor, increased Sequestosome-1 (SQSTM1/P62) and microtubule-associated protein-1 light chain 3 (LC3B) after 24h of treatment. Also, ROC-325 treatment sustained the elevated mitochondrial number after LY344864 pre-treatment and removal. Conclusion: These data revealed that inhibition of autophagy extends elevated mitochondrial number and function by preventing the lysosomal degradation of mitochondria after the removal of LY344864.

Decreased pyruvate dehydrogenase activity in Tafazzin-deficient cells is caused by dysregulation of pyruvate dehydrogenase phosphatase 1 (PDP1)

Cardiolipin (CL), the signature lipid of the mitochondrial inner membrane, is critical for maintaining optimal mitochondrial function and bioenergetics. Disruption of CL metabolism, caused by mutations in the CL remodeling enzyme TAFAZZIN, results in the rare and life-threatening disorder Barth syndrome (BTHS). While the clinical manifestations of BTHS, such as dilated cardiomyopathy and skeletal myopathy, point to defects in mitochondrial bioenergetics, the disorder is also characterized by broad metabolic dysregulation, including abnormal levels of metabolites associated with the tricarboxylic acid (TCA) cycle. In line with this, recent studies have identified inhibition of pyruvate dehydrogenase (PDH), the gatekeeper enzyme for TCA cycle carbon influx, as a key deficiency in various BTHS model systems. However, the molecular mechanisms linking aberrant CL remodeling, particularly the primary, direct consequence of reduced tetralinoleoyl-CL (TLCL) levels, to PDH activity deficiency are not yet understood. This knowledge gap has limited our understanding of lipid-mediated metabolic regulation in BTHS and hindered the development of effective treatment strategies. In the current study, we provide evidence that remodeled TLCL promotes PDH function by directly binding to and enhancing the activity of PDH phosphatase 1 (PDP1). This is supported by our findings that TLCL uniquely activates PDH in a dose-dependent manner, TLCL binds to PDP1 in vitro, TLCL-mediated PDH activation is attenuated in the presence of phosphatase inhibitor, and PDP1 activity is decreased in Tafazzin-knockout (TAZ-KO) C2C12 myoblasts. Additionally, we observed decreased mitochondrial calcium levels in TAZ-KO cells, which may affect the calcium-sensitive activity of PDP1. Treating TAZ-KO cells with calcium lactate (CaLac) increases mitochondrial calcium and restores PDH activity and mitochondrial oxygen consumption rate. Based on our findings, we conclude that reduced mitochondrial calcium levels and decreased binding of PDP1 to TLCL contribute to decreased PDP1 activity in TAZ-KO cells.

ROS-Suppression Nanoplatform Combined Activation of STAT3/Bcl-2 Pathway for Preventing Myocardial Infarction in Mice.

Myocardial infarction (MI) is the leading cause of death worldwide. The most effective way to treat myocardial infarction is to rescue ischemic cardiomyocytes. After an ischemic event, the overproduction of reactive oxygen species (ROS) is a key driver of myocardial injury. The produced ROS affects mitochondrial function and induces apoptosis in cardiomyocytes. This was accomplished by constructing platelet-membrane-encapsulated ROS-responsive drug-releasing nanoparticles (PMN@NIC-MalNPs) to deliver malonate and niclosamide (NIC). The results revealed that PMN@NIC-MalNPs degraded and released malonate and niclosamide in a high-level ROS microenvironment, effectively reducing the oxidative stress and apoptosis rate. By enhancing basal mitochondrial oxygen consumption rate (OCR), adenosine triphosphate (ATP) production, and spare respiratory capacity (SRC) in vitro, reduced the oxidative stress levels and restored mitochondrial function. In vivo studies revealed that the PMN@NIC-MalNPs improved cardiac dysfunction, inhibited succinate dehydrogenase (SDH) activity, increased ATP production, and reduced the myocardial infarct size in myocardial infarction model mice. Further, transcriptome analysis and Western blot revealed that PMN@NIC-MalNPs prevented apoptosis by activating the expressions of the signal transducer and activator of transcription 3 (STAT3) and Bcl-2, and inhibiting the expression of Bax. Thus, this study provides a novel therapeutic solution for treating myocardial infarction and predicting the viability of an antioxidant and antiapoptotic therapeutic solution in the treatment of myocardial injury.