Oxidative Phosphorylation Capacity Research Articles

Inhibition of mammalian mtDNA transcription acts paradoxically to reverse diet-induced hepatosteatosis and obesity.

The oxidative phosphorylation system1 in mammalian mitochondria plays a key role in transducing energy from ingested nutrients2. Mitochondrial metabolism is dynamic and can be reprogrammed to support both catabolic and anabolic reactions, depending on physiological demands or disease states. Rewiring of mitochondrial metabolism is intricately linked to metabolic diseases and promotes tumour growth3-5. Here, we demonstrate that oral treatment with an inhibitor of mitochondrial transcription (IMT)6 shifts whole-animal metabolism towards fatty acid oxidation, which, in turn, leads to rapid normalization of body weight, reversal of hepatosteatosis and restoration of normal glucose tolerance in male mice on a high-fat diet. Paradoxically, the IMT treatment causes a severe reduction of oxidative phosphorylation capacity concomitant with marked upregulation of fatty acid oxidation in the liver, as determined by proteomics and metabolomics analyses. The IMT treatment leads to a marked reduction of complex I, the main dehydrogenase feeding electrons into the ubiquinone (Q) pool, whereas the levels of electron transfer flavoprotein dehydrogenase and other dehydrogenases connected to the Q pool are increased. This rewiring of metabolism caused by reduced mtDNA expression in the liver provides a principle for drug treatment of obesity and obesity-related pathology.

PARKIN is not required to sustain OXPHOS function in adult mammalian tissues

Loss-of-function variants in the PRKN gene encoding the ubiquitin E3 ligase PARKIN cause autosomal recessive early-onset Parkinson’s disease (PD). Extensive in vitro and in vivo studies have reported that PARKIN is involved in multiple pathways of mitochondrial quality control, including mitochondrial degradation and biogenesis. However, these findings are surrounded by substantial controversy due to conflicting experimental data. In addition, the existing PARKIN-deficient mouse models have failed to faithfully recapitulate PD phenotypes. Therefore, we have investigated the mitochondrial role of PARKIN during ageing and in response to stress by employing a series of conditional Parkin knockout mice. We report that PARKIN loss does not affect oxidative phosphorylation (OXPHOS) capacity and mitochondrial DNA (mtDNA) levels in the brain, heart, and skeletal muscle of aged mice. We also demonstrate that PARKIN deficiency does not exacerbate the brain defects and the pro-inflammatory phenotype observed in mice carrying high levels of mtDNA mutations. To rule out compensatory mechanisms activated during embryonic development of Parkin-deficient mice, we generated a mouse model where loss of PARKIN was induced in adult dopaminergic (DA) neurons. Surprisingly, also these mice did not show motor impairment or neurodegeneration, and no major transcriptional changes were found in isolated midbrain DA neurons. Finally, we report a patient with compound heterozygous PRKN pathogenic variants that lacks PARKIN and has developed PD. The PARKIN deficiency did not impair OXPHOS activities or induce mitochondrial pathology in skeletal muscle from the patient. Altogether, our results argue that PARKIN is dispensable for OXPHOS function in adult mammalian tissues.

High OXPHOS efficiency in RA-FUdr-differentiated SH-SY5Y cells: involvement of cAMP signalling and respiratory supercomplexes

Neurons are highly dependent on mitochondria to meet their bioenergetic needs and understanding the metabolic changes during the differentiation process is crucial in the neurodegeneration context. Several in vitro approaches have been developed to study neuronal differentiation and bioenergetic changes. The human SH-SY5Y cell line is a widely used cellular model and several differentiation protocols have been developed to induce a neuron-like phenotype including retinoic acid (RA) treatment. In this work we obtained a homogeneous functional population of neuron-like cells by a two-step differentiation protocol in which SH-SY5Y cells were treated with RA plus the mitotic inhibitor 2-deoxy-5-fluorouridine (FUdr). RA-FUdr treatment induced a neuronal phenotype characterized by increased expression of neuronal markers and electrical properties specific to excitable cells. In addition, the RA-FUdr differentiated cells showed an enrichment of long chain and unsaturated fatty acids (FA) in the acyl chain composition of cardiolipin (CL) and the bioenergetic analysis evidences a high coupled and maximal respiration associated with high mitochondrial ATP levels. Our results suggest that the observed high oxidative phosphorylation (OXPHOS) capacity may be related to the activation of the cyclic adenosine monophosphate (cAMP) pathway and the assembly of respiratory supercomplexes (SCs), highlighting the change in mitochondrial phenotype during neuronal differentiation.

Influence of epicardial adipose tissue inflammation and adipocyte size on postoperative atrial fibrillation in patients after cardiovascular surgery.

Epicardial adipose tissue (EAT) is an active endocrine organ that is closely associated with occurrence of atrial fibrillation (AF). However, the role of EAT in the development of postoperative AF (POAF) remains unclear. We aimed to investigate the association between EAT profile and POAF occurrence in patients who underwent cardiovascular surgery. We obtained EAT samples from 53 patients to evaluate gene expression, histological changes, mitochondrial oxidative phosphorylation (OXPHOS) capacity in the EAT, and protein secretion in EAT-conditioned medium. EAT volume was measured using computed tomography scan. Eighteen patients (34%) experienced POAF within 7 days after surgery. Although no significant difference was observed in EAT profile between patients with and without POAF, logistic regression analysis identified that the mRNA expression levels of tumor necrosis factor-alpha (TNF-α) were positively correlated and adipocyte size in the EAT was inversely correlated with onset of POAF, respectively. Mitochondrial OXPHOS capacity in the EAT was not associated with POAF occurrence; however, it showed an inverse correlation with adipocyte size and a positive correlation with adiponectin secretion. In conclusion, changes in the secretory profile and adipocyte morphology of the EAT, which represent qualitative aspects of the adipose tissue, were present before the onset of AF.

The Predictive Value of Graft Viability and Bioenergetics Testing Towards the Outcome in Liver Transplantation.

Donor organ biomarkers with sufficient predictive value in liver transplantation (LT) are lacking. We herein evaluate liver viability and mitochondrial bioenergetics for their predictive capacity towards the outcome in LT. We enrolled 43 consecutive patients undergoing LT. Liver biopsy samples taken upon arrival after static cold storage were assessed by histology, real-time confocal imaging analysis (RTCA), and high-resolution respirometry (HRR) for mitochondrial respiration of tissue homogenates. Early allograft dysfunction (EAD) served as primary endpoint. HRR data were analysed with a focus on the efficacy of ATP production or P-L control efficiency, calculated as 1-L/P from the capacity of oxidative phosphorylation P and non-phosphorylating respiration L. Twenty-two recipients experienced EAD. Pre-transplant histology was not predictive of EAD. The mean RTCA score was significantly lower in the EAD cohort (-0.75 ± 2.27) compared to the IF cohort (0.70 ± 2.08; p = 0.01), indicating decreased cell viability. P-L control efficiency was predictive of EAD (0.76 ± 0.06 in IF vs. 0.70 ± 0.08 in EAD-livers; p = 0.02) and correlated with the RTCA score. Both RTCA and P-L control efficiency in biopsy samples taken during cold storage have predictive capacity towards the outcome in LT. Therefore, RTCA and HRR should be considered for risk stratification, viability assessment, and bioenergetic testing in liver transplantation.

High-dose atorvastatin therapy progressively decreases skeletal muscle mitochondrial respiratory capacity in humans.

BACKGROUNDWhile the benefits of statin therapy on atherosclerotic cardiovascular disease are clear, patients often experience mild to moderate skeletal myopathic symptoms, the mechanism for which is unknown. This study investigated the potential effect of high-dose atorvastatin therapy on skeletal muscle mitochondrial function and whole-body aerobic capacity in humans.METHODSEight overweight (BMI, 31.9 ± 2.0) but otherwise healthy sedentary adults (4 females, 4 males) were studied before (day 0) and 14, 28, and 56 days after initiating atorvastatin (80 mg/d) therapy.RESULTSMaximal ADP-stimulated respiration, measured in permeabilized fiber bundles from muscle biopsies taken at each time point, declined gradually over the course of atorvastatin treatment, resulting in > 30% loss of skeletal muscle mitochondrial oxidative phosphorylation capacity by day 56. Indices of in vivo muscle oxidative capacity (via near-infrared spectroscopy) decreased by 23% to 45%. In whole muscle homogenates from day 0 biopsies, atorvastatin inhibited complex III activity at midmicromolar concentrations, whereas complex IV activity was inhibited at low nanomolar concentrations.CONCLUSIONThese findings demonstrate that high-dose atorvastatin treatment elicits a striking progressive decline in skeletal muscle mitochondrial respiratory capacity, highlighting the need for longer-term dose-response studies in different patient populations to thoroughly define the effect of statin therapy on skeletal muscle health.FUNDINGNIH R01 AR071263.

Immortalised murine R349P desmin knock-in myotubes exhibit a reduced proton leak and decreased ADP/ATP translocase levels in purified mitochondria

Desmin gene mutations cause myopathies and cardiomyopathies. Our previously characterised R349P desminopathy mice, which carry the ortholog of the common human desmin mutation R350P, showed marked alterations in mitochondrial morphology and function in muscle tissue. By isolating skeletal muscle myoblasts from offspring of R349P desminopathy and p53 knock-out mice, we established an immortalised cellular disease model. Heterozygous and homozygous R349P desmin knock-in and wild-type myoblasts could be well differentiated into multinucleated spontaneously contracting myotubes. The desminopathy myoblasts showed the characteristic disruption of the desmin cytoskeleton and desmin protein aggregation, and the desminopathy myotubes showed the characteristic myofibrillar irregularities. Long-term electrical pulse stimulation promoted myotube differentiation and markedly increased their spontaneous contraction rate. In both heterozygous and homozygous R349P desminopathy myotubes, this treatment restored a regular myofibrillar cross-striation pattern as seen in wild-type myotubes. High-resolution respirometry of mitochondria purified from myotubes by density gradient ultracentrifugation revealed normal oxidative phosphorylation capacity, but a significantly reduced proton leak in mitochondria from the homozygous R349P desmin knock-in cells. Consistent with a reduced proton flux across the inner mitochondrial membrane, our quantitative proteomic analysis of the purified mitochondria revealed significantly reduced levels of ADP/ATP translocases in the homozygous R349P desmin knock-in genotype. As this alteration was also detected in the soleus muscle of R349P desminopathy mice, which, in contrast to the mitochondria purified from cultured cells, showed a variety of other dysregulated mitochondrial proteins, we consider this finding to be an early step in the pathogenesis of secondary mitochondriopathy in desminopathy.

Restricting bioenergetic efficiency enhances longevity and mitochondrial redox capacity in Drosophila melanogaster.

Mitochondria are essential for survival and as such, impairments in organelle homeostasis significantly accelerate age-related morbidity and mortality. Here, we determined the contribution of bioenergetic efficiency to life span and health span in Drosophila melanogaster utilizing the mitochondrial uncoupler BAM15. Life span was determined in flies fed a normal diet (ND) or high fat diet (HFD) supplemented with vehicle or BAM15. Locomotor function was determined by negative geotaxis assay in middle-aged flies fed vehicle or BAM15 under ND or HFD conditions. Redox capacity (high-resolution respirometry/fluorometry), citrate synthase (enzyme activity), mtDNA content (qPCR), gene expression (qPCR), and protein expression (western blot) were assessed in flight muscle homogenates of middle-aged flies fed vehicle or BAM15 ND. The molar ratio of H2 O2 and O2 (H2 O2 :O2 ) in a defined respiratory state was calculated as a measure of redox balance. BAM15 extended life span by 9% on ND and 25% on HFD and improved locomotor activity by 125% on ND and 53% on HFD. Additionally, BAM15 enhanced oxidative phosphorylation capacity supported by pyruvate + malate, proline, and glycerol 3-phosphate. Concurrently, BAM15 enhanced the mitochondrial H2 O2 production rate, reverse electron flow from mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) to Complex I, mGPDH, and Complex I without altering the H2 O2 :O2 ratio. BAM15 upregulated transcriptional signatures associated with mitochondrial function and fitness as well as antioxidant defense. BAM15-mediated restriction of bioenergetic efficiency prolongs life span and health span in Drosophila fed a ND or HFD. Improvements in life span and health span in ND were supported by synergistic enhancement of muscular redox capacity.

Effects of 30 days bed rest and exercise countermeasures on PBMC bioenergetics.

Altered mitochondrial function across various tissues is a key determinant of spaceflight-induced physical deconditioning. In comparison to tissue biopsies, blood cell bioenergetics holds promise as a systemic and more readily accessible biomarker, which was evaluated during head-down tilt bed rest (HDTBR), an established ground-based analog for spaceflight-induced physiological changes in humans. More specifically, this study explored the effects of HDTBR and an exercise countermeasure on mitochondrial respiration in peripheral blood mononuclear cells (PBMCs). We subjected 24 healthy participants to a strict 30-day HDTBR protocol. The control group (n = 12) underwent HDTBR only, while the countermeasure group (n = 12) engaged in regular supine cycling exercise followed by veno-occlusive thigh cuffs post-exercise for 6 h. We assessed routine blood parameters 14 days before bed rest, the respiratory capacity of PBMCs via high-resolution respirometry, and citrate synthase activity 2 days before and at day 30 of bed rest. We confirmed PBMC composition by flow cytometry. The change of the PBMC maximal oxidative phosphorylation capacity (OXPHOS) amounted to an 11% increase in the countermeasure group, while it decreased by 10% in the control group (p = 0.04). The limitation of OXPHOS increased in control only while other respiratory states were not affected by either intervention. Correlation analysis revealed positive associations between white blood cells, lymphocytes, and basophils with PBMC bioenergetics in both groups. This study reveals that a regular exercise countermeasure has a positive impact on PBMC mitochondrial function, confirming the potential application of blood cell bioenergetics for human spaceflight.

Functional and Morphological Differences of Muscle Mitochondria in Chronic Fatigue Syndrome and Post-COVID Syndrome.

Patients suffering from chronic fatigue syndrome (CFS) or post-COVID syndrome (PCS) exhibit a reduced physiological performance capability. Impaired mitochondrial function and morphology may play a pivotal role. Thus, we aimed to measure the muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity and assess mitochondrial morphology in CFS and PCS patients in comparison to healthy controls (HCs). Mitochondrial OXPHOS capacity was measured in permeabilized muscle fibers using high-resolution respirometry. Mitochondrial morphology (subsarcolemmal/intermyofibrillar mitochondrial form/cristae/diameter/circumference/area) and content (number and proportion/cell) were assessed via electron microscopy. Analyses included differences in OXPHOS between HC, CFS, and PCS, whereas comparisons in morphology/content were made for CFS vs. PCS. OXPHOS capacity of complex I, which was reduced in PCS compared to HC. While the subsarcolemmal area, volume/cell, diameter, and perimeter were higher in PCS vs. CFS, no difference was observed for these variables in intermyofibrillar mitochondria. Both the intermyofibrillar and subsarcolemmal cristae integrity was higher in PCS compared to CFS. Both CFS and PCS exhibit increased fatigue and impaired mitochondrial function, but the progressed pathological morphological changes in CFS suggest structural changes due to prolonged inactivity or unknown molecular causes. Instead, the significantly lower complex I activity in PCS suggests probably direct virus-induced alterations.

OXPHOS capacity is diminished and the phosphorylation system inhibited during diapause in an extremophile, embryos of Artemia franciscana.

Diapause exhibited by embryos of Artemia franciscana is accompanied by severe arrest of respiration. A large fraction of this depression is attributable to downregulation of trehalose catabolism that ultimately restricts fuel to mitochondria. This study now extends knowledge on the mechanism by revealing metabolic depression is heightened by inhibitions within mitochondria. Compared with that in embryo lysates during post-diapause, oxidative phosphorylation (OXPHOS) capacity P is depressed during diapause when either NADH-linked substrates (pyruvate and malate) for electron transfer (electron transfer capacity, E) through respiratory Complex I or the Complex II substrate succinate are used. When pyruvate, malate and succinate were combined, respiratory inhibition by the phosphorylation system in diapause lysates was discovered as judged by P/E flux control ratios (two-way ANOVA; F1,24=38.78; P<0.0001). Inhibition was eliminated as the diapause extract was diluted (significant interaction term; F2,24=9.866; P=0.0007), consistent with the presence of a diffusible inhibitor. One candidate is long-chain acyl-CoA esters known to inhibit the adenine nucleotide translocator. Addition of oleoyl-CoA to post-diapause lysates markedly decreased the P/E ratio to 0.40±0.07 (mean±s.d.; P=0.002) compared with 0.79±0.11 without oleoyl-CoA. Oleoyl-CoA inhibits the phosphorylation system and may be responsible for the depressed P/E in lysates from diapause embryos. With isolated mitochondria, depression of P/E by oleoyl-CoA was fully reversed by addition of l-carnitine (control versus recovery with l-carnitine, P=0.338), which facilitates oleoyl-CoA transport into the matrix and elimination by β-oxidation. In conclusion, severe metabolic arrest during diapause promoted by restricting glycolytic carbon to mitochondria is reinforced by depression of OXPHOS capacity and the phosphorylation system.

SARM1 regulates NAD+-linked metabolism and select immune genes in macrophages

Sterile alpha and HEAT/armadillo motif-containing protein (SARM1) was recently described as a NAD+-consuming enzyme and has previously been shown to regulate immune responses in macrophages. Neuronal SARM1 is known to contribute to axon degeneration due to its NADase activity. However, how SARM1 affects macrophage metabolism has not been explored. Here, we show that macrophages from Sarm1-/- mice display elevated NAD+ concentrations and lower cyclic ADP-ribose, a known product of SARM1-dependent NAD+ catabolism. Further, SARM1-deficient macrophages showed an increase in the reserve capacity of oxidative phosphorylation and glycolysis compared to WT cells. Stimulation of macrophages to a proinflammatory state by lipopolysaccharide (LPS) revealed that SARM1 restricts the ability of macrophages to upregulate glycolysis and limits the expression of the proinflammatory gene interleukin (Il) 1b, but boosts expression of anti-inflammatory Il10. In contrast, we show macrophages lacking SARM1 induced to an anti-inflammatory state by IL-4 stimulation display increased oxidative phosphorylation and glycolysis, and reduced expression of the anti-inflammatory gene, Fizz1. Overall, these data show that SARM1 fine-tunes immune gene transcription in macrophages via consumption of NAD+ and altered macrophage metabolism.

Quantitative Assessment of Mitochondrial Morphology and Electrophysiological Function in the Diabetic Heart.

Mitochondria within a cardiomyocyte form a highly dynamic network that undergoes fusion and fission events in response to acute and chronic stressors, such as hyperglycemia and diabetes mellitus. Changes in mitochondrial architecture and morphology not only reflect their capacity for oxidative phosphorylation and ATP synthesis but also impact their subcellular localization and interaction with other organelles. The role of these ultrastructural abnormalities in modulating electrophysiological properties and excitation-contraction coupling remains largely unknown and warrants direct investigation considering the growing appreciation of the functional and structural coupling between the mitochondrial network, the calcium cycling machinery, and sarcolemmal ion channels in the cardiac myocyte. In this Methods in Molecular Biology chapter, we provide a protocol that allows for a quantitative assessment of mitochondrial shape and morphology in control and diabetic hearts that had undergone detailed electrophysiological measurements using high resolution optical action potential (AP) mapping.

IRW improves diet-induced non-alcoholic fatty liver disease by reducing steatosis associated with increased capacity for oxidative phosphorylation

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, remains without approved pharmacological treatment. Food-derived bioactive peptides can aid in the management of metabolic conditions including hypertension, obesity and insulin resistance. IRW (isoleucine-arginine-tryptophan) is a tripeptide produced from egg white with angiotensin converting enzyme-inhibitory properties. IRW supplementation elicits antihypertensive effects, improves skeletal muscle insulin signaling and glucose tolerance while reducing body weight gain. In this study, we hypothesized that IRW supplementation would prevent high-fat diet (HFD)-induced NAFLD by modulating hepatic lipid metabolism and increasing mitochondrial content. We found that IRW prevents diet-induced NAFLD, while rosiglitazone (ROSI) treatment worsens it. IRW decreases hepatic triglyceride content and lipid droplet size compared to HFD and ROSI. IRW increases the hepatic mitochondrial complexes and citrate synthase activity, phosphorylation of 5'-AMP-activated protein kinase and microsomal triglyceride transfer protein abundance compared to HFD. Compared with ROSI, IRW increases phosphorylated acetyl CoA carboxylase and mitochondrial complexes. Overall, the hypothesis is supported by these findings.

Molecular mechanisms underpinning favourable physiological adaptations to exercise prehabilitation for urological cancer surgery.

Surgery for urological cancers is associated with high complication rates and survivors commonly experience fatigue, reduced physical ability and quality of life. High-intensity interval training (HIIT) as surgical prehabilitation has been proven effective for improving the cardiorespiratory fitness (CRF) of urological cancer patients, however the mechanistic basis of this favourable adaptation is undefined. Thus, we aimed to assess the mechanisms of physiological responses to HIIT as surgical prehabilitation for urological cancer. Nineteen male patients scheduled for major urological surgery were randomised to complete 4-weeks HIIT prehabilitation (71.6 ± 0.75 years, BMI: 27.7 ± 0.9 kg·m2) or a no-intervention control (71.8 ± 1.1 years, BMI: 26.9 ± 1.3 kg·m2). Before and after the intervention period, patients underwent m. vastus lateralis biopsies to quantify the impact of HIIT on mitochondrial oxidative phosphorylation (OXPHOS) capacity, cumulative myofibrillar muscle protein synthesis (MPS) and anabolic, catabolic and insulin-related signalling. OXPHOS capacity increased with HIIT, with increased expression of electron transport chain protein complexes (C)-II (p = 0.010) and III (p = 0.045); and a significant correlation between changes in C-I (r = 0.80, p = 0.003), C-IV (r = 0.75, p = 0.008) and C-V (r = 0.61, p = 0.046) and changes in CRF. Neither MPS (1.81 ± 0.12 to 2.04 ± 0.14%·day-1, p = 0.39) nor anabolic or catabolic proteins were upregulated by HIIT (p > 0.05). There was, however, an increase in phosphorylation of AS160Thr642 (p = 0.046) post-HIIT. A HIIT surgical prehabilitation regime, which improved the CRF of urological cancer patients, enhanced capacity for skeletal muscle OXPHOS; offering potential mechanistic explanation for this favourable adaptation. HIIT did not stimulate MPS, synonymous with the observed lack of hypertrophy. Larger trials pairing patient-centred and clinical endpoints with mechanistic investigations are required to determine the broader impacts of HIIT prehabilitation in this cohort, and to inform on future optimisation (i.e., to increase muscle mass).

Skeletal muscle mitochondria demonstrate similar respiration per cristae surface area independent of training status and sex in healthy humans.

The impact of training status and sex on intrinsic skeletal muscle mitochondrial respiratory capacity remains unclear. We examined this by analysing human skeletal muscle mitochondrial respiration relative to mitochondrial volume and cristae density across training statuses and sexes. Mitochondrial cristae density was estimated in skeletal muscle biopsies originating from previous independent studies. Participants included females (n=12) and males (n=41) across training statuses ranging from untrained (UT, n=8), recreationally active (RA, n=9), active-to-elite runners (RUN, n=27) and cross-country skiers (XC, n=9). The XC and RUN groups demonstrated higher mitochondrial volume density than the RA and UT groups while all active groups (RA, RUN and XC) displayed higher mass-specific capacity of oxidative phosphorylation (OXPHOS) and mitochondrial cristae density than UT. Differences in OXPHOS diminished between active groups and UT when normalising to mitochondrial volume density and were lost when normalising to muscle cristae surface area density. Moreover, active females (n=6-9) and males (n=15-18) did not differ in mitochondrial volume and cristae density, OXPHOS, or when normalising OXPHOS to mitochondrial volume density and muscle cristae surface area density. These findings demonstrate: (1) differences in OXPHOS between active and untrained individuals may be explained by both higher mitochondrial volume and cristae density in active individuals, with no difference in intrinsic mitochondrial respiratory capacity (OXPHOS per muscle cristae surface area density); and (2) no sex differences in mitochondrial volume and cristae density or mass-specific and normalised OXPHOS. This highlights the importance of normalising OXPHOS to muscle cristae surface area density when studying skeletal muscle mitochondrial biology. KEY POINTS: Oxidative phosphorylation is the mitochondrial process by which ATP is produced, governed by the electrochemical gradient across the inner mitochondrial membrane with infoldings named cristae. In human skeletal muscle, the mass-specific capacity of oxidative phosphorylation (OXPHOS) can change independently of shifts in mitochondrial volume density, which may be attributed to variations in cristae density. We demonstrate that differences in skeletal muscle OXPHOS between healthy females and males, ranging from untrained to elite endurance athletes, are matched by differences in cristae density. This suggests that higher OXPHOS in skeletal muscles of active individuals is attributable to an increase in the density of cristae. These findings broaden our understanding of the variability in human skeletal muscle OXPHOS and highlight the significance of cristae, specific to mitochondrial respiration.

TRAF3-Inactivated Chronic Lymphocytic Leukemia Cells Show an Enhanced Metabolic Plasticity That Can be Attenuated By Glutaminolysis and Mitochondrial Pyruvate Import Inhibition

I ntroduction: TRAF3 gene is located in 14q32.32 and has been identified as a tumor suppressor gene involved in B-cell survival, immune response, and cellular metabolism. TRAF3 is frequently encompassed within 14q deletion and affected by inactivating mutations in 4% of chronic lymphocytic leukemia (CLL) patients. Recently, our group found that CLL patients harboring biallelic TRAF3 inactivation (deletion and mutation) showed a dismal prognosis, with a short time to first treatment (Pérez Carretero, AJH 2022). However, the biological role of TRAF3 alterations in CLL pathogenesis remains to be elucidated. Thus, the aims of this study wereto assess the functional consequences of these alterations and to determine their impact on therapy response. Methods: Previously reported TRAF3 mutations were mimicked in a CRISPR/Cas9-edited CLL model by using the PGA1 cell line (PGA1- TRAF3 mut). Metabolomic (liquid chromatography-mass spectrometry), transcriptomic (RNAseq) and functional analyses were carried out. To evaluate the metabolic specificities of PGA1- TRAF3 mut cells, oxidative phosphorylation (OXPHOS) and glycolytic capacity were studied by assessing the mitochondrial oxygen consumption rate (OCR) and extracellular acidification (ECAR), respectively. Additionally, we assessed the metabolite profile of 25 primary CLL cells (n=3 TRAF3 mut cells, n=22 TRAF3 wt cells) and their response to a library of drugs (CLL drugs and metabolic inhibitors), in the presence and absence of microenvironment (with and without HS-5 co-culture and IL-2 and CpG stimulation). Results: Metabolomic analysis of CRISPR/Cas9-edited CLL models showed an enrichment of metabolites involved in Warburg effect (pyruvate, phosphoenolpyruvate, and lactate), citric acid cycle (TCA- acetyl-CoA, α-ketoglutarate, succinate) and glutamate metabolism (α-ketoglutarate) in PGA1- TRAF3 mut cells. Moreover, PGA1- TRAF3 mut cells exhibited higher basal and maximal respiration levels and a more important spare capacity and generation of ATP-linked to mitochondrial respiration. Glycolysis was also slightly enhanced in the TRAF3 mut cells. Remarkably, primary CLL cells with TRAF3 mutations presented a higher concentration of phosphoenolpyruvate, glutamate and the TCA intermediates fumarate and malate, as well as higher levels of NADH and ATP, proving an increased energy production in these cells. Altogether, these results reveal that TRAF3 mut cells showed an enhanced mitochondrial glycolytic metabolism. Given the metabolic changes identified in TRAF3 mut cells, we subsequently assessed their OCR and ECAR after treatment with metabolic inhibitors. Unlike in WT cells, UK5099 administration (inhibitor of the mitochondrial pyruvate transporter that regulates mitochondrial glycolysis) induced an increase in the maximal respiration of TRAF3 mut cells, suggesting a metabolic reprogramming towards an alternative metabolic way fueling mitochondrial metabolism. Therefore, we tested the effect of C968 (glutaminolysis inhibitor), finding the same effect on TRAF3 mut cells, which reveals a specific metabolic plasticity (mitochondrial glycolysis/glutaminolysis). Notably, the transcriptomic analysis revealed an upregulation of the glutamine transporter SLC1A4 (p-adj=0.007), what could be favoring glutamine uptake for an enhanced glutaminolysis. Interestingly, by blocking pyruvate import and glutaminolysis simultaneously, a decrease in TRAF3 mut CLL cells proliferation was observed (p=0.03), with equal levels of the previously dysregulated metabolites to those of the WT, which may indicate that this treatment sensitizes TRAF3 mut cells through its relieving effect on metabolic reprogramming. Moreover, primary CLL cells with TRAF3 mutations were also sensitive to the combination, in the presence and absence of microenvironment (p=0.01, p=0.04 respectively), demonstrating the potential of the combination of metabolic inhibitors as a new therapeutic approach in TRAF3 mut patients. Conclusion: Our work identifies a novel role of TRAF3 alterations in the CLL cellular metabolism. Specifically, TRAF3 inactivation induced an increased mitochondrial glycolytic activity and distinct metabolic dependencies in CLL cells, contributing to an enhanced metabolic plasticity (mitochondrial glycolysis/glutaminolysis) potentially targetable in CLL. Funding: PI21/00983 FEHH-GELLC(CPC), JYCL(ARS)

Restoration of LAT Signaling in CD33-Directed CAR T Cells Improves Potency and Persistence Against Acute Myeloid Leukemia

Despite the development of novel therapies, the majority of patients with acute myeloid leukemia (AML) will not achieve long-term remissions. Patients with relapsed/refractory (r/r) AML have particularly poor outcomes, as conventional chemotherapy regimens are often unable to elicit long-term remissions. Allogeneic hematopoietic stem cell transplant (HSCT) is potentially curative in eligible patients, however, bridging therapies which can induce deep remissions prior to HSCT are needed. Second generation chimeric antigen receptor (CAR) T cells targeting lineage-restricted antigens, such as CD19, have induced remissions in up to 80 - 90% of pediatric patients with r/r B-lineage acute lymphoblastic leukemia (ALL), however, post-CAR relapses are common and only ~50% of treated patients achieve durable remissions. Similar strategies targeting myeloid-restricted antigens with 2 nd generation CAR T cells in AML have not replicated the success seen in ALL. As clinical experience has highlighted the impact of CAR T cell potency, persistence, and antigen-sensitivity to long-term efficacy in ALL, strategies to boost these properties in myeloid-directed CAR T cells are necessary to extend the clinical benefit of this therapy to patients with AML. Utilizing a global phosphoproteomics screen, we recently identified that inefficient phosphorylation of the Linker for activation of T cells (LAT) and the subsequent decrease in the downstream signaling pathways results in suboptimal CAR T cell activation. To overcome inefficient LAT activation, we developed a novel bicistronic CAR platform, consisting of a 2 nd generation CAR along with an Adjunctive LAT-Activating CAR (ALA-CART) which improved sensitivity to low-antigen leukemia cells, increased in vivo potency, and enhanced persistence in xenograft models of ALL. We hypothesized that the restoration of LAT signaling via the ALA-CART platform would also improve potency and persistence in pre-clinical models of AML. We found that while CD33 ALA-CART cells responded to CD33+ AML cells in vitro, they were less cytotoxic and produced lower levels of cytokines than CD33-28z or CD33-BBz 2 ndgeneration CAR T cells. However, CD33 ALA-CART cells demonstrated increased potency in xenograft models, eradicating AML cells at much lower doses than CD33-28z and CD33-BBz CAR T cells (Fig. 1A, p&amp;lt;0.0001). This in vivo potency correlated with an enrichment of T stem cell memory cells in the ALA-CART product, resulting in increased persistence of CD33 ALA-CART cells after leukemia clearance (Fig. 1B, p=0.0139). The ALA-CART platform was associated with increased mitochondrial mass and function, suggesting that the increased signaling through LAT results in an enhanced capacity for oxidative phosphorylation, potentially contributing to the increased persistence of CD33 ALA-CART cells in vivo. Thus, the ALA-CART platform, designed to restore LAT signaling in CAR T cells, demonstrates that CAR molecules can be rationally designed to enhance both in vivo potency and persistence, and represents a promising strategy for the development for more effective AML-directed CAR T cells.

Parental cardiorespiratory fitness influences early life energetics and metabolic health.

High cardiorespiratory fitness (CRF) is associated with a reduced risk of metabolic disease and is linked to superior mitochondrial respiratory function. This study investigated how intrinsic CRF affects bioenergetics and metabolic health in adulthood and early life. Adult rats selectively bred for low and high running capacity [low capacity runners (LCR) and high capacity runners (HCR), respectively] underwent metabolic phenotyping before mating. Weanlings were evaluated at 4-6 wk of age, and whole body energetics and behavior were assessed using metabolic cages. Mitochondrial respiratory function was assessed in permeabilized tissues through high-resolution respirometry. Proteomic signatures of adult and weanling tissues were determined using mass spectrometry. The adult HCR group exhibited lower body mass, improved glucose tolerance, and greater physical activity compared with the LCR group. The adult HCR group demonstrated higher mitochondrial respiratory capacities in the soleus and heart compared with the adult LCR group, which coincided with a greater abundance of proteins involved in lipid catabolism. HCR and LCR weanlings had similar body mass, but HCR weanlings displayed reduced adiposity. In addition, HCR weanlings exhibited better glucose tolerance and higher physical activity levels than LCR weanlings. Higher respiratory capacities were observed in the soleus, heart, and liver tissues of HCR weanlings compared with LCR weanlings, which were not owed to greater mitochondrial content. Proteomic analyses indicated a greater potential for lipid oxidation in the contractile muscles of HCR weanlings. In conclusion, offspring born to parents with high CRF possess an enhanced capacity for lipid catabolism and oxidative phosphorylation, thereby influencing metabolic health. These findings highlight that intrinsic CRF shapes the bioenergetic phenotype with implications for metabolic resilience in early life.NEW & NOTEWORTHY Inherited cardiorespiratory fitness (CRF) influences early life bioenergetics and metabolic health. Higher intrinsic CRF was associated with reduced adiposity and improved glucose tolerance in early life. This metabolic phenotype was accompanied by greater mitochondrial respiratory capacity in skeletal muscle, heart, and liver tissue. Proteomic profiling of these three tissues further revealed potential mechanisms linking inherited CRF to early life metabolism.

Hyperbaric Oxygen Therapy Alleviates Memory and Motor Impairments Following Traumatic Brain Injury via the Modulation of Mitochondrial-Dysfunction-Induced Neuronal Apoptosis in Rats.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality in young adults, characterized by primary and secondary injury. Primary injury is the immediate mechanical damage, while secondary injury results from delayed neuronal death, often linked to mitochondrial damage accumulation. Hyperbaric oxygen therapy (HBOT) has been proposed as a potential treatment for modulating secondary post-traumatic neuronal death. However, the specific molecular mechanism by which HBOT modulates secondary brain damage through mitochondrial protection remains unclear. Spatial learning, reference memory, and motor performance were measured in rats before and after Controlled Cortical Impact (CCI) injury. The HBOT (2.5 ATA) was performed 4 h following the CCI and twice daily (12 h intervals) for four consecutive days. Mitochondrial functions were assessed via high-resolution respirometry on day 5 following CCI. Moreover, IHC was performed at the end of the experiment to evaluate cortical apoptosis, neuronal survival, and glial activation. The current result indicates that HBOT exhibits a multi-level neuroprotective effect. Thus, we found that HBOT prevents cortical neuronal loss, reduces the apoptosis marker (cleaved-Caspase3), and modulates glial cell proliferation. Furthermore, HBO treatment prevents the reduction in mitochondrial respiration, including non-phosphorylation state, oxidative phosphorylation, and electron transfer capacity. Additionally, a superior motor and spatial learning performance level was observed in the CCI group treated with HBO compared to the CCI group. In conclusion, our findings demonstrate that HBOT during the critical period following the TBI improves cognitive and motor damage via regulating glial proliferation apoptosis and protecting mitochondrial function, consequently preventing cortex neuronal loss.