Photodegradation measured by high-resolution respirometry in leaf preparations

The acute systemic inflammatory response syndrome (SIRS) and multiorgan dysfunction (MOD) that occur in large burn injuries may be attributed, in part, to immunosuppressive responses such as decreased lymphocytes. However, the mitochondrial bioenergetics of lymphocytes after severe burn injury are poorly understood. The purpose of this study was to examine mitochondrial function of lymphocytes following severe burns in a swine model. Anesthetized Yorkshire swine (n = 17) sustained 40% total body surface area full-thickness contact burns. Blood was collected at pre-injury (Baseline; BL) and at 24 and 48 h after injury for complete blood cell analysis, flow cytometry, cytokine analysis, and ficoll separation of intact lymphocytes for high-resolution mitochondrial respirometry analysis. While neutrophil numbers increased, a concomitant decrease was found in lymphocytes (P < 0.001) after burn injury, which was not specific to CD4+ or CD8+ lymphocytes. No changes in immune cell population were observed from 24 h to 48 h post-injury. IL 12-23 decreased while a transient increase in IL 4 was found from BL to 24h (P < 0.05). CRP progressively increased from BL to 24h (P < 0.05) and 48h (P < 0.001) post-injury. Routine and maximal mitochondrial respiration progressively increased from BL to 24h (P < 0.05) and 48 h post-injury (P < 0.001). No changes were found in leak respiration or residual oxygen consumption. When considering the reduction in lymphocyte number, the total peripheral lymphocyte bioenergetics per volume of blood significantly decreased from BL to 24h and 48h (P < 0.05). For the first time, we were able to measure mitochondrial activity in intact lymphocyte mitochondria through high-resolution respirometry in a severely burned swine model. Our data showed that the non-specific reduction in peripheral T cells after injury was larger than the increased mitochondrial activity in those cells, which may be a compensatory mechanism for the total reduction in lymphocytes. Additional studies in the metabolic activation of T cell subpopulations may provide diagnostic or therapeutic targets after severe burn injury.

Background: Spinal muscular atrophy (SMA) is a children’s neuromuscular disorder. Although motor neuron loss is a major feature of the disease, we have identified fatty acid abnormalities in SMA patients and in preclinical animal models, suggesting metabolic perturbation is also an important component of SMA. Methods: Biochemical, histological, proteomic, and high resolution respirometry were used. Results: SMA patients are more susceptible to dyslipidemia than the average population as determined by a standard lipid profile in a cohort of 72 pediatric patients. As well, we observed a non-alcoholic liver disease phenotype in apreclinical mouse model. Denervation alone was not sufficient to induce liver steatosis, as a mouse model of ALS, did not develop fatty liver. Hyperglucagonemia in Smn2B/-mice could explain the hepatic steatosis by increasing plasma substrate availability via glycogen depletion and peripheral lipolysis. Proteomic analysis identified mitochondrion and lipid metabolism as major clusters. Alterations in mitochondrial function were revealed by high-resolution respirometry. Finally, low-fat diets led to increased survival in Smn2B/-mice. Conclusions: These results provide strong evidence for lipid metabolism defects in SMA. Further investigation will be required to establish the primary mechanism of these alterations and understand how they lead to additional co-morbidities in SMA patients.

IntroductionDuring racing, mitochondrial oxidative phosphorylation (OXPHOS) is a major source of energy for muscle contraction. Assessment of muscle OXPHOS capacity by high‐resolution respirometry (HRR) might identify animals with altered energy metabolism and consequently increased risk of exertional rhabdomyolysis (ER).MethodsProspective cohort study. Ten healthy French Standardbreds in active training, aged 4–7 years, recruited at the start of a competitive racing season, underwent a standardised field exercise test with determination of speed at onset of lactate accumulation (VLa4) and measurement of serum creatine kinase activity (CK) two hours post‐exercise. Triceps brachii muscle microbiopsies were obtained for determination of OXPHOS and electron transfer system (ETS) capacities with HRR. Associations between HRR variables and occurrence of ER over the following four months of racing were analysed using univariate logistic regression.ResultsTwo horses developed ER during the study period: one within one month of recruitment, another presented multiple minor episodes over the season. For these ER horses OXPHOS/ETS ratio was lower, and Complex I/Complex I + II ratio and Complex I /Complex II ratio were higher than non‐affected horses in the study (perfect prediction of occurrence of ER with ratio of ≤0.74, ≥0.60 and ≥0.715 respectively). VLa4 for ER horses was within the range of non‐affected horses. Post‐exercise CK was within normal limits for all horses in the study.ConclusionsOXPHOS and ETS capacities and their derived ratios indicate dysfunctional muscle energy metabolism whereas VLa4 and CK were unremarkable in horses subsequently affected by ER.Ethical Animal ResearchThe study was approved by Liège University's Animal Ethics Committee (agreement N° 07‐629); owner informed consent was obtained. Sources of funding: Part of the study protocol was funded by Les Haras Nationaux (France). Competing interests: The Oxygraph‐2k HRR apparatus was provided on loan by OROBOROS Instruments (Innsbruck, Austria); there are no patents, products in development or marketed products to declare.

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm mitochondrial activity, so the measurement of sperm mitochondrial function is an indicator of sperm quality. High-resolution respirometry is a method of measuring the oxygen consumption of cells or tissues in a closed-chamber system. This technique can be implemented to measure respiration in human sperm and provides information about the quality and integrity of the sperm mitochondria. High-resolution respirometry allows the cells to move freely, which is an a priori advantage in the case of sperm. This technique can be applied with intact or permeabilized spermatozoa and allows for the study of intact sperm mitochondrial function and the activity of individual respiratory chain complexes. The high-resolution oxygraph instrument uses sensors to measure the oxygen concentration coupled with sensitive software to calculate the oxygen consumption. The data are used to calculate respiratory indices based on the oxygen consumption ratios. Consequently, the indices are the proportions of two oxygen consumption rates and are internally normalized to the cell number or protein mass. The respiratory indices are an indicator of sperm mitochondrial function and dysfunction.

The lifetime risk of developing heart failure is approximately 20%, and survival rates remain poor. Myocardial mitochondrial function has been suggested to play a pivotal role in heart failure pathophysiology. Human studies on ex vivo mitochondrial function have mostly been limited to atrial tissue obtained during open heart surgery and have provided contradictory results. This study aimed at measuring myocardial mitochondrial function in transcatheter ventricular endomyocardial biopsies and assessing the relationship between oxidative capacity and heart function. We enrolled 40 heart failure patients undergoing ventricular assist device surgery or heart transplantation (34 males, age 57 ± 11 years, body mass index 26.6 ± 4.8 kg/m2) and 29 heart transplant recipients of comparable age and body mass index with normal left ventricular function undergoing surveillance biopsies (23 males, 57 ± 12 years, body mass index 26.2 ± 4.1 kg/m2). High-resolution respirometry was established in the myocardium to measure oxidative capacity ex vivo. The mitochondrial oxidative capacity was 90% higher in ventricular compared to atrial tissues (n = 11, p < 0.01) of explanted hearts. Respiration rates were comparable in ventricular samples of heart failure patients obtained during open heart surgery by standard tissue preparation or ex vivo endomyocardial biopsy (r = 0.9988, p < 0.0001, n = 8), and the mitochondrial oxidative capacity in samples from these patients remained stable for 8 h when stored in either of two common preservation buffers. The oxidative capacity was 44% lower in heart failure than in transplant recipients (67 ± 3 vs. 97 ± 5 pmol/[s mg], p < 0.0001) and correlated positively with heart function (r = 0.49, p < 0.01). High-resolution respirometry of ventricular tissue is feasible in transcatheter biopsies, facilitating clinical studies on myocardial mitochondrial function in patients not undergoing heart surgery.

Alterations in mitochondrial respiration are an important hallmark of Huntington’s disease (HD), one of the most common monogenetic causes of neurodegeneration. The ubiquitous expression of the disease causing mutant huntingtin gene raises the prospect that mitochondrial respiratory deficits can be detected in skeletal muscle. While this tissue is readily accessible in humans, transgenic animal models offer the opportunity to cross-validate findings and allow for comparisons across organs, including the brain. The integrated respiratory chain function of the human vastus lateralis muscle was measured by high-resolution respirometry (HRR) in freshly taken fine-needle biopsies from seven pre-manifest HD expansion mutation carriers and nine controls. The respiratory parameters were unaffected. For comparison skeletal muscle isolated from HD knock-in mice (HdhQ111) as well as a broader spectrum of tissues including cortex, liver and heart muscle were examined by HRR. Significant changes of mitochondrial respiration in the HdhQ knock-in mouse model were restricted to the liver and the cortex. Mitochondrial mass as quantified by mitochondrial DNA copy number and citrate synthase activity was stable in murine HD-model tissue compared to control. mRNA levels of key enzymes were determined to characterize mitochondrial metabolic pathways in HdhQ mice. We demonstrated the feasibility to perform high-resolution respirometry measurements from small human HD muscle biopsies. Furthermore, we conclude that alterations in respiratory parameters of pre-manifest human muscle biopsies are rather limited and mirrored by a similar absence of marked alterations in HdhQ skeletal muscle. In contrast, the HdhQ111 murine cortex and liver did show respiratory alterations highlighting the tissue specific nature of mutant huntingtin effects on respiration.

The aim of the present study was to examine whether improvements in pulmonary oxygen uptake (V̇o2) kinetics following a short period of high-intensity training (HIT) would be associated with improved skeletal muscle mitochondrial function. Ten untrained male volunteers (age 26 ± 2 yr; mean ± SD) performed six HIT sessions (8-12 × 60 s at incremental test peak power; 271 ± 52 W) over a 2-wk period. Before and after the HIT period, V̇o2 kinetics was modeled during moderate-intensity cycling (110 ± 19 W). Mitochondrial function was assessed with high-resolution respirometry (HRR), and maximal activities of oxidative enzymes citrate synthase (CS) and cytochrome c oxidase (COX) were accordingly determined. In response to HIT, V̇o2 kinetics became faster (τ: 20.4 ± 4.4 vs. 28.9 ± 6.1 s; P < 0.01) and fatty acid oxidation (ETFP) and leak respiration (LN) both became elevated (P < 0.05). Activity of CS and COX did not increase in response to training. Both before and after the HIT period, fast V̇o2 kinetics (low τ values) was associated with large values for ETFP, electron transport system capacity (ETS), and electron flow specific to complex II (CIIP) (P < 0.05). Collectively, these findings support that selected measures of mitochondrial function obtained with HRR are important for fast V̇o2 kinetics and better markers than maximal oxidative enzyme activity in describing the speed of the V̇o2 response during moderate-intensity exercise.

Phosphocreatine attenuates doxorubicin-induced nephrotoxicity through inhibition of apoptosis, and restore mitochondrial function via activation of Nrf2 and PGC-1α pathways

Multiple system atrophy (MSA) is a fatal neurodegenerative disease that belongs to the family of α-synucleinopathies. At post mortem examination, intracellular inclusions of misfolded α-synuclein are found in neurons and oligodendrocytes and are considered to play a significant role in the pathogenesis. However, the early steps of the disease process are unknown and difficult to study in tissue derived from end-stage disease. Induced pluripotent stem cells (iPSCs) were generated from patients' and control skin fibroblasts and differentiated into NCAM-positive neural progenitor cells (NPCs). The mitochondrial morphology and function were assessed by immunocytochemistry and high resolution respirometry. The ability to cope with exogenous oxidative stress was tested by exposure to different doses of luperox. The expression of α-synuclein was studied by immunocytochemistry. We identified increased tubulation of mitochondria with preserved respiration profile in MSA-derived NPCs. Exposure of these cells to exogenous oxidative stress even at low doses, triggered an excessive generation of reactive oxygen species (ROS) and cleavage of caspase-3. MSA-derived NPCs did not present changed levels of SNCA gene expression nor intracellular aggregates of α-synuclein. However, we identified disease-related translocation of α-synuclein to the nucleus. Our results show early cellular dysfunction in MSA-derived NPCs. We identified changes in the redox homeostasis which are functionally compensated at baseline but cause increased susceptibility to exogenous oxidative stress. In addition, nuclear translocation of α-synuclein in MSA-derived NPCs supports an early cellular stress response which may precede the neurodegenerative process in this disorder.

The effects of exercise training on the skeletal muscle (SKM) lipidome and mitochondrial function have not been thoroughly explored in individuals with type 2 diabetes (T2D). We investigated the effects of exercise training on molecular signatures within SKM of T2D, employing integrated multi-omics data analyses including lipidomics [MS/MS-shotgun], global DNA methylation and transcriptomics [RNA-Seq]. Muscle tissue and cellular mitochondrial respiration was quantified by high resolution respirometry. Ten weeks of aerobic training increased monolysocardiolipin (MLCL), a marker of mitochondrial remodeling, reduced total and specific species of sphingomyelin (SM), phosphatidylserine (PS) and ceramides lipid content and improved intrinsic mitochondrial respiration in SKM of T2D without alterations in mitochondrial DNA copy number or cardiolipin content. Exercise training differentially modified 50 DNA methylation sites. Among these genes, NOX4, acts as an oxygen sensor and catalyzes the reduction of reactive oxygen species (ROS), and PLCE1, catalyzes the hydrolysis of phosphoinositol 1,4,5-triphosphate (IP3) and DAG, were hypo-methylated and hyper-methylated, respectively, following training. Exercise training differentially regulated 70 transcripts in SKM of T2D that resulted in enrichments of lipid and amino acid metabolism and β-oxidation pathways. In summary, aerobic training improves intrinsic and cell autonomous SKM mitochondrial function and modifies SKM lipid composition in men with T2D, with potentially advantageous augmentations of oxidative stress and insulin signaling pathways. Disclosure M.F. Pino: None. N. Stephens: None. F. Yi: None. A.P. Hodges: None. R.E. Pratley: Other Relationship; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eisai Inc., GlaxoSmithKline plc., Janssen Pharmaceuticals, Inc., Lexicon Pharmaceuticals, Inc., Ligand Pharmaceuticals, Inc., Eli Lilly and Company, Merck &amp; Co., Inc., Novo Nordisk Inc., Pfizer Inc., Sanofi-Aventis, Takeda Development Center Americas, Inc.. S.R. Smith: None. B.H. Goodpaster: None. L.M. Sparks: None.

Heat stress can negatively impact pig health and performance but the effects of heat stress on skeletal muscle mitochondrial function are largely unknown. We hypothesized that mitochondrial function and capacity would be impaired in heat stressed (HS) compared to thermoneutral (TN) pigs but mitochondrially-targeted coenzyme Q (MitoQ) supplementation would rescue the impairment. Oxidative portions of the semitendinosus muscle were evaluated from TN and HS gilts receiving no supplementation (CON) or MitoQ for 2 d prior to and during the 24h environmental heat treatment (n = 8 per group). Mitochondrial oxidative phosphorylation (P) and electron transfer (E) capacities were determined via high resolution respirometry and mitochondrial volume density and function were quantified by citrate synthase (CS) and cytochrome c oxidase activities, respectively. Data were analyzed using linear models in SAS v9.4 with fixed effects of heat, MitoQ treatment (trt), and heat×trt interaction. There were trends for the interaction of trt and heat (P≤0.1) on integrative (per mg tissue) and intrinsic (relative to CS) P with complexes I and II (PCI+II), maximum noncoupled E (ECI+II), and E with complex II only (ECII), in which all measures were greater in HS-MitoQ than TN-MitoQ (P≤0.03), but measures did not differ due to HS in CON pigs. The contribution of leak to total E (flux control ratio, FCRLeak) was lesser in HS-MitoQ than HS-CON, TN-CON, and TN-MitoQ (P≤0.02). The FCRPCI was greater (P≤0.05) while the FCRPCI+II was lesser (P=0.01) in TN compared to HS pigs. Finally, the FCRPCI+II was greater (P=0.02) while the FCRECII tended to be lesser (P=0.09) for CON than MitoQ pigs. Neither mitochondrial volume density nor function were affected by HS or MitoQ supplementation. In total, these data indicate improved mitochondrial capacities following heat stress in pigs receiving MitoQ but no difference in mitochondrial capacities in unsupplemented, HS pigs.

Glucagon, an essential regulator of glucose and lipid metabolism, also promotes weight loss. We have identified that the hepatic Farnesol X Receptor (FXR) plays an important role in an anti-obesogenic effect of glucagon receptor (GcgR) agonism. Chronic administration of IUB288, a potent GcgR agonist, significantly reduced diet-induced obesity (DIO) in wild type (WT, 17%) mice compared to (FXR∆liver, 6%) liver-specific FXR knock-out mice. Weight loss was associated with increased energy expenditure (p&amp;lt;0.05) and fat oxidation (reduced RQ, p&amp;lt;0.05). We hypothesized that GcgR agonism alters mitochondrial bioenergetics, at least in part, through hepatic FXR signaling. IUB288 treatment increased body temperature (p&amp;lt;0.05) and [14C]-palmitate oxidation capacity of liver homogenate (p&amp;lt;0.05) to a greater extent in DIO WT mice than in FXR∆liver mice. RNA-Seq analysis of DIO liver samples revealed that the mitochondrial oxidative phosphorylation pathway, fatty acid metabolism, and bile acid metabolism were enriched in WT treated with IUB288, but not in FXR∆liver mice. Liver slices isolated from WT mice treated with IUB288 for two days significantly increased state 3 respiration rates compared to FXR∆liver mice using high resolution respirometry. Respiration was increased utilizing both carbohydrate (pyruvate, malate, glutamate, and succinate) and lipid substrates (palmitoyl-carnitine), suggesting GcgR-agonism modulates hepatic mitochondrial respiration via FXR. Our study elucidates a molecular mechanism of the anti-obesogenic effect of GcgR signaling. This heretofore-unappreciated aspect of glucagon biology has implications for the use of GcgR agonism in therapeutic strategies for multiple components of the metabolic syndrome. Disclosure T. Kim: None. S. Nason: None. J.P. Antipenko: None. C.S. Vestri: None. K. Smith-Johnston: None. M.E. Pepin: None. A.R. Wende: None. B. Finan: Employee; Self; Novo Nordisk Inc. R. DiMarchi: Employee; Self; Novo Nordisk Inc. D. Perez-Tilve: Research Support; Self; Novo Nordisk A/S, Cohbar. D.R. Moellering: None. K.M. Habegger: Consultant; Self; Glyscend, Inc.. Research Support; Self; Glyscend, Inc.. Consultant; Self; Intarcia Therapeutics, Inc..

Introduction: Peripheral artery disease (PAD) is an atherosclerotic condition that impairs blood flow to the lower extremities, resulting in mitochondrial myopathy in the affected leg muscles and significant walking impairment. Previous studies using a porcine model of PAD have shown that injection of adipose-derived mesenchymal stem cells (ADMSC) around the aortic trifurcation in the pelvis improves ischemic limb hemodynamics and walking performance. However, the effect of ADMSC treatment on skeletal muscle mitochondrial function remains unknown. Hypothesis: We hypothesized that ADMSC injection around the aortic trifurcation in the pelvis improves skeletal muscle mitochondrial function in a swine model of PAD. Methods: Ten Ossabaw miniswine (age 6 months) with metabolic syndrome were used. Right hindlimb ischemia was induced at week 0 (T0) by coil occlusion of the right external iliac, superficial femoral, and popliteal arteries via carotid artery access. At week 4 (T4), ADMSC (20 x 106 cells) were isolated using stromal vascular fractionation of autologous abdominal wall fat. Using jugular vein access, a catheter with a retractable side-deploying needle was inserted in the right iliac vein. The needle was deployed to access the retroperitoneum, and ADMSC (N=5) or vehicle (N=5) were infused around the aortic trifurcation. Gastrocnemius samples from the ischemic hindlimb were obtained at T4 (before treatment) and week 8 (T8, 4 weeks after treatment) to assess mitochondrial function using high resolution respirometry. Results: ADMSC treatment significantly increased mitochondrial respiration at T8 compared to T4 in complex I (T8: 27.92 ± 1.96; T4: 20.22 ± 2.01 pmols·s-1·mg-1; p&lt;0.05), complex II (T8: 35.71 ± 1.79; T4: 24.01 ± 2.44 pmols·s-1·mg-1; p&lt;0.05) and complex I+II state 3 (T8: 62.75 ± 3.25; T4: 44.23 ± 4.37 pmols·s-1·mg-1; p&lt;0.05), and state 4 (T8: 22.13 ± 1.16; T4: 16.45 ± 1.3 pmols·s-1·mg-1; p&lt;0.05) in the ischemic hindlimbs of ADMSC-treated swine. Conclusions: Our study, using a clinically relevant porcine model of PAD, similar to humans in size, anatomy, physiology, and comorbidities, demonstrates that ADMSC therapy can effectively improve mitochondrial function in ischemic hindlimbs. These findings suggest that ADMSC may be a promising treatment for mitochondrial myopathy in patients with PAD. Funding sources: R01 AG062198, R01 AG077803, R01 HD106911, P20 GM152301, and the Nebraska Research Initiative This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Mitochondrial and immune dysfunctions are often implicated in the aetiology of autism spectrum disorder (ASD). Here, we studied for the first time the relationship between ASD severity measures and mitochondrial respiratory rates in freshly isolated platelets as well as the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) in isolated neutrophils. We also verified the impact of hyperbaric oxygen therapy (HBOT) on mitochondrial and immune functions as well as on ASD severity measures. Blood samples were collected from three age-matched male groups (Control (Norm-N), autistic (Aut-N), and autistic + HBOT (Aut-H); N = 10 per group). Using high resolution respirometry, we found that routine basal respiration, complex I- and complex I + II-dependent oxidative phosphorylation rate were significantly impaired in Aut-N platelets. Similarly, deficits in immune response of neutrophils were evidenced through lower rates of oxygen consumption and reactive oxygen species (ROS) production by phagocytic NOX. ASD-related behavioural outcomes were found to moderately correlate with platelets’ mitochondrial bioenergetic parameters as well as with NOX-mediated activity in neutrophils. HBOT was not able to improve mitochondrial dysfunctions or to counteract ASD-related behavioral deficits. Although HBOT improved one measure of the immune response; namely, NOX-mediated superoxide burst, this was not associated with significant changes in trends of recurrent infections between groups. Taken together, our data suggest that ASD-associated mitochondria and immune deficits are detectable in platelets and neutrophils. We also found no evidence that HBOT confers any significant improvement of ASD-associated physiological or behavioural phenotypes.

Northern elephant seals (NES, Mirounga angustirostris) undergo an annual molt during which they spend ∼40 days fasting on land with reduced activity and lose approximately one-quarter of their body mass. Reduced activity and muscle load in stereotypic terrestrial mammalian models results in decreased muscle mass and capacity for force production and aerobic metabolism. However, the majority of lost mass in fasting female NES is from fat while muscle mass is largely preserved. Although muscle mass is preserved, potential changes to the metabolic and contractile capacity are unknown. To assess potential changes in NES skeletal muscle during molt, we collected muscle biopsies from 6 adult female NES before the molt and after ∼30 days at the end of the molt. Skeletal muscle was assessed for respiratory capacity using high resolution respirometry, and RNA was extracted to assess changes in gene expression. Despite a month of reduced activity, fasting, and weight loss, skeletal muscle respiratory capacity was preserved with no change in OXPHOS respiratory capacity. Molt was associated with 162 upregulated genes including those favoring lipid metabolism. We identified 172 downregulated genes including those coding for ribosomal proteins and genes associated with skeletal muscle force transduction and glucose metabolism. Following ∼30 days of molt, NES skeletal muscle metabolic capacity is preserved although mechanotransduction may be compromised. In the absence of exercise stimulus, fasting-induced shifts in muscle metabolism may stimulate pathways associated with preserving the mass and metabolic capacity of slow oxidative muscle.