Mitochondrial Respiratory Capacity Research Articles

Evidence of acrylamide-induced behavioral deficit, mitochondrial dysfunction and cell death in Drosophila melanogaster

Acrylamide (ACR), a ubiquitous compound with diverse route of exposure, has been demonstrated to have detrimental effects on human and animal health. The mechanisms underlying its toxicity is multifaceted and not fully elucidated. This study aims to provide further insight into novel pathways underlying ACR toxicity by leveraging on Drosophila melanogaster as a model organism. The concentrations of acrylamide (25, 50 and 100 mg/kg) and period of exposure (7-days) used in this study was established through a concentration response curve. ACR exposure demonstrably reduced organismal viability, evidenced by decline in survival rate, offspring emergence and deficits in activity, sleep and locomotory behaviors. Using a high-resolution respirometry assay, the role of mitochondria respiratory system in ACR-mediated toxicity in the flies was investigated. Acrylamide caused dysregulation in mitochondrial bioenergetics and respiratory capacity leading to an impaired OXPHOS activity and electron transport, ultimately contributing to the pathological process of ACR-toxicity. Furthermore, ACR exacerbated apoptosis and induced oxidative stress in D. melanogaster. The up-regulation of mRNA transcription of Reaper, Debcl and Dark genes and down-regulation of DIAP1, an ubiquitylation catalyzing enzyme, suggests that ACR promotes apoptosis through disruption of caspase and pro-apoptotic protein ubiquitination and a mitochondria-dependent pathway in Drosophila melanogaster. Conclusively, this study provides valuable insights into the cellular mechanism underlying ACR-mediated toxicity. Additionally, our study reinforces the utility of D. melanogaster as a translational tool for elucidating the complex mechanisms of ACR toxicity.

Circulating B Cell-Derived Small RNA Delivered by Extracellular Vesicles: A Dialogue Mechanism for Long-Range Targeted Renal Mitochondrial Injury in Obesity.

The intricate processes that govern the interactions between peripatetic immune cells and distal renal injury in obesity are not fully understood. Employing transcriptomic analysis of circulating extracellular vesicles (EVs), a marked amplification of small RNA (miR-3960) is discerned within CD3-CD19+ B cells. This RNA is found to be preferentially augmented in kidney tissues, contrasting with its subdued expression in other organs. By synthesizing dual-luciferase reporter assay with co-immunoprecipitation analysis, it is pinpointed that miR-3960 specifically targets the nuclear gene TRMT5, a pivotal actor in the methylation of mitochondrial tRNA. This liaison instigates aberrations in the post-transcriptional modifications of mitochondrial tRNA, engendering deficiencies within the electron respiratory chain, primarily attributable to the diminution of the mitochondrial bioenergetic compound (NDUFA7) complex I. Such perturbations lead to a compromised mitochondrial respiratory capacity in renal tubular cells, thereby exacerbating tubular injury. In contrast, EV blockade or miR-3960 depletion markedly alleviates renal tubular injury in obesity. This investigation unveils a hitherto unexplored pathway by which obesity-induced circulating immune cells remotely manipulate mitochondrial metabolism in target organs. The strategic targeting of obese EVs or infiltrative immune cells and their specifically secreted RNAs emerges as a promising therapeutic avenue to forestall obesity-related renal afflictions.

Rv0547c, a functional oxidoreductase, supports Mycobacterium tuberculosis persistence by reprogramming host mitochondrial fatty acid metabolism

Mycobacterium tuberculosis (Mtb) successfully thrives in the host by adjusting its metabolism and manipulating the host environment. In this study, we investigated the role of Rv0547c, a protein that carries mitochondria-targeting sequence (MTS), in mycobacterial persistence. We show that Rv0547c is a functional oxidoreductase that targets host-cell mitochondria. Interestingly, the localization of Rv0547c to mitochondria was independent of the predicted MTS but depended on specific arginine residues at the N- and C-terminals. As compared to the mitochondria-localization defective mutant, Rv0547c-2SDM, wild-type Rv0547c increased mitochondrial membrane fluidity and spare respiratory capacity. To comprehend the possible reason, comparative lipidomics was performed that revealed a reduced variability of long-chain and very long-chain fatty acids as well as altered levels of phosphatidylcholine and phosphatidylinositol class of lipids upon expression of Rv0547c, explaining the increased membrane fluidity. Additionally, the over representation of propionate metabolism and β-oxidation intermediates in Rv0547c-targeted mitochondrial fractions indicated altered fatty acid metabolism, which corroborated with changes in oxygen consumption rate (OCR) upon etomoxir treatment in HEK293T cells transiently expressing Rv0547c, resulting in enhanced mitochondrial fatty acid oxidation capacity. Furthermore, Mycobacterium smegmatis over expressing Rv0547c showed increased persistence during infection of THP-1 macrophages, which correlated with its increased expression in Mtb during oxidative and nutrient starvation stresses. This study identified for the first time an Mtb protein that alters mitochondrial metabolism and aids in survival in host macrophages by altering fatty acid metabolism to its benefit and, at the same time increases mitochondrial spare respiratory capacity to mitigate infection stresses and maintain cell viability.

Carboxylesterase 1 mediates a distinctive metabolic profile of dendritic cells to attain an inflammatory phenotype.

The metabolic profile of dendritic cells (DCs) shapes their phenotype and functions. Carboxylestrase 1 (CES1) enzyme is highly expressed in mononuclear myeloid cells however its exact role in DCs is elusive. We used a CES1 inhibitor (WWL113) and genetic overexpression to explore the role of CES1 in DCs differentiation in inflammatory models. CES1 expression was analyzed during CD14+ monocytes differentiation to DCs (MoDCs) using quantitative PCR. CES1 Inhibitor (WWL113) was applied during MoDCs differentiation. Surface markers, secreted cytokines, lactic acid production, phagocytic and T cell polarization capacity were analyzed. Transcriptomic and metabolic profile were assessed with RNA-sequencing and mass spectrometry. Cellular respiration was assessed with seahorse respirometry. Transgenic mice were used to assess CES1 overexpression in DCs in inflammatory models. CES1 expression peaks early during MoDCs differentiation. Pharmacological inhibition of CES1 led to higher expression of CD209, CD86 and MHCII. WWL113 treated MoDCs secreted higher quantities of IL6, IL8, TNF and IL10 and demonstrated stronger phagocytic ability and higher capacity to polarize Th17 differentiation in autologous DCs-T cells co-culture model. Transcriptomic profiling revealed enrichment of multiple inflammatory and metabolic pathways. Functional metabolic analysis shows impaired maximal mitochondrial respiration capacity, increased lactate production and decreased intracellular amino acids and TCA intermediates. Transgenic human CES1 overexpression in murine DCs generated less inflammatory phenotype and increased resistance to T cell mediated colitis. In conclusion, CES1 inhibition directs DCs differentiation towards more inflammatory phenotype, that shows stronger phagocytic capacity and supports Th17 skewing. This is associated with disrupted mitochondrial respiration and amino acids depletion.

Integrative multi-omics analysis reveals ortho-topolin riboside exhibits anticancer activity by regulating metabolic pathways in radio-resistant triple negative breast cancer cells

Radio-resistant triple negative breast cancer (TNBC) is resistant to conventional drugs and radiation therapy. ortho-topolin riboside (oTR) has been evaluated for its anticancer activity in several types of cancer cells. However, its anti-proliferative activity in radio-resistant TNBC cells has not yet been reported. Therefore, we investigated the anti-proliferative activity of oTR in radio-resistant TNBC cells, and performed metabolome, lipidome, transcriptome, and proteome profiling to reveal the mechanisms of the anticancer activity of oTR. oTR showed cytotoxicity against radio-resistant TNBC cells with an inhibitory concentration (IC50) value of 7.78 μM. Significantly decreased (p value < 0.05) basal and compensatory glycolysis were observed in the oTR-treated group than untreated group. Mitochondrial spare respiratory capacity, which is relevant to cell fitness and flexibility, was significantly decreased (p value < 0.05) in the oTR-treated group. The major metabolic pathways significantly altered by oTR according to metabolome, transcriptome, and proteome profiles were the glycerolipid/glycerophospholipid pathway (log2(FC) of MGLL = −0.13, log2(FC) of acylglycerol lipase = −1.35, log2(FC) of glycerol = −0.81), glycolysis (log2(FC) of EGLN1 = 0.16, log2(FC) of EGLN1 = 0.62, log2(FC) of glucose = −0.76, log2(FC) of lactate = −0.81), and kynurenine pathway (log2(FC) of KYNU = 0.29, log2(FC) of kynureninase = 0.55, log2(FC) of alanine = 0.72). Additionally, proline metabolism (log2(FC) of PYCR1 = −0.17, log2(FC) of proline = −0.73) was significantly altered in the metabolomic and transcriptomic profiles. The MAPK signaling pathway (log2(FC) of CCN1 = −0.15, log2(FC) of CCN family member 1 = −1.02) and Rap 1 signaling pathway (log2(FC) of PARD6B = −0.28, log2(FC) of PAR6B = −3.13) were also significantly altered in transcriptomic and proteomic profiles. The findings of this study revealed that oTR has anticancer activity in radio-resistant TNBC cells by affecting various metabolic pathways, suggesting the potential of oTR as a novel anticancer agent for radio-resistant TNBC patients.

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.

Exploring the mechanisms of metabolic adaptations to cardiotoxic doxorubicin

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Leducq Foundation Background Doxorubicin (DOX) is an anthracycline chemotherapeutic agent whose clinical application is hindered by the emergence of early and late cardiotoxic effects. Recent findings demonstrated that DOX damages cardiac mitochondria, with subsequent metabolic perturbation and energetic imbalance. We previously observed that phosphoinositide 3-kinase γ (PI3Kγ) contributes to DOX-induced cardiotoxicity, mediating mitophagy inhibition and accumulation of damaged mitochondria into cardiac cells. Objective Here we intend to describe the cardiotoxic metabolic phenotype of DOX-treated hearts, unravelling the contribution of PI3Kγ signalling to this process. Methods Wild-type (WT) and knock-in mice expressing a kinase-inactive PI3Kγ (kinase-dead; KD) were treated with DOX at day 0, 7 and 14 (cumulative dose 12 mg/kg). To explore the contribution of PI3Kγ on the early cardiac metabolic rewiring, metabolomic analysis, mitochondrial respiration capacity and glycolytic enzymes activity were evaluated at day 3. To investigate the role of autophagy, mice were infected with an adeno-associated virus 9 (AAV9) carrying a vector expressing a short-hairpin RNA against ATG7 (ATG7sh). To test the role of PI3Kγ in glycolysis regulation, glucose uptake and plasma membrane exposure of GLUT-4 were measured in neonatal mouse cardiomyocytes with DOX acute treatment (1µM for 3h). To evaluate the contribution of different substrates to oxygen consumption, OROBOROS analysis were performed on myocardial slices from mice sacrificed at day 3 and 42. Results DOX-treated cardiomyocytes exhibited a metabolic phenotype characterized by glucose as the primary energy substrate, with increased uptake and augmented glycolytic enzymes activity. Metabolomic analysis of DOX-treated hearts indicated diminished pyruvate levels, the glycolysis end-product, unaltered lactate but increased Acetyl-CoA quantity, suggesting enhanced glucose processing for the TCA cycle. Accordingly, oxygen consumption after pyruvate supplementation significantly increased in DOX-treated condition, resulting in the generation of cytotoxic ROS rather than energy production. This was mainly due to DOX-induced mitochondrial damage, with impaired fatty acid oxidation and electron transport chain activity, resulting in TCA cycle slow-down, with accumulation of cardiotoxic glucose-derived intermediates. These metabolic alterations were completely prevented in KD hearts. In vitro experiments demonstrated that inhibiting PI3Kγ reduced the activity of pyruvate dehydrogenase (PDH), the key enzyme of Randle cycle, regulating the switch from fatty acids to glucose usage, while decreasing DOX-induced mobilization of GLUT-4-carrying vesicles to the plasma membrane, limiting subsequent glucose uptake. Conclusion These results demonstrate that DOX promotes an early metabolic rewiring with PI3Kγ-dependent increased glucose uptake and modulation of the Randle cycle towards augmented glucose utilization.

Effects of sodium glucose co-transporter-2 inhibitor on atrial tachyarrhythmia burden in patients with cardiovascular implantable electronic device

Abstract Introduction Previous studies showed that sodium glucose co-transporter-2 inhibitors (SGLT2i) were associated with a lower atrial fibrillation (AF) incidence. Purpose Since mitochondrial dysfunction has been linked with AF, we aimed to examine the effects of SGLT2i on AF burden and mitochondrial function in patients with cardiovascular implantable electronic device (CIED). Methods Patients with CIED were randomly assigned to receive either dapagliflozin 10 mg daily or placebo for 3 months. The AF burden was measured as atrial high-rate episodes (AHRE) via CIED interrogations, and data were collected at baseline and 3-months post-treatment. Oxidative stress and mitochondrial function were determined in peripheral blood mononuclear cells (PBMCs). Results The study enrolled 54 CIED patients. There were 9 (16.7%) patients with diabetes mellitus and 36 (66.7%) patients with history of clinical AF. The changes of AHRE burden after 3-month treatments did not differ between dapagliflozin and placebo groups. However, in those with clinical AF, dapagliflozin significantly decreased the number of AHRE per month as compared to placebo (-2.2 vs. +0.6 episodes per month, p=0.048) (Table 1). Mitochondrial spare respiratory capacity (SRC) percentage was significantly increased after treatment with dapagliflozin (162.1% to 203.2%, p=0.003), whereas this was not found in the placebo group (Figure 1). In the subgroup analysis of patients with clinical AF, dapagliflozin showed significant improvement in both the SRC percentage (165.7 to 201.7%, p=0.016) and a reduction in cellular oxidative stress (26,840.5 to 18,164.0 arbitrary unit, p=0.049). Conclusions Dapagliflozin significantly reduced the number of AHRE per month in patients with history of clinical AF. The improvement in PBMC’s mitochondrial function and reduced cellular oxidative stress among dapagliflozin users could be the mechanisms responsible for its anti-atrial arrhythmic effect.Table 1Figure 1

SIRT3 protects endometrial receptivity in patients with polycystic ovary syndrome.

The sirtuin family is well recognized for its crucial involvement in various cellular processes. Nevertheless, studies on its role in the human endometrium are limited. This study aimed to explore the expression and localization of the sirtuin family in the human endometrium, focusing on sirtuin 3 (SIRT3) and its potential role in the oxidative imbalance of the endometrium in polycystic ovary syndrome (PCOS). Endometrial specimens were collected from both patients with PCOS and controls undergoing hysteroscopy at the Center for Reproductive Medicine, Peking University Third Hospital, from July to August 2015 and used for cell culture. The protective effects of SIRT3 were investigated, and the mechanism of SIRT3 in improving endometrial receptivity of patients with PCOS was determined using various techniques, including cellular bioenergetic analysis, small interfering ribonucleic acid (siRNA) silencing, real-time quantitative polymerase chain reaction, Western blot, immunofluorescence, immunohistochemistry, and flow cytometry analysis. The sirtuin family was widely expressed in the human endometrium, with SIRT3 showing a significant increase in expression in patients with PCOS compared with controls (P <0.05), as confirmed by protein and gene assays. Concurrently, endometrial antioxidant levels were elevated, while mitochondrial respiratory capacity was reduced, in patients with PCOS (P <0.05). An endometrial oxidative stress (OS) model revealed that the downregulation of SIRT3 impaired the growth and proliferation status of endometrial cells and reduced their receptivity to day 4 mouse embryos. The results suggested that SIRT3 might be crucial in maintaining normal cellular state by regulating antioxidants, cell proliferation, and apoptosis, thereby contributing to enhanced endometrial receptivity. Our findings proposed a significant role of SIRT3 in improving endometrial receptivity in patients with PCOS by alleviating OS and regulating the balance between cell proliferation and apoptosis. Therefore, SIRT3 could be a promising target for predicting and improving endometrial receptivity in this patient population.

Cilia-deficient renal tubule cells are primed for injury with mitochondrial defects and aberrant tryptophan metabolism.

The exocyst and Ift88 are necessary for primary ciliogenesis. Overexpression of Exoc5 (OE), a central exocyst component, resulted in longer cilia and enhanced injury recovery. Mitochondria are involved in acute kidney injury (AKI). To investigate cilia and mitochondria, basal respiration and mitochondrial maximal and spare respiratory capacity were measured in Exoc5 OE, Exoc5 knockdown (KD), Exoc5 ciliary targeting sequence mutant (CTS-mut), control Madin-Darby canine kidney (MDCK), Ift88 knockout (KO), and Ift88 rescue cells. In Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells, these parameters were decreased. In Exoc5 OE and Ift88 rescue cells they were increased. Reactive oxygen species were higher in Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells compared with Exoc5 OE, control, and Ift88 rescue cells. By electron microscopy, mitochondria appeared abnormal in Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells. A metabolomics screen of control, Exoc5 KD, Exoc5 CTS-mut, Exoc5 OE, Ift88 KO, and Ift88 rescue cells showed a marked increase in tryptophan levels in Exoc5 CTS-mut (113-fold) and Exoc5 KD (58-fold) compared with control cells. A 21% increase was seen in Ift88 KO compared with rescue cells. In Exoc5 OE compared with control cells, tryptophan was decreased 59%. To determine the effects of ciliary loss on AKI, we generated proximal tubule-specific Exoc5 and Ift88 KO mice. These mice had loss of primary cilia, decreased mitochondrial ATP synthase, and increased tryptophan in proximal tubules with greater injury following ischemia-reperfusion. These data indicate that cilia-deficient renal tubule cells are primed for injury with mitochondrial defects in tryptophan metabolism.NEW & NOTEWORTHY Mitochondria are centrally involved in acute kidney injury (AKI). Here, we show that cilia-deficient renal tubule cells both in vitro in cell culture and in vivo in mice are primed for injury with mitochondrial defects and aberrant tryptophan metabolism. These data suggest therapeutic strategies such as enhancing ciliogenesis or improving mitochondrial function to protect patients at risk for AKI.

Impact of Reduced Activity on Endurance of Rat Diaphragm Muscle

The diaphragm muscle (DIAm) as the major inspiratory pump is highly active. During breathing, fatigue resistant DIAm motor units are recruited that comprise type I and IIa fibers. Accordingly, these fibers have substantially higher mitochondrial volume density (MVD) and mitochondrial respiratory capacity (SDHmax) compared to more fatigable type IIx/IIb fibers. Cervical spinal cord injury disrupts descending inspiratory drive and markedly reduces eupneic activity of the DIAm. With C2 spinal cord hemisection (C2SH) DIAm activity on the ipsilateral side is reduced. Following a two-week period of C2SH, we found reduced MVD and SDHmax in type I and IIa DIAm fibers. As a result, we hypothesize that endurance of the DIAm is reduced. Female and male Sprague Dawley rats were used. EMG electrodes were placed in the left and right DIAm 3 days prior to C2SH surgery, and baseline recordings of eupneic DIAm activity were obtained in awake animals. The right C2 spinal cord was hemisected, and the extent of injury was confirmed by the absence of eupneic DIAm activity on the ipsilateral side while animals were anesthetized. Subsequently, DIAm EMG activity was recorded at 3, 7 and 14 days post C2SH. After two weeks, rats were anesthetized and the DIAm was excised. DIAm strips were cut and suspended in a tissue chamber containing Rees-Simpson solution maintained at 26oC, with thecostal margin clamped and the central tendon attached to a Cambridge Dual-Mode force/length transducer. DIAm strips were stimulated using platinum plate electrodes with supramaximal current (~250 mA) pulses (1 ms), and maximum specific force was determined at 75 Hz. The force-velocity relationship was then determined by systematically changing load, and maximum velocity (Vmax) was estimated based on curve fitting with the Hill-equation. Based on the force-velocity measurements peak power of the DIAm was calculated. Subsequently, the DIAm was repetitively stimulated and allowed to shorten at 30% maximum velocity (isovelocity approximating peak power output). Endurance of the DIAm was then determined as the duration that peak power output was sustained. Finally, the DIAm was rapidly frozen, and cross-sections were cut for fiber type and cross-sectional area (CSA) measurements. C2SH resulted in a marked decrease in eupneic DIAm EMG activity across the two-week period, which disproportionately affected type I and IIa DIAm fibers. However, there was no impact of C2SH on DIAm fiber type proportions or CSA. Following two-weeks C2SH, maximum specific force, Vmax, and peak power output of the DIAm were comparable between intact and injured sides. However, endurance of the DIAm was reduced on the injured side compared to the intact side. These results support our hypothesis that reduced activity of DIAm, reduces oxidative capacity of type I and IIa fibers and thereby reduces endurance. In clinical conditions such as mechanical ventilation DIAm activity is reduced and if maintained for longer periods of time this may affect DIAm endurance post weaning. Research supported by NIH grant: HL146114 (GCS). 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.

Assessment of Mitochondrial Function in a Mouse Model of Mild Chronic Kidney Disease Induced by High-Fat and High-Salt Diet

Intro: Kidneys are known for high energy demands involved in active transport and are thus highly oxidative organs, consuming the second-highest amount of oxygen per gram of tissue at rest (only exceeded by the heart). Chronic kidney disease (CKD) is a complex disorder that presents substantial challenges in understanding its pathophysiological mechanisms, including mitochondrial alterations. However, there are no established mouse models that recapitulate the CKD phenotype observed in humans.Hypothesis: We hypothesized that a high-fat, high-salt diet would induce a mild CKD phenotype and mitochondrial dysfunction in mice. Methods: We subjected C57Bl/6 mice to a high-fat (42%) and high-salt (8% NaCl) diet (HF/HNaCl) or standard chow diet for 16 weeks (n=4 per group). Additionally, we conducted comprehensive investigations into mitochondrial function, focusing on state 3 mitochondrial oxygen consumption (Oroboros O2K) and adenosine triphosphate (ATP) production (Horiba Fluoromax). Differences between groups were determined using Student’s t-tests. Results: HF/HNaCl feeding resulted in polyurea indicated by an increase in 24-hour urine output (Chow: 1.5 ± 0.2 mL; HF/HNaCl: 3.7 ± 0.8 mL; p=0.05) and a 30% reduction in glomerular filtration rate compared to chow-fed animals (Chow: 911.2 ± 107.8 μL/min/100g; Hf/HNaCl: 639.5 ± 25.9 μL/min/100g; p=0.08), indicative of the development of mild kidney disease. Although subtle changes were observed in mitochondrial oxygen consumption and ATP production, the alterations were not statistically significant. Discussion: Our findings suggest that the induced mild CKD state via the dietary regimen led to moderate renal impairment without substantial impacts on mitochondrial respiratory capacity and ATP synthesis. The establishment of this mouse model provides a valuable platform for further investigations into the complex interplay between renal dysfunction and mitochondrial dynamics in the context of mild CKD, offering insights that may pave the way for the development of targeted therapeutic interventions for this prevalent and debilitating condition. This project was supported by grants from the National Institutes of Health. 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.

Unraveling the evolutionary marathon: improved mitochondrial bioenergetics in CMP-Neu5Ac hydroxylase gene deleted (Cmah−/−) mouse skeletal myofibers

Objective: Humans are unique among hominids in that they are endurance athletes. Inactivation of the CMP-Neu5Ac hydroxylase (CMAH) gene, which encodes a hydroxylase converting sialic acid Neu5Ac to Neu5Gc, is thought to contribute to this exceptional capacity for endurance running. Recently, our group demonstrated improved fatigue resistance and preserved intracellular O2, estimated by NAD(P)H levels in contracting mouse Cmah−/− flexor digitorum brevis (FDB) single fibers under low oxygen tension. Hypothesis: Effcient regulation of mitochondrial oxygen consumption contributes to improved skeletal myofiber fatigue resistance in Cmah−/− mice. Methods: The mitochondrial bioenergetic potential of permeabilized FDB and soleus fiber bundles from wild type (WT) and Cmah−/− mice was evaluated using a physiological creatine kinase clamp method and high-resolution respirometer. Experiments were conducted with pyruvate and malate with or without branched chain amino acids (BCAAs) as the substrates. Respiratory chain conductance and maximal respiration were measured. Results: FDB Cmah−/− fibers from male but not female mice revealed an increase conductance above WT fibers independent of substrate (male: genotype, p=0.006, substrate, ns; female: genotype, ns, substrate, ns). Maximal respiration was also increased only in male FDBs fibers and was even greater when BCAAs were included in the respiration media. (male: genotype, p=0.003, substrate, p=0.0007, interaction, p=0.002.; female: genotype, ns, substrate, ns). Soleus Cmah−/− fibers from male and female mice revealed an increase conductance above WT fiber bundles that was also greater in the presence of BCAAs but only for male mice (male: genotype, p=0.003, substrate, p=0.05.; female: genotype, p=0.009, substrate, ns). Maximal respiration was increased only in male Cmah−/− soleus fibers independent of substrate. (male: genotype, p=0.002, substrate, ns.; female: genotype, ns, substrate, ns). Summary: Fibers from both the FDB and soleus of male Cmah−/− mice have a greater capacity for mitochondrial respiration under a metabolically active state that is similar to myofibers stimulated to repeatedly contraction in a hypoxic environment. Conclusion: These data suggest that effcient regulation of mitochondrial respiration utilizing BCAAs as a substrate, particularly in males, could contribute to an elite endurance phenotype. This project was funded through the UC San Diego Academic Senate Grant. 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.

Disrupted mitochondrial transcription factor A expression promotes mitochondrial dysfunction and enhances ocular surface inflammation by activating the absent in melanoma 2 inflammasome

PurposeSevere dry eye disease causes ocular surface damage, which is highly associated with mitochondrial dysfunction. Mitochondrial transcription factor A (TFAM) is essential for packaging mitochondrial DNA (mtDNA) and is crucial for maintaining mitochondrial function. Herein, we aimed to explore the effect of a decreased TFAM expression on ocular surface damage. MethodsFemale C57BL/6 mice were induced ocular surface injury by topical administrating benzalkonium chloride (BAC). Immortalized human corneal epithelial cells (HCECs) were stimulated by tert-butyl hydroperoxide (t-BHP) to create oxidative stress damage. HCECs with TFAM knockdown were established. RNA sequencing was employed to analyze the whole-genome expression. Mitochondrial changes were measured by transmission electron microscopy, Seahorse metabolic flux analysis, mitochondrial membrane potential, and mtDNA copy number. TFAM expression and inflammatory cytokines were determined using RT-qPCR, immunohistochemistry, immunofluorescence, and immunoblotting. ResultsIn both the corneas of BAC-treated mice and t-BHP-induced HCECs, we observed impaired TFAM expression, accompanied by mitochondrial structure and function defects. TFAM downregulation in HCECs suppressed mitochondrial respiratory capacity, reduced mtDNA content, induced mtDNA leakage into the cytoplasm, and led to inflammation. RNA sequencing revealed the absent in melanoma 2 (AIM2) inflammasome was activated in the corneas of BAC-treated mice. The AIM2 inflammasome activation was confirmed in TFAM knockdown HCECs. TFAM knockdown in t-BHP-stimulated HCECs aggravated mitochondrial dysfunction and the AIM2 inflammasome activation, thereby further triggering the secretion of inflammatory factors such as interleukin (IL) -1β and IL-18. ConclusionsTFAM reduction impaired mitochondrial function, activated AIM2 inflammasome and promoted ocular surface inflammation, revealing an underlying molecular mechanism for ocular surface disorders.

A Mitochondrial Targeted Ubiquinol (MitoQ) Improves Immune Cell Bioenergetics in Patients with Heart Failure with Preserved Ejection Fraction

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is a proinflammatory syndrome. Mitochondrial dysfunction that is characteristic of HFpEF could alter immunometabolism, attenuating the functional reserve of immune cells. In addition, immune cell mitochondrial respiration is a biomarker of systemic mitochondrial health. The aim of this study was to assess the impact of a chronic oral mitochondrial targeted ubiquinol (MitoQ) on the immune cell bioenergetic profile in patients with HFpEF. We hypothesized that MitoQ administration in patients with HFpEF, would improve mitochondrial respiration in peripheral blood mononuclear cells (PBMC’s). METHODS: In this double-blind, crossover trial 8 patients with a clinical diagnosis of HFpEF (6 female, 2 male; 6 African American/Black, 2 white; mean ± SEM, Age, 58±5 years; BMI, 37±1 kg/m2) received a 4 week oral dose of MitoQ (MTQ, 20 mg/day) and placebo (PLB) in random order separated by a 2 week wash out period. Following completion of each trial arm, a venous blood sample was obtained and PBMCs were isolated using density gradient centrifugation. High resolution respirometry coupled with modulators of cellular respiration was performed to obtain basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity of the immune cells. Paired samples t-tests were used to compare variables between conditions. RESULTS: MitoQ was safe, well tolerated, and compliance was high (MTQ, 99 ± 1%; PLB, 89±4%). Basal respiration (MTQ, 106.4 ± 11.5 pmol/min; PLB, 66.5±5.9 pmol/min; p=0.002), ATP-linked respiration (MTQ, 41.4± 16.9 pmol/min; PLB, 16.3±6.7 pmol/min; p=0.04), maximal respiration (MTQ, 323.9 ± 50.0 pmol/min; PLB, 65.4±23.1 pmol/min; p=0.01), spare respiratory capacity (MTQ, 217.5 ± 39.6 pmol/min; PLB, 149.6 ±18.2 pmol/min; p=0.02), were higher following MTQ compared to PLB. CONCLUSIONS: In patients with HFpEF, administration of a mitochondrial targeted ubiquinol improved the immune cell mitochondrial respiration capacity and effciency. An increase in spare respiratory capacity following MTQ reflects an improvement in functional reserve that would augment the ability to respond to an acute stressor. These findings have positive implications for the ability to target and improve mitochondrial function in patients with HFpEF. Future work should investigate if the observed improvements in immune cell mitochondrial function are mirrored in other tissues. Supported by AHA 19CDA3474002. 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 Health, Physical Functioning, and Daily Affect: Bioenergetic Mechanisms of Dementia Caregiver Well-Being.

Chronic stress adversely affects mental and physical well-being. However, health outcomes vary among people experiencing the same stressor. Individual differences in physical and emotional well-being may depend on mitochondrial biology, as energy production is crucial for stress regulation. This study investigated whether mitochondrial respiratory capacity corresponds to individual differences in dementia spousal caregivers' mental and physical health. Spousal caregivers of individuals with Alzheimer's disease and related dementias ( N = 102, mean age = 71, 78% female, 83% White) provided peripheral blood samples and completed self-report questionnaires on quality of life, caregiver burden, and a 7-day affect scale. Multiple and mixed linear regressions were used to test the relationship between mitochondrial biology and well-being. Spare respiratory capacity ( b = 12.76, confidence interval [CI] = 5.23-20.28, p = .001), maximum respiratory capacity ( b = 8.45, CI = 4.54-12.35, p < .0001), and ATP-linked respiration ( b = 10.11, CI = 5.05-15.18, p = .0001) were positively associated with physical functioning. At average ( b = -2.23, CI = -3.64 to -0.82, p = .002) and below average ( b = -4.96, CI = -7.22 to 2.70, p < .0001) levels of spare respiratory capacity, caregiver burden was negatively associated with daily positive affect. At above average levels of spare respiratory capacity, caregiver burden was not associated with positive affect ( p = .65). Findings suggest that higher mitochondrial respiratory capacity is associated with better psychological and physical health-a pattern consistent with related research. These findings provide some of the earliest evidence that cellular bioenergetics are related to well-being.

Platelet Bioenergetics and Associations With Delirium and Coma in Patients With Sepsis: A Prospective Cohort Study

BackgroundAcute brain dysfunction during sepsis, which manifests as delirium or coma, is common and associated with multiple adverse outcomes including longer periods of mechanical ventilation, prolonged hospital stays, and increased mortality. Delirium and coma during sepsis may be manifestations of alteration in systemic metabolism. Since access to brain mitochondrial is a limiting factor, measurement of peripheral platelet bioenergetics offers a potential opportunity to understand metabolic changes associated with acute brain dysfunction during sepsis. Research QuestionAre altered platelet mitochondrial bioenergetics associated with acute brain dysfunction during sepsis? Study Design and MethodsWe assessed participants with critical illness in the ICU for the presence of delirium or coma via validated assessment measures. Blood samples were collected and processed to isolate and measure platelet mitochondrial oxygen consumption. We used Seahorse extracellular flux to directly measure baseline, proton leak, maximal oxygen consumption rate, and extracellular acidification rate. We calculated ATP-linked, spare respiratory capacity, and non-mitochondrial oxygen consumption rate from the measured values. ResultsMaximal oxygen consumption was highest in patients with coma, as was spare respiratory capacity and ECAR in unadjusted analysis. After adjusting for age, sedation, modified Sequential Organ Failure Assessment (SOFA) without the neurological component, and pre-existing cognitive function, increased spare respiratory capacity remained associated with coma. Delirium was not associated with any platelet mitochondrial bioenergetics. InterpretationIn this single-center, exploratory, prospective cohort study, we found increased platelet mitochondrial spare respiratory capacity was associated with coma in patients with sepsis. Future studies powered to determine any relationship between delirium and mitochondrial respiration bioenergetics are needed.

Inhibitory Regulation of FOXO1 in PPARδ Expression Drives Mitochondrial Dysfunction and Insulin Resistance.

Forkhead box protein O1 (FoxO1) regulates muscle growth, but the metabolic role of FoxO1 in skeletal muscle and its mechanisms remain unclear. To explore the metabolic role of FoxO1 in skeletal muscle, we generated skeletal muscle-specific FoxO1 inducible knockout (mFoxO1 iKO) mice and fed them a high-fat diet to induce obesity. We measured insulin sensitivity, fatty acid oxidation, mitochondrial function, and exercise capacity in obese mFoxO1 iKO mice, and assessed the correlation between FoxO1 and mitochondrial-related protein in the skeletal muscle of diabetic patients. Obese mFoxO1 iKO mice exhibited improved mitochondrial respiratory capacity, which was followed by attenuated insulin resistance, enhanced fatty acid oxidation, and improved skeletal muscle exercise capacity. Transcriptional inhibition of FoxO1 in peroxisome proliferator-activated receptor δ (PPARδ) expression was confirmed in skeletal muscle and deletion of PPARδ abolished the beneficial effects of FoxO1 deficiency. FoxO1 protein levels were higher in the skeletal muscle of diabetic patients and negatively correlated with PPARδ and electron transport chain protein levels. These findings highlight FoxO1 as a new repressor in PPARδ gene expression in skeletal muscle and suggest that FoxO1 links insulin resistance and mitochondrial dysfunction in skeletal muscle via PPARδ.

Low-Volume Speed Endurance Training with Reduced Volume Improves Short-Term Exercise Performance in Highly Trained Cyclists.

We investigated the effects of low and high volume speed endurance training (SET), with a reduced training volume, on sprint ability, short- and long-term exercise capacity, muscle mitochondrial properties, ion transport proteins and maximal enzyme activity in highly trained athletes. Highly-trained male cyclists (V̇O2max: 68.3 ± 5.0 mL × min-1 × kg-1, n = 24) completed six weeks of either low (SET-L; 6x30-s intervals, n = 8) or high (SET-H; 12 × 30-s intervals, n = 8) volume SET twice per week with a 30%-reduction in training volume. A control group (CON, n = 8) maintained their training. Exercise performance was evaluated by i) 6-s sprinting, ii) a 4-min time trial, iii) a 60-min preload at 60% V̇O2max followed by a 20-min time trial. A biopsy of m. vastus lateralis was collected before and after the training intervention. In SET-L, 4-min time trial performance was improved (P < 0.05) by 3.8%, with no change in SET-H and CON. Sprint ability, prolonged endurance exercise capacity, V̇O2max, muscle mitochondrial respiratory capacity, maximal citrate synthase activity, fiber-type specific mitochondrial proteins (complex I - V) and PFK content did not change in any of the groups. In SET-H, maximal activity of muscle PFK and abundance of Na+-K+ pump-subunit α1, α2, β1, and phospholemman (FXYD1) were 20%, 50%, 19%, 24%, and 42 % higher (P < 0.05), respectively after compared to before the intervention, with no changes in SET-L or CON. Low SET volume combined with a reduced aerobic low and moderate intensity training volume does improve short duration intense exercise performance and maintain sprinting ability, V̇O2max, endurance exercise performance and muscle oxidative capacity, whereas, high volume of SET appears necessary to upregulate muscle ion transporter content and maximal PFK activity in highly trained cyclists.

Metabolic reprogramming regulated by TRAF6 contributes to the leukemia progression

TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that has been implicated in myeloid malignancies. Although altered TRAF6 expression is observed in human acute myeloid leukemia (AML), its role in the AML pathogenesis remains elusive. In this study, we showed that the loss of TRAF6 in AML cells significantly impairs leukemic function in vitro and in vivo, indicating its functional importance in AML subsets. Loss of TRAF6 induces metabolic alterations, such as changes in glycolysis, TCA cycle, and nucleic acid metabolism as well as impaired mitochondrial membrane potential and respiratory capacity. In leukemic cells, TRAF6 expression shows a positive correlation with the expression of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of O-GlcNAc to target proteins involved in metabolic regulation. The restoration of growth capacity and metabolic activity in leukemic cells with TRAF6 loss, achieved through either forced expression of OGT or pharmacological inhibition of O-GlcNAcase (OGA) that removes O-GlcNAc, indicates the significant role of O-GlcNAc modification in the TRAF6-related cellular and metabolic dynamics. Our findings highlight the oncogenic function of TRAF6 in leukemia and illuminate the novel TRAF6/OGT/O-GlcNAc axis as a potential regulator of metabolic reprogramming in leukemogenesis.