Respiratory Capacity Research Articles

The Effects of Chest Cages Expansion Exercises on Respiratory Capacity and Quality of Life in the Elderly

The Effects of Chest Cages Expansion Exercises on Respiratory Capacity and Quality of Life in the Elderly

Praseodymium and warming interactions in mussels: Comparison between observed and predicted results

Being a crucial element for technological development, praseodymium (Pr) has been increasingly used, leading to a rise in its concentration in aquatic systems. However, its potential threats to organisms remain poorly understood. Besides contamination, organisms are also threatened by climate change-related factors, including warming. It is important to evaluate how climate change-related factors may influence the effects of contaminants. To address this, histopathological and biochemical analyses were performed in adult mussels of Mytilus galloprovincialis, following a 28-day exposure to Pr (10 μg/L) and warming (4 °C increase) separately, and in combination. Additionally, biochemical and physiological alterations were analysed in the sperm of mussels after 30-min exposure to the same treatments. Furthermore, it was used the Independent Action model to predict the interaction between Pr and warming. The results showed, in the case of adults exposed to Pr, an increase in superoxide dismutase (SOD) and glutathione S-transferases (GSTs) activities. However, it was insufficient, leading to histopathological injuries, redox imbalance, and cellular damage. In the case of sperm, Pr induced an increase of mitochondrial activity and respiration rate, in response to the increase in systemic metabolic rate and oxygen demand. Warming increased the metabolism, and induced redox imbalance and cellular damage in adults. In sperm, a rise in temperature induced lipid peroxidation and a decrease in velocity. Warming induced some alterations in how adult mussels responded to Pr, activating catalase instead of SOD, and in addition to GSTs, also activated carboxylesterases. However, it was not enough to avoid redox imbalance and cellular damage. In the case of sperm, the combination induced a decrease in H2O2 production, and higher oxygen demand, which prevented the decrease in motility and velocity. This study highlights the limitations of using models and emphasizes the importance of studying the impacts of emerging contaminants, such as rare earth elements, and their combination with climate change-related factors. Under environmental conditions, chronic exposure to the combined effect of different stressors might generate impacts at higher biological levels. This may affect organisms' respiratory and filtration capacity, nutrient absorption, defence capacity against infections or diseases, and sperm viability, ultimately resulting in reduced growth and reproduction, with consequences at the population level.

The Effects of Various Approaches to Lobectomies on Respiratory Muscle Strength, Diaphragm Thickness, and Exercise Capacity in Lung Cancer

BackgroundThe most common surgery for non-small cell lung cancer is lobectomy, which can be performed through either thoracotomy or video-assisted thoracic surgery (VATS). Insufficient research has examined respiratory muscle function and exercise capacity in lobectomy performed using conventional thoracotomy (CT), muscle-sparing thoracotomy (MST), or VATS. This study aimed to assess and compare respiratory muscle strength, diaphragm thickness, and exercise capacity in lobectomy using CT, MST, and VATS.MethodsThe primary outcomes were changes in respiratory muscle strength, diaphragm thickness, and exercise capacity. Maximal inspiratory pressure (MIP) and maximal expiratory pressure (MEP) were recorded for respiratory muscle strength. The 6-min walk test (6MWT) was used to assess functional exercise capacity. Diaphragm thickness was measured using B-mode ultrasound.ResultsThe study included 42 individuals with lung cancer who underwent lobectomy via CT (n = 14), MST (n = 14), or VATS (n = 14). Assessments were performed on the day before surgery and on postoperative day 20 (range 17–25 days). The decrease in MIP (p < 0.001), MEP (p = 0.003), 6MWT (p < 0.001) values were lower in the VATS group than in the CT group. The decrease in 6MWT distance was lower in the MST group than in the CT group (p = 0.012). No significant differences were found among the groups in terms of diaphragmatic muscle thickness (p > 0.05).ConclusionThe VATS technique appears superior to the CT technique in terms of preserving respiratory muscle strength and functional exercise capacity. Thoracic surgeons should refer patients to physiotherapists before lobectomy, especially patients undergoing CT. If lobectomy with VATS will be technically difficult, MST may be an option preferable to CT because of its impact on exercise capacity.

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.

Hypoxia exposure blunts angiogenic signaling and upregulates the antioxidant system in endothelial cells derived from elephant seals

BackgroundElephant seals exhibit extreme hypoxemic tolerance derived from repetitive hypoxia/reoxygenation episodes they experience during diving bouts. Real-time assessment of the molecular changes underlying protection against hypoxic injury in seals remains restricted by their at-sea inaccessibility. Hence, we developed a proliferative arterial endothelial cell culture model from elephant seals and used RNA-seq, functional assays, and confocal microscopy to assess the molecular response to prolonged hypoxia.ResultsSeal and human endothelial cells exposed to 1% O2 for up to 6 h respond differently to acute and prolonged hypoxia. Seal cells decouple stabilization of the hypoxia-sensitive transcriptional regulator HIF-1α from angiogenic signaling. Rapid upregulation of genes involved in glutathione (GSH) metabolism supports the maintenance of GSH pools, and intracellular succinate increases in seal but not human cells. High maximal and spare respiratory capacity in seal cells after hypoxia exposure occurs in concert with increasing mitochondrial branch length and independent from major changes in extracellular acidification rate, suggesting that seal cells recover oxidative metabolism without significant glycolytic dependency after hypoxia exposure.ConclusionsWe found that the glutathione antioxidant system is upregulated in seal endothelial cells during hypoxia, while this system remains static in comparable human cells. Furthermore, we found that in contrast to human cells, hypoxia exposure rapidly activates HIF-1 in seal cells, but this response is decoupled from the canonical angiogenesis pathway. These results highlight the unique mechanisms that confer extraordinary tolerance to limited oxygen availability in a champion diving mammal.

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 (maximal oxygen consumption (V̇O 2max ): 68.3 ± 5.0 mL·min -1 ·kg -1 , n = 24) completed 6 wk of either low (SET-L; 6 × 30-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, and iii) a 60-min preload at 60% V̇O 2max 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, V̇O 2max , muscle mitochondrial respiratory capacity, maximal citrate synthase activity, fiber type-specific mitochondrial proteins (complexes I-V), and phosphofructokinase (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 α 1 , α 2 , β 1 , and phospholemman (FXYD1) were 20%, 50%, 19%, 24%, and 42% higher ( P < 0.05), respectively after compared with 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, V̇O 2max , endurance exercise performance, and muscle oxidative capacity, whereas, high volume of SET seems necessary to upregulate muscle ion transporter content and maximal PFK activity in highly trained cyclists.

Aberrant expression of thyroidal hormone receptor α exasperating mitochondrial dysfunction induced sarcopenia in aged mice.

Disrupted mitochondrial dynamics and mitophagy contribute to functional deterioration of skeletal muscle (SM) during aging, but the regulatory mechanisms are poorly understood. Our previous study demonstrated that the expression of thyroid hormone receptor α (TRα) decreased significantly in aged mice, suggesting that the alteration of thyroidal elements, especially the decreased TRα, might attenuate local THs action thus to cause the degeneration of SM with aging, while the underlying mechanism remains to be further explored. In this study, decreased expression of myogenic regulators Myf5, MyoD1, mitophagy markers Pink1, LC3II/I, p62, as well as mitochondrial dynamic factors Mfn1 and Opa1, accompanied by increased reactive oxygen species (ROS), showed concomitant changes with reduced TRα expression in aged mice. Further TRα loss- and gain-of-function studies in C2C12 revealed that silencing of TRα not only down-regulated the expression of above-mentioned myogenic regulators, mitophagy markers and mitochondrial dynamic factors, but also led to a significant decrease in mitochondrial activity and maximum respiratory capacity, as well as more mitochondrial ROS and damaged mitochondria. Notedly, overexpression of TRα could up-regulate the expression of those myogenic regulators, mitophagy markers and mitochondrial dynamic factors, meanwhile also led to an increase in mitochondrial activity and number. These results confirmed that TRα could concertedly regulate mitochondrial dynamics, autophagy, and activity, and myogenic regulators rhythmically altered with TRα expression. Summarily, these results suggested that the decline of TRα might cause the degeneration of SM with aging by regulating mitochondrial dynamics, mitophagy and myogenesis.

Effectiveness of Rehabilitation Interventions in Individuals With Emerging Virtual Respiratory Tract Infectious Disease: A Systematic Review and Meta-Analysis

Objective Assessing rehabilitation effectiveness for persistent symptoms post-infection with emerging viral respiratory diseases. Data sources Systematic review of seven databases (MEDLINE, EMBASE, Cochrane Library, PEDro, MedRxiv, CNKI, Wanfang) until 30 December 2023. Review methods Evaluated 101 studies (9593 participants) on respiratory function, exercise capacity, and quality of life. Methodological quality was assessed using the Cochrane Collaboration's Risk of Bias tool for randomized controlled trials (RCTs), the Newcastle-Ottawa Scale (NOS) for observational studies and non-RCTs, and the NIH Quality Assessment Tools for before-after studies. Results The most common rehabilitation program combined breathing exercises with aerobic exercise or strength training. Rehabilitation interventions significantly enhanced respiratory function, as evidenced by improvements on the Borg Scale (MD, −1.85; 95% CI, −3.00 to −0.70, low certainty), the mMRC Dyspnea Scale (MD, −0.45; 95% CI, −0.72 to −0.18, low certainty), and the Multidimensional Dyspnoea-12 Scale (MD, −4.64; 95% CI, −6.54 to −2.74, moderate certainty). Exercise capacity also improved, demonstrated by results from the Six-Minute Walk Test (MD, 38.18; 95% CI, 25.33–51.03, moderate certainty) and the Sit-to-Stand Test (MD, 3.04; 95% CI, 1.07–5.01, low certainty). Conclusion Rehabilitation interventions are promising for survivors of viral respiratory diseases, yet gaps in research remain. Future investigations should focus on personalizing rehabilitation efforts, utilizing remote technology-assisted programs, improving research quality, and identifying specific subgroups for customized rehabilitation strategies to achieve the best outcomes for survivors.

Lactate oxidation in Paracoccus denitrificans

Paracoccus denitrificans has a classical cytochrome-dependent electron transport chain and two alternative oxidases. The classical transport chain is very similar to that in eukaryotic mitochondria. Thus, P. denitrificans can serve as a model of the mammalian mitochondrion that may be more tractable in elucidating mechanisms of regulation of energy production than are mitochondria. In a previous publication we reported detailed studies on respiration in P. denitrificans grown aerobically on glucose or malate. We noted that P. denitrificans has large stores of lactate under various growth conditions. This is surprising because P. denitrificans lacks an NAD+-dependent lactate dehydrogenase. The aim of this study was to investigate the mechanisms of lactate oxidation in P. denitrificans. We found that the bacterium grows well on either d-lactate or l-lactate. Growth on lactate supported a rate of maximum respiration that was equal to that of cells grown on glucose or malate. We report proteomic, metabolomic, and biochemical studies that establish that the metabolism of lactate by P. denitrificans is mediated by two non-NAD+-dependent lactate dehydrogenases. One prefers d-lactate over l-lactate (D-iLDH) and the other prefers l-lactate (L-iLDH). We cloned and produced the D-iLDH and characterized it. The Km for d-lactate was 34 μM, and for l-lactate it was 3.7 mM. Pyruvate was not a substrate, rendering the reaction unidirectional with lactate being converted to pyruvate for entry into the TCA cycle. The intracellular lactate was ∼14 mM such that both isomers could be metabolized by the enzyme. The enzyme has 1 FAD per molecule and utilizes a quinone rather than NAD + as an electron acceptor. D-iLDH provides a direct entry of lactate reducing equivalents into the cytochrome chain, potentially explaining the high respiratory capacity of P. denitrificans in the presence of lactate.

The effect of respiratory muscle training on children and adolescents with cystic fibrosis: a systematic review and meta-analysis

BackgroundCystic fibrosis is a chronic genetic disease that can affect the function of the respiratory system. Previous reviews of the effects of respiratory muscle training in people with cystic fibrosis are uncertain and do not consider the effect of age on disease progression. This systematic review aims to determine the effectiveness of respiratory muscle training in the clinical outcomes of children and adolescents with cystic fibrosis.MethodsUp to July 2023, electronic databases and clinical trial registries were searched. Controlled clinical trials comparing respiratory muscle training with sham intervention or no intervention in children and adolescents with cystic fibrosis. The primary outcomes were respiratory muscle strength, respiratory muscle endurance, lung function, and cough. Secondary outcomes included exercise capacity, quality of life and adverse events. Two review authors independently extracted data and assessed study quality using the Cochrane Risk of Bias Tool 2. The certainty of the evidence was assessed according to the GRADE approach. Meta-analyses where possible; otherwise, take a qualitative approach.ResultsSix studies with a total of 151 participants met the inclusion criteria for this review. Two of the six included studies were published in abstract form only, limiting the available information. Four studies were parallel studies and two were cross-over designs. There were significant differences in the methods and quality of the methodology included in the studies. The pooled data showed no difference in respiratory muscle strength, lung function, and exercise capacity between the treatment and control groups. However, subgroup analyses suggest that inspiratory muscle training is beneficial in increasing maximal inspiratory pressure, and qualitative analyses suggest that respiratory muscle training may benefit respiratory muscle endurance without any adverse effects.ConclusionsThis systematic review and meta-analysis indicate that although the level of evidence indicating the benefits of respiratory muscle training is low, its clinical significance suggests that we further study the methodological quality to determine the effectiveness of training.Trial registrationThe protocol for this review was recorded in the International Prospective Register of Systematic Reviews (PROSPERO) under registration number CRD42023441829.

RANKL signaling drives skeletal muscle into the oxidative profile.

The bone-muscle unit refers to the reciprocal regulation between bone and muscle by mechanical interaction and tissue communication via soluble factors. The RANKL stimulation induces mitochondrial biogenesis and increases the oxidative capacity in osteoclasts and adipocytes. RANKL may bind to the membrane bound RANK or to osteoprotegerin (OPG), a decoy receptor that inhibits RANK-RANKL activation. RANK is highly expressed in skeletal muscle, but the contribution of RANKL to healthy skeletal muscle fiber remains elusive. Here we show that RANKL stimulation in C2C12-derived myotubes induced activation of mitochondrial biogenesis pathways as detected by RNA-seq and western blot. RANKL expanded the mitochondrial reticulum, as shown by mitochondrial DNA quantification and MitoTracker staining, and boosted the spare respiratory capacity. Using MEK and MAPK inhibitors, we found that RANKL signals via ERK and p38 to induce mitochondrial biogenesis. The soleus from OPG-/- and OPG+/- mice showed higher respiratory rates compared to C57BL6/J WT mice, which correlates with high serum RANKL levels. RANKL infusion using a mini-osmotic pump in WT mice increased the number of mitochondria, boosted the respiratory rate, increased succinate dehydrogenase activity in skeletal muscle, and improved the fatigue resistance of gastrocnemius. Therefore, our findings reveal a new role of RANKL as an osteokine-like protein that impacts muscle fiber metabolism.

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.

β-hydroxybutyrate exposure restores mitochondrial function in skeletal muscle satellite cells of critically ill patients

Background and AimDysfunction of skeletal muscle satellite cells might impair muscle regeneration and prolong ICU-acquired weakness, a condition associated with disability and delayed death. This study aimed to elucidate the distinct metabolic effects of critical illness and β-OHB on satellite cells isolated from these patients. MethodsSatellite cells were extracted from vastus lateralis muscle biopsies of patients with ICU-acquired weakness (n = 10) and control group of healthy volunteers or patients undergoing elective hip replacement surgery (n = 10). The cells were exposed to standard culture media supplemented with β-OH-butyrate to assess its influence on cell proliferation by ELISA, mitochondrial functions by extracellular flux analysis, electron transport chain complexes by high resolution respirometry, and ROS production by confocal microscopy. ResultsCritical illness led to a decline in maximal respiratory capacity, ATP production and glycolytic capacity and increased ROS production in ICU patients’ cells. Notably, the function of complex II was impaired due to critical illness but restored to normal levels upon exposure to β-OH-butyrate. While β-OH-butyrate significantly reduced ROS production in both control and ICU groups, it had no significant impact on global mitochondrial functions. ConclusionCritical illness induces measurable bioenergetic dysfunction of skeletal muscle satellite cells. β-OH-butyrate displayed a potential in rectifying complex II dysfunction caused by critical illness and this warrants further exploration.

Identification of mulberry leaf flavonoids and evaluating their protective effects on H2O2-induced oxidative damage in equine skeletal muscle satellite cells.

Introduction: Horses are susceptible to oxidative stress during strenuous endurance exercise, leading to muscle fatigue and damage. Mulberry leaf flavonoids (MLFs) possess significant antioxidant properties. However, the antioxidant efficacy of MLFs can be influenced by the extraction process, and their impact on H2O2-induced oxidative stress in equine skeletal muscle satellite cells (ESMCs) remains unexplored. Methods: Our study employed three extraction methods to obtain MLFs: ultrasound-assisted extraction (CEP), purification with AB-8 macroporous resin (RP), and n-butanol extraction (NB-EP). We assessed the protective effects of these MLFs on H2O2-induced oxidative stress in ESMCs and analyzed the MLF components using metabolomics. Results: The results revealed that pre-treatment with MLFs dose-dependently protected ESMCs against H2O2-induced oxidative stress. The most effective concentrations were 0.8mg/mL of CEP, 0.6mg/mL of RP, and 0.6mg/mL of NB-EP, significantly enhancing EMSC viability (p < 0.05). These optimized MLF concentrations promoted the GSH-Px, SOD and T-AOC activities (p < 0.05), while reducing MDA production (p < 0.05) in H2O2-induced ESMCs. Furthermore, these MLFs enhanced the gene expression, including Nrf2 and its downstream regulatory genes (TrxR1, GPX1, GPX3, SOD1, and SOD2) (p < 0.05). In terms of mitochondrial function, ESMCs pre-treated with MLFs exhibited higher basal respiration, spare respiratory capacity, maximal respiration, ATP-linked respiration compared to H2O2-induced ESMCs (p < 0.05). Additionally, MLFs enhanced cellular basal glycolysis, glycolytic reserve, and maximal glycolytic capacity (p < 0.05). Metabolomics analysis results revealed significant differences in mulberrin, kaempferol 3-O-glucoside [X-Mal], neohesperidin, dihydrokaempferol, and isobavachalcone among the three extraction processes (p < 0.05). Discussion: Our study revealed that MLFs enhance antioxidant enzyme activity, alleviate oxidative damage in ESMCs through the activation of the Nrf2 pathway, and improve mitochondrial respiration and cell energy metabolism. Additionally, we identified five potential antioxidant flavonoid compounds, suggesting their potential incorporation into the equine diet as a strategy to alleviate exercise-induced oxidative stress.

Enhancing CAR-T cell metabolism to overcome hypoxic conditions in the brain tumor microenvironment.

The efficacy of chimeric antigen receptor T cell (CAR-T) therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28 EGFRvIII gliomas revealed impaired mitochondrial ATP production and a markedly hypoxic status compared with ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of the AMPK activator metformin and the mTOR inhibitor rapamycin (Met+Rap). Met+Rap-pretreated mouse CAR-T cells showed activated PPAR-γ coactivator 1α (PGC-1α) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap-pretreated CAR-T cells demonstrated persistent and effective antiglioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap-pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28 EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group, with fewer Ly6c+CD11b+ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions under in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap-pretreated CAR-T cells in human trials.

Abstract LB334: Mechanisms of endocrine resistance to letrozole, fulvestrant, and palbociclib are associated with increased sensitivity to hexyl-cuban-1-yl biguanide inhibitors of CYP3A4 mediated epoxyeicosatrienoic acid biosynthesis

Abstract Introduction: Eicosanoid mediated mechanisms of hormone therapy resistance in ER+HER2- breast cancer are poorly understood. Cytochrome P450 arachidonic acid (AA) epoxygenase-derived epoxyeicosatrienoic acids (EETs) contribute to breast cancer progression by promoting mitochondrial oxidative phosphorylation (OXPHOS). However, it remains unclear how EETs contribute to hormonal therapy resistance. In this study, the well-known letrozole resistant (LR) MCF-7 AC1 cell line overexpressing aromatase was selected for fulvestrant resistance (FR). A clonal cell line resistant to letrozole and fulvestrant (LR/FR) also exhibited resistance to palbociclib (PR) (MCF-7 AC1 LR/FR/PR). Xenograft tumors of this cell line were resistant to letrozole, fulvestrant, and palbociclib. Structure activity studies and modeling led to the development of CYP3A4 AA epoxygenase inhibiting biguanides hexyl cuban-1-yl biguanide (HCB) and fluorinated hexyl derivatives C5F2-HCB and C6F3-HCB. Fluorination on the hexyl moiety led to broader inhibition of EET regioisomer biosynthesis. These agents were tested for inhibition of proliferation, signaling, and OXPHOS/glycolysis balance in the MCF-7 AC1 LR/FR/PR cell line. Results: In the MCF-7 AC1 LR/FR/PR clonal cell line cyclin D1 and estrogen receptor (ER) expression were undetectable, while cyclin E1, CDK4, CDK6, CYP3A4, and c-MYC were upregulated 11, 40, 13, 10, and 12- fold (n=3, p&amp;lt;0.001) compared to the MCF-7 cell line. LC-MS analysis demonstrated 1.8, 1.4, and 1.2- fold higher total cellular levels of (±)8,9, (±)11,12, and (±)14,15-EET in the MCF-7 AC1 LR/FR/PR cell line compared to the MCF-7 cell line (n=3, p&amp;lt;0.05). C6F3-HCB suppressed cellular levels of EETs: (±)8,9-EET by 55% (n=3, p&amp;lt;0.031), (±)11,12-EET by 53% (n=3, p&amp;lt;0.029), and (±)14,15-EET by 60% (n=3, P&amp;lt;0.0001). The MCF-7 AC1 LR/FR/PR cell line was more potently inhibited by hexyl-cuban-1-yl biguanides compared to the MCF-7 cell line, exhibiting lower IC50 values (MCF-7 AC1 LR/FR/PR IC50 vs. MCF-7 IC50; HCB 4.1 vs. 7.0 uM; for C5F2-HCB 10 vs. 25 uM; C6F3-HCB: 5.4 vs. 12 uM; P values all &amp;lt;0.05). Measurement of spare respiratory capacity revealed that the MCF-7 AC1 LR/FR/PR cell line had 4.2% spare respiratory capacity compared to 34% for MCF-7, indicating a lower OXPHOS reserve for the resistant cells. Additionally, the MCF-7 AC1 LR/FR/PR cell line displayed a 2.4-fold higher extracellular acidification rate (ECAR) than the MCF-7 cell line, indicating a higher glycolysis rate (Warburg effect) in the resistant cells. Conclusion: Selection for fulvestrant resistance of MCF-7 AC1 cells resulted in upregulation of CYP3A4 and biosynthesis of hormone therapy resistance associated EETs while correlating with greater sensitivity to fluorinated hexyl-cuban-1-yl biguanides that inhibit EET biosynthesis. Citation Format: Zhijun Guo, Jianxun Lei, Allison Makovec, Swaathi Jayaraman, John R. Hawse, Carol Lange, Elizabeth Ambrose, Gunda I. Georg, Tony D'Assoro, Goetz P. Matthew, David A. Potter. Mechanisms of endocrine resistance to letrozole, fulvestrant, and palbociclib are associated with increased sensitivity to hexyl-cuban-1-yl biguanide inhibitors of CYP3A4 mediated epoxyeicosatrienoic acid biosynthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB334.

Abstract SY35-02: Spatial mapping of mitochondrial networks in lung cancer

Abstract Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional profiling of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform comprised of positron emission tomography (PET) imaging, respirometry and 3-dimensional scanning block-face electron microscopy (3D SBEM). Interestingly, the diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumors align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumor cells. In tumors with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. In contrast, we discovered that in tumors with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated i) peri-nuclear localization of mitochondria, ii) structural remodeling of cristae and iii) mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumors. Structure-and-function studies defining the relationship between mitochondrial architecture and metabolic dependencies may hold promise as strategies to profile metabolic liabilities unique to lung cancer subtypes. We anticipate that coupling PET imaging with 3D SBEM will have dynamic applications beyond that of lung cancer and enrich our understanding of how mitochondrial bioenergetics impact human disease. Citation Format: David B. Shackelford. Spatial mapping of mitochondrial networks in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY35-02.

Targeting Mitochondrial Dynamics during Lower-Limb Ischemia Reperfusion in Young and Old Mice: Effect of Mitochondrial Fission Inhibitor-1 (mDivi-1).

Peripheral arterial disease (PAD) strikes more than 200 million people worldwide and has a severe prognosis by potentially leading to limb amputation and/or death, particularly in older patients. Skeletal muscle mitochondrial dysfunctions and oxidative stress play major roles in this disease in relation with ischemia-reperfusion (IR) cycles. Mitochondrial dynamics through impairment of fission-fusion balance may contribute to skeletal muscle pathophysiology, but no data were reported in the setting of lower-limb IR despite the need for new therapeutic options. We, therefore, investigated the potential protective effect of mitochondrial division inhibitor-1 (mDivi-1; 50 mg/kg) in young (23 weeks) and old (83 weeks) mice submitted to two-hour ischemia followed by two-hour reperfusion on systemic lactate, muscle mitochondrial respiration and calcium retention capacity, and on transcripts specific for oxidative stress and mitochondrial dynamics. At the systemic levels, an IR-related increase in circulating lactate was still major despite mDivi-1 use (+305.9% p < 0.0001, and +269.4% p < 0.0001 in young and old mice, respectively). Further, IR-induced skeletal muscle mitochondrial dysfunctions (more severely impaired mitochondrial respiration in old mice (OXPHOS CI state, -68.2% p < 0.0001 and -84.9% p < 0.0001 in 23- and 83-week mice) and reduced calcium retention capacity (-46.1% p < 0.001 and -48.2% p = 0.09, respectively) were not corrected by mDivi-1 preconditioning, whatever the age. Further, mDivi-1 treatment did not oppose superoxide anion production (+71.4% p < 0.0001 and +37.5% p < 0.05, respectively). At the transcript level, markers of antioxidant enzymes (SOD 1, SOD 2, catalase, and GPx) and fission markers (Drp1, Fis) remained unchanged or tended to be decreased in the ischemic leg. Fusion markers such as mitofusin 1 or 2 decreased significantly after IR in both groups. In conclusion, aging enhanced the deleterious effects or IR on muscle mitochondrial respiration, and in this setting of lower-limb IR, mDivi-1 failed to protect the skeletal muscle both in young and old mice.