High-resolution Respirometry Research Articles

Differences in Brain Region Mitochondrial Bioenergetics Detected Using CK Clamp

The brain is a non-uniform organ comprised of discrete structures of varying function and cell composition. The differing function of brain regions ostensibly would necessitate disparate energetic demands, thereby suggesting brain bioenergetics would be different among brain regions. This study aimed to address whether brain regions indeed have different bioenergetics by evaluating substrate preference, fuel-specific mitochondrial function at physiologically relevant energy demands as well as associated membrane potential and electron conductance using high-resolution respirometry (Oroboros O2k) in the frontal cortex, hypothalamus, and hippocampus. We hypothesize there will be different bioenergetics among brain regions. Substrate preference was established using a substrate-uncoupler-inhibitor-titration (SUIT) with additions, in order: ADP, Pyruvate/Malate, UK5099, Octanoylcarnitine, Rotenone, Succinate, and Antimycin A. There was no statistically significant interaction between region and substrate (p=0.15) nor a main effect of region (p=0.91), however, there was a main effect of substrate in which pyruvate/malate (+275%) and succinate/rotenone (+170%) had significantly greater respiration than octanoylcarnitine, independent of region (p<0.001). Given the results of the SUIT test, pyruvate/malate was used for subsequent tests. The SUIT method relies upon saturating ADP levels and non-physiological ATP:ADP concentration ratios, the extent to which this test obscures the interpretation of brain region respiration is unclear. Instead, an alternative approach that leverages the enzymatic reaction of creatine kinase and phosphocreatine to assess mitochondrial respiration and membrane potential at clamped physiological ATP:ADP ratios, “CK Clamp,” was used. Function of mitochondria with a pyruvate fuel was then evaluated in all three brain regions in which energetic demand was clamped at ΔGATP = -12.87 (high demand), -14.08, -14.5, and -14.85 (low demand). There was a statistically significant interaction between brain region and clamped ΔGATP values (p = 0.02). Frontal cortex displayed higher respiration than hippocampus at -12.87 ΔGATP (33%). There was no statistically significant interaction between region and clamp for mitochondrial membrane potential assessed during the CK clamp (p=0.16), however there was a significant main effect of clamped ΔGATP (p<0.001) in which membrane potential increased (i.e., hyperpolarized) as energetic demand decreased. This agrees with the bioenergetic model that a decrease in energetic demand increases the proton motive force (aka, hyperpolarization), thereby applying a backpressure on the electron transport chain, slowing respiration. Electron conductance was then assessed by calculating the slope of respiration versus clamped ΔGATP. Electron conductance was greater in the frontal cortex than hypothalamus (+70%, p = 0.015). The conclusion of this study is there are differences in mitochondrial bioenergetics among brain regions and the CK clamp was more sensitive at detecting these differences than the SUIT method. None. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Peripheral artery disease patients with chronic kidney disease have lower muscle strength and mitochondrial function compared to those with normal kidney function

Introduction: Chronic kidney disease is a significant risk factor for both the development of peripheral artery disease (PAD) and major adverse limb events. However, little is known about how the coalescence of these two conditions impacts limb muscle pathophysiology. The objective of the current study was to determine the impact of CKD on skeletal muscle strength and mitochondrial health in patients with and without PAD. Methods: A cross-sectional prospective study design was employed to assess plantar flexor muscle strength, muscle fiber cross-sectional area (CSA), and mitochondrial health in four groups of patients (61 males and 29 females): PAD only (Toe brachial index, TBI<0.5 and eGFR>60 mL/min), CKD only (TBI>0.75 and eGFR:15-45 mL/min), PAD with CKD (TBI<0.5 and eGFR:15-45 mL/min), and adult controls without PAD and CKD (TBI>0.75 and eGFR>60 mL/min). Results: Plantar flexor muscle strength was lowest in PAD patients with CKD compared to adult controls ( P<0.0001), PAD only ( P=0.001), and CKD only ( P=0.003). No differences in the mean muscle fiber CSA were detected. The percentage of COX+ fibers was significantly lower in PAD patients with CKD compared to adult controls ( P=0.03). High resolution respirometry analysis of mitochondrial oxidative phosphorylation (OXPHOS) function demonstrated that CKD has a significant negative impact on muscle bioenergetics. PAD patients with CKD had lower OXPHOS function than adult controls ( P=0.0016) and PAD patients without CKD ( P=0.0015). Notably, CKD patients without PAD also had lower OXPHOS function than adult controls ( P=0.0054) and PAD patients without CKD ( P=0.005). Conclusion: The current results demonstrate a profound negative impact of CKD on the associated myopathy of PAD patients. This study was supported by National Institutes of Health (NIH) grant R01-HL149704 (T.E.R.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is a proinflammatory syndrome. Mitochondrial dysfunction that is characteristic of HFpEF could alter immunometabolism, attenuating the functional reserve of immune cells. In addition, immune cell mitochondrial respiration is a biomarker of systemic mitochondrial health. The aim of this study was to assess the impact of a chronic oral mitochondrial targeted ubiquinol (MitoQ) on the immune cell bioenergetic profile in patients with HFpEF. We hypothesized that MitoQ administration in patients with HFpEF, would improve mitochondrial respiration in peripheral blood mononuclear cells (PBMC’s). METHODS: In this double-blind, crossover trial 8 patients with a clinical diagnosis of HFpEF (6 female, 2 male; 6 African American/Black, 2 white; mean ± SEM, Age, 58±5 years; BMI, 37±1 kg/m2) received a 4 week oral dose of MitoQ (MTQ, 20 mg/day) and placebo (PLB) in random order separated by a 2 week wash out period. Following completion of each trial arm, a venous blood sample was obtained and PBMCs were isolated using density gradient centrifugation. High resolution respirometry coupled with modulators of cellular respiration was performed to obtain basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity of the immune cells. Paired samples t-tests were used to compare variables between conditions. RESULTS: MitoQ was safe, well tolerated, and compliance was high (MTQ, 99 ± 1%; PLB, 89±4%). Basal respiration (MTQ, 106.4 ± 11.5 pmol/min; PLB, 66.5±5.9 pmol/min; p=0.002), ATP-linked respiration (MTQ, 41.4± 16.9 pmol/min; PLB, 16.3±6.7 pmol/min; p=0.04), maximal respiration (MTQ, 323.9 ± 50.0 pmol/min; PLB, 65.4±23.1 pmol/min; p=0.01), spare respiratory capacity (MTQ, 217.5 ± 39.6 pmol/min; PLB, 149.6 ±18.2 pmol/min; p=0.02), were higher following MTQ compared to PLB. CONCLUSIONS: In patients with HFpEF, administration of a mitochondrial targeted ubiquinol improved the immune cell mitochondrial respiration capacity and effciency. An increase in spare respiratory capacity following MTQ reflects an improvement in functional reserve that would augment the ability to respond to an acute stressor. These findings have positive implications for the ability to target and improve mitochondrial function in patients with HFpEF. Future work should investigate if the observed improvements in immune cell mitochondrial function are mirrored in other tissues. Supported by AHA 19CDA3474002. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Skeletal Muscle Mitochondrial Function and Mitochondria-related Protein Expression in Men and Women During Acute Exercise-Heat Stress and Acclimation

INTRODUCTION: Military personnel can exhibit improved performance and reduced physiological strain in hot environments with the induction of heat adaptations. Mild heat exposure at 40°C heat serves therapeutic purposes, enhancing metabolic flexibility, and promoting positive muscle adaptations. Research indicates that heat acclimation (HA) leads to increased mitochondrial biogenesis and reduced reactive oxygen species in skeletal muscle, improving thermal tolerance. However, understanding sex differences in mitochondrial function and related protein expression affected by heat acclimation remain limited. Therefore, this study aimed to explore mitochondrial respiration and the expression of mitochondria-related proteins to better understand the impact of heat stress on mitochondrial adaptations in skeletal muscle in both males and females. METHODS: Healthy men (M:n = 7, 22±2yo, VO2max 52.86±2.35ml·kg−1·min−1) and women (F:n = 7, 24±1yo, VO2max 45.97±2.57 ml·kg−1·min−1) participated in a 5-day heat acclimation (HA) at 40°C and 40% relative humidity. Before and after HA, participants completed a heat tolerance test (HTT) measuring time to reach a rectal temperature of 39.5°C or volitional failure. Biopsies from the vastus lateralis were taken immediately post-HTT for musculoskeletal analysis. Mitochondrial respiratory function was assessed using Oroboros O2K, employing high-resolution respirometry. Paired sample t-tests were used to determine statistical significance between groups (p<0.05). Proteomics data were mined for Gene Ontology (GO) terms related to mitochondrial function (STRING V.12.0) (adjusted p<0.05). RESULTS: Both men and women exhibited significant enhancements in complex I-mediated respiration and complex I+II maximal coupled respiration after HA (PRE-HA: M:P=0.009, P=0.026; F:P=0.016, P=0.011). No sex differences in mitochondria respiration were observed before or after HA. Before HA, skeletal muscle from women showed higher expression in 4 proteins (ND61, NDUFB1, COX1, ATP5MF) associated with 9 GO terms related to mitochondrial function compared to men. However, after HA, men exhibited higher expression in 14 proteins (MTX2, APOOL, DNAJC11, TIMM13, NDUFAB1, DNAJC11, MAVS, CHCHD4, NDUFAF4, ATP5PF, NDUFAS, UQCRH, SSBP1, ND3) associated with 21 mitochondria-related GO terms compared to women. CONCLUSION: Acclimation to heat stress is associated with positive mitochondrial adaptation in both men and women. Although enhancements in mitochondrial respiratory function in both men and women after HA, there were different skeletal muscle responses between men and women regarding the regulation of mitochondria-related proteins after a bout of exercise-heat stress. Funding: DoD BA200299. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1151: Concurrent Obesity And Chronic Alcohol Consumption Exacerbates Skeletal Muscle Mitochondrial Dysfunction In A Murine Model Of Peripheral Artery Disease (PAD).

PAD causes ischemic mitochondrial and consequent muscle damage in the legs, amplifying patient morbidity and mortality. While obesity and alcoholism are common comorbidities, their impact on PAD myopathy is unclear. We sought to determine the effect of concurrent hindlimb ischemia (HLI), obesity and chronic alcohol consumption on muscle oxidative stress, and mitochondrial and limb function. Methods: Twenty-four male, 24 female, 8 month old C57BL/6 mice received high-fat-sucrose (HFS) or low-fat-sucrose (LFS) diets for 16 weeks, followed by either 20% ethanol (EtOH) added to the drinking water / continued water access for another 12 weeks ( n =12 mice/4 groups). The left femoral artery was then ligated to induce HLI, and mice continued their assigned diets for another 4 weeks. The quadriceps from ischemic and non-ischemic limbs were excised at euthanasia. High-resolution respirometry measured muscle mitochondrial respiration and reactive oxygen species (H 2 O 2 ) output. Muscle oxidative stress and mitochondrial quality control (mitophagy) were assessed enzymatically and by western blot. Results: Compared to LFS animals with/without EtOH access, two-way ANOVA showed post-HLI limb function was lower in HFS+water and HFS+EtOH animals (p<0.05). Mitochondrial respiration was lower in LFS+EtOH, HFS+water, and HFS+EtOH ischemic muscle during electron transport chain Complex I, state 2 (I.2), Complex I, state 3 (I.3) and Complex III versus LFS+water controls (p<0.05), and was further reduced during Complex II and IV in LFS and HFS-ischemic muscle exposed to EtOH (p<0.01). Mitochondrial H 2 O 2 yield was increased in HFS+water and HFS+EtOH ischemic muscle at complexes I.2, I.3, II, III and IV (p<0.05), but not in LFS+EtOH muscle. Oxidative damage (carbonyl accumulation) (p<0.0001) and reduced antioxidant levels (superoxide dismutase (p<0.05) and PGC-1alpha (p<0.001)) was only evident in HFS ischemic muscle with/without EtOH exposure. However, the mitochondrial detoxifying enzyme, aldehyde dehydrogenase-2 (p=0.03), and mitophagy markers LC3II:I ratio (p=0.002) and mitofusins 1 (p=0.02) and 2 (p=0.002), were only reduced in HFS+EtOH muscle. Conclusion: Obesity and chronic alcohol consumption exacerbates muscle mitochondriopathy in mice with HLI.

Smoking cessation only partially reverses cardiac metabolic and structural remodeling in mice.

Active cigarette smoking is a major risk factor for chronic obstructive pulmonary disease that remains elevated after cessation. Skeletal muscle dysfunction has been well documented after smoking, but little is known about cardiac adaptations to cigarette smoking. The underlying cellular and molecular cardiac adaptations, independent of confounding lifestyle factors, and time course of reversibility by smoking cessation remain unclear. We hypothesized that smoking negatively affects cardiac metabolism and induces local inflammation in mice, which do not readily reverse upon 2-week smoking cessation. Mice were exposed to air or cigarette smoke for 14 weeks with or without 1- or 2-week smoke cessation. We measured cardiac mitochondrial respiration by high-resolution respirometry, cardiac mitochondrial density, abundance of mitochondrial supercomplexes by electrophoresis, and capillarization, fibrosis, and macrophage infiltration by immunohistology, and performed cardiac metabolome and lipidome analysis by mass spectrometry. Mitochondrial protein, supercomplex content, and respiration (all p < 0.03) were lower after smoking, which were largely reversed within 2-week smoking cessation. Metabolome and lipidome analyses revealed alterations in mitochondrial metabolism, a shift from fatty acid to glucose metabolism, which did not revert to control upon smoking cessation. Capillary density was not different after smoking but increased after smoking cessation (p = 0.02). Macrophage infiltration and fibrosis (p < 0.04) were higher after smoking but did not revert to control upon smoking cessation. While cigarette-impaired smoking-induced cardiac mitochondrial function was reversed by smoking cessation, the remaining fibrosis and macrophage infiltration may contribute to the increased risk of cardiovascular events after smoking cessation.

Specific NOX4 Inhibition Preserves Mitochondrial Function and Dampens Kidney Dysfunction Following Ischemia-Reperfusion-Induced Kidney Injury.

Background: Acute kidney injury (AKI) is a sudden episode of kidney failure which is frequently observed at intensive care units and related to high morbidity/mortality. Although AKI can have many different causes, ischemia-reperfusion (IR) injury is the main cause of AKI. Mechanistically, NADPH oxidases (NOXs) are involved in the pathophysiology contributing to oxidative stress following IR. Previous reports have indicated that knockout of NOX4 may offer protection in cardiac and brain IR, but there is currently less knowledge about how this could be exploited therapeutically and whether this could have significant protection in IR-induced AKI. Aim: To investigate the hypothesis that a novel and specific NOX4 inhibitor (GLX7013114) may have therapeutic potential on kidney and mitochondrial function in a mouse model of IR-induced AKI. Methods: Kidneys of male C57BL/6J mice were clamped for 20 min, and the NOX4 inhibitor (GLX7013114) was administered via osmotic minipump during reperfusion. Following 3 days of reperfusion, kidney function (i.e., glomerular filtration rate, GFR) was calculated from FITC-inulin clearance and mitochondrial function was assessed by high-resolution respirometry. Renal histopathological evaluations (i.e., hematoxylin-eosin) and TUNEL staining were performed for apoptotic evaluation. Results: NOX4 inhibition during reperfusion significantly improved kidney function, as evidenced by a better-maintained GFR (p < 0.05) and lower levels of blood urea nitrogen (p < 0.05) compared to untreated IR animals. Moreover, IR caused significant tubular injuries that were attenuated by simultaneous NOX4 inhibition (p < 0.01). In addition, the level of renal apoptosis was significantly reduced in IR animals with NOX4 inhibition (p < 0.05). These favorable effects of the NOX4 inhibitor were accompanied by enhanced Nrf2 Ser40 phosphorylation and conserved mitochondrial function, as evidenced by the better-preserved activity of all mitochondrial complexes. Conclusion: Specific NOX4 inhibition, at the time of reperfusion, significantly preserves mitochondrial and kidney function. These novel findings may have clinical implications for future treatments aimed at preventing AKI and related adverse events, especially in high-risk hospitalized patients.

Muscle Mitochondrial Bioenergetic Capacities Are Associated With Multimorbidity Burden in Older Adults: The Study of Muscle, Mobility and Aging.

The geroscience hypothesis posits that aging biological processes contribute to many age-related deficits, including the accumulation of multiple chronic diseases. Though only one facet of mitochondrial function, declines in muscle mitochondrial bioenergetic capacities may contribute to this increased susceptibility to multimorbidity. The Study of Muscle, Mobility and Aging (SOMMA) assessed ex vivo muscle mitochondrial energetics in 764 older adults (mean age = 76.4, 56.5% women, and 85.9% non-Hispanic White) by high-resolution respirometry of permeabilized muscle fibers. We estimated the proportional odds ratio (POR [95% CI]) for the likelihood of greater multimorbidity (4 levels: 0 conditions, N = 332; 1 condition, N = 299; 2 conditions, N = 98; or 3+ conditions, N = 35) from an index of 11 conditions, per SD decrement in muscle mitochondrial energetic parameters. Distribution of conditions allowed for testing the associations of maximal muscle energetics with some individual conditions. Lower oxidative phosphorylation supported by fatty acids and/or complex I- and II-linked carbohydrates (eg, Max OXPHOSCI+CII) was associated with a greater multimorbidity index score (POR = 1.32 [1.13, 1.54]) and separately with diabetes mellitus (OR = 1.62 [1.26, 2.09]), depressive symptoms (OR = 1.45 [1.04, 2.00]) and possibly chronic kidney disease (OR = 1.57 [0.98, 2.52]) but not significantly with other conditions (eg, cardiac arrhythmia, chronic obstructive pulmonary disease). Lower muscle mitochondrial bioenergetic capacities were associated with a worse composite multimorbidity index score. Our results suggest that decrements in muscle mitochondrial energetics may contribute to a greater global burden of disease and are more strongly related to some conditions than others.

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.

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

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

Mitochondrial respiration in peripheral arterial disease depends on stage severity.

Peripheral arterial disease (PAD) is an increasing cause of morbidity and its severity is graded based on clinical manifestation. To investigate the influence of the different stages on myopathy of ischemic muscle we analysed severity-dependent effects of mitochondrial respiration in PAD. Eighteen patients with severe PAD, defined as chronic limb-threatening ischemia, 47 patients with intermittent claudication (IC) and 22 non-ischemic controls were analysed. High-resolution respirometry (HRR) was performed on muscle biopsies of gastrocnemius and vastus lateralis muscle of patients in different PAD stages to investigate different respiratory states. Results from HRR are given as median and interquartile range and were normalized to citrate synthase activity (CSA), a marker for mitochondrial content. In order to account for inter-individual differences between patients and controls, we calculated the ratio of O₂-flux in gastrocnemius muscle over vastus muscle ('GV ratio'). CSA of the gastrocnemius muscle as a proxy for mitochondrial content was significantly lower in critical ischemia compared to controls. Mitochondrial respiration normalized to CSA was higher in IC compared to controls. Likewise, the GV ratio was significantly higher in IC compared to control. Mitochondrial respiration and CSA of PAD patients showed stage-dependent modifications with greater changes in the mild PAD stage group (IC).

Microcirculation-driven mitochondrion dysfunction during the progression of experimental sepsis

Sepsis is accompanied by a less-known mismatch between hemodynamics and mitochondrial respiration. We aimed to characterize the relationship and time dependency of microcirculatory and mitochondrial functions in a rodent model of intraabdominal sepsis. Fecal peritonitis was induced in rats, and multi-organ failure (MOF) was evaluated 12, 16, 20, 24 or 28 h later (n = 8/group, each) using rat-specific organ failure assessment (ROFA) scores. Ileal microcirculation (proportion of perfused microvessels (PPV), microvascular flow index (MFI) and heterogeneity index (HI)) was monitored by intravital video microscopy, and mitochondrial respiration (OxPhos) and outer membrane (mtOM) damage were measured with high-resolution respirometry. MOF progression was evidenced by increased ROFA scores; microcirculatory parameters followed a parallel time course from the 16th to 28th h. Mitochondrial dysfunction commenced with a 4-h time lag with signs of mtOM damage, which correlated significantly with PPV, while no correlation was found between HI and OxPhos. High diagnostic value was demonstrated for PPV, mtOM damage and lactate levels for predicting MOF. Our findings indicate insufficient splanchnic microcirculation to be a possible predictor for MOF that develops before the start of mitochondrial dysfunction. The adequate subcellular compensatory capacity suggests the presence of mitochondrial subpopulations with differing sensitivity to septic insults.

Abstract 1790: Pyruvate carboxylase regulates tumor progression through central carbon metabolism and immunosuppression

Abstract The tumor microenvironment (TME) is characterized by metabolic reprogramming and immune evasion, essential features in tumor progression. Tumor metabolic dysregulation, particularly the high production of lactate, plays a pivotal role in suppressing antitumor immunity within the TME. Pyruvate carboxylase (PC), responsible for converting pyruvate to oxaloacetate, is a critical determinant for lung metastasis in triple-negative breast cancer (TNBC). Herein we test the hypothesis that PC-mediated regulation of central carbon metabolism controls TME immunosurrveilance. Wild-type C57BL6/J and NSG mice received orthotopic injections of E0771 or M-Wnt tumor cells with shRNA targeting PC or a scramble control. RNA from tumors underwent transcriptomic profiling using Affymetrix Clariom D microarray (n=3-6/group per experiment). Gene set enrichment analysis identified pathways enriched in each condition, including a PC-associated hypoxia signature. Immunohistochemistry with anti-CD3 antibody and full spectrum flow cytometry assessed T cell infiltration and TME immune cell composition. Untargeted metabolomics, extracellular flux analysis, and high-resolution respirometry determined how loss of PC altered cellular metabolism. We found that suppressing PC expression increased primary tumor growth (~2 fold), potently drove immunosuppression, and remodeled cellular metabolism towards lactate production. Injection of PC-suppressed cells increased regulatory T cell levels in wild type mice. Importantly, PC-suppressed cells injected into NSG mice, which lack T, B, and NK cells, demonstrated slower, rather than accelerated, tumor growth. PC-suppression reduced oxygen consumption rate (OCR), but supply of anaplerotic carbon through exogenous malate or oxaloacetate restored OCR. Conversely, both the rate of and reliance on lactate production was increased by PC suppression. Notably, loss of immunosurveillance following downregulation of PC was reversed by inhibiting lactate transport. Additionally, lower PC expression was associated with increased hypoxia in tumors, supporting in vitro evidence of PC-repression by hypoxia. In summary, hypoxia-induced PC suppression emerges as a crucial mechanism through which primary tumors hinder antitumor immunity, partly via increased lactate production. These findings underscore the significance of PC-directed tumor metabolism as an understudied nexus in the interplay between tumor progression and antitumor immunity. Citation Format: Michael F. Coleman, Eylem Kulkoyluoglu Cotul, Alexander J. Pfeil, Alyssa N. Ho, Emily N. Devericks, Muhammad H. Safdar, Hao Chen, Numair Attaar, Violet A. Kiesel, Dorothy Teegarden, Stephen D. Hursting, Michael K. Wendt. Pyruvate carboxylase regulates tumor progression through central carbon metabolism and immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1790.

Targeting of mitochondrial fission through natural flavanones elicits anti-myeloma activity.

Mitochondrial alterations, often dependent on unbalanced mitochondrial dynamics, feature in the pathobiology of human cancers, including multiple myeloma (MM). Flavanones are natural flavonoids endowed with mitochondrial targeting activities. Herein, we investigated the capability of Hesperetin (Hes) and Naringenin (Nar), two aglycones of Hesperidin and Naringin flavanone glycosides, to selectively target Drp1, a pivotal regulator of mitochondrial dynamics, prompting anti-MM activity. Molecular docking analyses were performed on the crystallographic structure of Dynamin-1-like protein (Drp1), using Hes and Nar molecular structures. Cell viability and apoptosis were assessed in MM cell lines, or in co-culture systems with primary bone marrow stromal cells, using Cell Titer Glo and Annexin V-7AAD staining, respectively; clonogenicity was determined using methylcellulose colony assays. Transcriptomic analyses were carried out using the Ion AmpliSeq™ platform; mRNA and protein expression levels were determined by quantitative RT-PCR and western blotting, respectively. Mitochondrial architecture was assessed by transmission electron microscopy. Real time measurement of oxygen consumption was performed by high resolution respirometry in living cells. In vivo anti-tumor activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells. Hes and Nar were found to accommodate within the GTPase binding site of Drp1, and to inhibit Drp1 expression and activity, leading to hyperfused mitochondria with reduced OXPHOS. In vitro, Hes and Nar reduced MM clonogenicity and viability, even in the presence of patient-derived bone marrow stromal cells, triggering ER stress and apoptosis. Interestingly, Hes and Nar rewired MM cell metabolism through the down-regulation of master transcriptional activators (SREBF-1, c-MYC) of lipogenesis genes. An extract of Tacle, a Citrus variety rich in Hesperidin and Naringin, was capable to recapitulate the phenotypic and molecular perturbations of each flavanone, triggering anti-MM activity in vivo. Hes and Nar inhibit proliferation, rewire the metabolism and induce apoptosis of MM cells via antagonism of the mitochondrial fission driver Drp1. These results provide a framework for the development of natural anti-MM therapeutics targeting aberrant mitochondrial dependencies.

Mitochondrial function and reactive oxygen species production during human sperm capacitation: Unraveling key players.

Sperm capacitation is a critical process for male fertility. It involves a series of biochemical and physiological changes that occur in the female reproductive tract, rendering the sperm competent for successful fertilization. The precise mechanisms and, specifically, the role of mitochondria, in sperm capacitation remain incompletely understood. Previously, we revealed that in mouse sperm mitochondrial activity (e.g., oxygen consumption, membrane potential, ATP/ADP exchange, and mitochondrial Ca2+ ) increases during capacitation. Herein, we studied mitochondrial function by high-resolution respirometry (HRR) and reactive oxygen species production in capacitated (CAP) and non-capacitated (NC) human spermatozoa. We found that in capacitated sperm from normozoospermic donors, the respiratory control ratio increased by 36%, accompanied by a double oxygen consumption rate (OCR) in the presence of antimycin A. Extracellular hydrogen peroxide (H2 O2 ) detection was three times higher in CAP than in NC sperm cells. To confirm that H2 O2 production depends on mitochondrial superoxide ( ) formation, we evaluated mitochondrial aconitase (ACO2) amount, activity, and role in the metabolic flux from the sperm tricarboxylic acid cycle. We estimated that CAP cells produce, on average by individual, (59 ± 22)% more in the steady-state compared to NC cells. Finally, we analyzed two targets of oxidative stress: lipid peroxidation by western blot against 4-hydroxynonenal and succinate dehydrogenase (SDH) activity by HRR. We did not observe modifications in lipoperoxidation nor the activity of SDH, suggesting that during capacitation, the increase in mitochondrial H2 O2 production does not damage sperm and it is necessary for the normal CAP process.

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.

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.

Spheroid architecture strongly enhances miR-221/222 expression and promotes oxidative phosphorylation in an ovarian cancer cell line through a mechanism that includes restriction of miR-9 expression.

Tumor cell spheroids are organized multicellular structures that form during the expansive growth of carcinoma cells. Spheroids formation is thought to contribute to metastasis by supporting growth and survival of mobile tumor cell populations. We investigated how spheroid architecture affects OXPHOS activity, microRNA expression, and intraperitoneal survival of an ovarian carcinoma cell line using high resolution respirometry, quantitative RT-PCR, and a rodent intraperitoneal growth model. Rates of oxidative phosphorylation/respiration per cell of cells growing as spheroids were nearly double those of a variant of the same cell type growing in suspension as loosely aggregated cells. Further, inhibition of spheroid formation by treatment with CDH2 (N-cadherin) siRNA reduced the rate of OXPHOS to that of the non-spheroid forming variant. Cells growing as spheroids showed greatly enhanced expression of miR-221/222, an oncomiR that targets multiple tumor suppressor genes and promotes invasion, and reduced expression of miR-9, which targets mitochondrial tRNA-modification enzymes and inhibits OXPHOS. Consistent with greater efficiency of ATP generation, tumor cells growing as spheroids injected into the nutrient-poor murine peritoneum survived longer than cells growing in suspension as loosely associated aggregates. The data indicate that growth in spheroid form enhances the OXPHOS activity of constituent tumor cells. In addition, spheroid architecture affects expression of microRNA genes involved in growth control and mitochondrial function. During the mobile phase of metastasis, when ovarian tumor cells disperse through nutrient-poor environments such as the peritoneum, enhanced OXPHOS activity afforded by spheroid architecture would enhance survival and metastatic potential.

Abstract A027: Targeting MYC-induced reprogramming of mitochondrial metabolism in breast cancer

Abstract Oncogenic MYC is frequently overexpressed in breast cancer, and its overexpression is associated with aggressiveness and unfavorable prognosis of breast cancer. MYC overexpression promotes tumor growth by stimulating cell cycle progression and rewiring metabolism – creating cancer vulnerabilities that can be therapeutically targeted via synthetic lethal strategies. A large part of our current understanding of the MYC overexpression biology and related synthetic lethal pathways is based on findings in various inducible MYC overexpression systems, which demonstrate the acute effects of MYC activation but may poorly represent long-term MYC overexpression status in real cancers. Here we classified 15 triple-negative breast cancer (TNBC) cell lines according to both MYC target gene set expression and MYC protein expression into MYC-high and MYC-low categories and explored the MYC-high status-coupled therapeutic responses and cancer-relevant metabolic changes. We show that mitochondrial respiration inhibiting biguanide drug metformin inhibits cell proliferation and generates senescence-like features in MYC-high TNBC cells. Furthermore, high-resolution respirometry experiments show that MYC-high TNBC cells have higher mitochondrial respiration rates than MYC-low TNBC cells, a phenotype not explained by a higher number of mitochondria. Also, targeting mitochondrial respiration with small molecule inhibitors specifically reduces proliferation in MYC-high TNBC cells and sensitizes them to apoptosis – indicating that MYC-high TNBC cells have higher dependency on mitochondrial respiration than the MYC-low cells. Targeted metabolomic flux experiments demonstrate that small molecule inhibitors of mitochondrial respiration rewire TCA cycle, resulting in suppression of MYC-driven glutamine anaplerosis, which offers a possible explanation for the apoptotic sensitization. In conclusion, our findings provide new insight into the long-term MYC overexpression-associated metabolic changes – highlighting mitochondrial respiration as a potential target for the development of MYC-dependent synthetic lethal strategies against breast cancer. Citation Format: Johanna M. Anttila, Mariel Savelius, Daniel Nicorici, Ryan Awadhpersad, Pauliina M. Munne, Linda Id, Topi A. Tervonen, Christopher Jackson, Anni I. Nieminen, Juha Klefström. Targeting MYC-induced reprogramming of mitochondrial metabolism in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A027.

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.