High-resolution Respirometry Research Articles

Muscle mitochondrial bioenergetic capacities are associated with multimorbidity burden in older adults: the Study of Muscle, Mobility and Aging (SOMMA).

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, 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 (four 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 (e.g., 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 (e.g., cardiac arrhythmia, chronic obstructive pulmonary disease). Lower muscle mitochondrial bioenergetic capacities was 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 is more strongly related to some conditions than others.

Enantiomer-Specific Cardiovascular Effects of the Ketone Body 3-Hydroxybutyrate.

The ketone body 3-hydroxybutyrate (3-OHB) increases cardiac output (CO) by 35% to 40% in healthy people and people with heart failure. The mechanisms underlying the effects of 3-OHB on myocardial contractility and loading conditions as well as the cardiovascular effects of its enantiomeric forms, D-3-OHB and L-3-OHB, remain undetermined. Three groups of 8 pigs each underwent a randomized, crossover study. The groups received 3-hour infusions of either D/L-3-OHB (racemic mixture), 100% L-3-OHB, 100% D-3-OHB, versusan isovolumic control. The animals were monitored with pulmonary artery catheter, left ventricle pressure-volume catheter, and arterial and coronary sinus blood samples. Myocardial biopsies were evaluated with high-resolution respirometry, coronary arteries with isometric myography, and myocardial kinetics with D-[11C]3-OHB and L-[11C]3-OHB positron emission tomography. All three 3-OHB infusions increased 3-OHB levels (P<0.001). D/L-3-OHB and L-3-OHB increased CO by 2.7 L/min (P<0.003). D-3-OHB increased CO nonsignificantly (P=0.2). Circulating 3-OHB levels correlated with CO for both enantiomers (P<0.001). The CO increase was mediated through arterial elastance (afterload) reduction, whereas contractility and preload were unchanged. Exvivo, D- and L-3-OHB dilated coronary arteries equally. The mitochondrial respiratory capacity remained unaffected. The myocardial 3-OHB extraction increased only during the D- and D/L-3-OHB infusions. D-[11C]3-OHB showed rapid cardiac uptake and metabolism, whereas L-[11C]3-OHB demonstrated much slower pharmacokinetics. 3-OHB increased CO by reducing afterload. L-3-OHB exerted a stronger hemodynamic response than D-3-OHB due to higher circulating 3-OHB levels. There was a dissocitation between the myocardial metabolism and hemodynamic effects of the enantiomers, highlighting L-3-OHB as a potent cardiovascular agent with strong hemodynamic effects.

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 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.

Passive exercise is an effective alternative to HRT for restoring OVX induced mitochondrial dysfunction in skeletal muscle.

Postmenopausal women are more prone to develop muscle weakness, which is strongly associated with impairment of mitochondrial function in skeletal muscle. This study aimed to examine the impact of a passive exercise modality, whole-body vibration training (WBVT), on muscle mitochondrial function in ovariectomized (OVX) mice, in comparison with 17β-estradiol (E2) replacement. Female C57BL/6J mice were assigned to four groups: sham operation control group (Sham), ovariectomized group (OVX), OVX with E2 supplement group (OVX+E), and OVX with WBVT group (OVX+W). The estrous cycle, body weight, body composition, and muscle strength of the mice were monitored after the operation. Serum E2 level was assessed by enzyme-linked immunosorbent assay (ELISA). The ATP levels were determined using a luciferase-catalyzed bioluminescence assay. The activity of mitochondrial respiration chain complexes was evaluated using high-resolution respirometry (O2K). Expression levels of oxidative phosphorylation (OXPHOS), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), and mitochondrial transcription factor A (TFAM) were detected using western blotting. We observed decreased muscle strength and impaired mitochondrial function in the skeletal muscle of OVX mice. The vibration training alleviated these impairments as much as the E2 supplement. In addition, the vibration training was superior to the ovariectomy and the estradiol replacement regarding the protein expression of PGC-1α and TFAM. WBVT improves the OVX-induced decline in muscle strength and impairment of mitochondrial function in the skeletal muscle. This passive exercise strategy may be useful as an alternative to E2 replacement for preventing menopausal muscular weakness. Further studies are needed to understand the effects of WBVT on various physiological systems, and precautions should be taken when implementing it in patient treatment.

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.

Uncovering the Therapeutic Potential of Phosphocreatine in Diabetic Retinopathy: Mitigating Mitochondrial Dysfunction and Apoptosis via JAK2/STAT3 Signaling Pathway.

Diabetic retinopathy (DR) stands as a prevalent complication of diabetes mellitus, causing damage to the delicate retinal capillaries and potentially leading to visual impairment. While the exact underlying cause of DR remains elusive, compelling research suggests that mitochondrial energy deficiency and the excessive generation of reactive oxygen species (ROS) play pivotal roles in its pathogenesis. Recognizing that controlling hyperglycemia alone fails to reverse the defects in retinal mitochondria induced by diabetes, current strategies seek to restore mitochondrial function as a means of safeguarding against DR. To address this pressing issue, a comprehensive study was undertaken to explore the potential of phosphocreatine (PCr) in bolstering mitochondrial bioenergetics and providing protection against DR via modulation of the JAK2/STAT3 signaling pathway. Employing rat mitochondria and RGC-5 cells, the investigation meticulously assessed the impact of PCr on ROS production, mitochondrial membrane potential, as well as the expression of crucial apoptotic and JAK2/STAT3 signaling pathway proteins, utilizing cutting-edge techniques such as high-resolution respirometry and western blotting. The remarkable outcomes revealed that PCr exerts a profound protective influence against DR by enhancing mitochondrial function and alleviating diabetes-associated symptoms and biochemical markers. Notably, PCr administration resulted in an upregulation of antiapoptotic proteins, concomitant with a downregulation of proapoptotic proteins and the JAK2/STAT3 signaling pathway. These significant findings firmly establish PCr as a potential therapeutic avenue for combating diabetic retinopathy. By augmenting mitochondrial function and exerting antiapoptotic effects via the JAK2/STAT3 signaling pathway, PCr demonstrates promising efficacy both in vivo and in vitro, particularly in counteracting the oxidative stress engendered by hyperglycemia. In summary, our study sheds light on the potential of PCr as an innovative therapeutic strategy for diabetic retinopathy. By bolstering mitochondrial function and exerting protective effects via the modulation of the JAK2/STAT3 signaling pathway, PCr holds immense promise in ameliorating the impact of DR in the face of oxidative stress induced by hyperglycemia.

Intracellular delivery of Parkin-RING0-based fragments corrects Parkin-induced mitochondrial dysfunction through interaction with SLP-2

BackgroundLoss-of-function mutations in the PRKN gene, encoding Parkin, are the most common cause of autosomal recessive Parkinson’s disease (PD). We have previously identified mitochondrial Stomatin-like protein 2 (SLP-2), which functions in the assembly of respiratory chain proteins, as a Parkin-binding protein. Selective knockdown of either Parkin or SLP-2 led to reduced mitochondrial and neuronal function in neuronal cells and Drosophila, where a double knockdown led to a further worsening of Parkin-deficiency phenotypes. Here, we investigated the minimal Parkin region involved in the Parkin-SLP-2 interaction and explored the ability of Parkin-fragments and peptides from this minimal region to restore mitochondrial function.MethodsIn fibroblasts, human induced pluripotent stem cell (hiPSC)-derived neurons, and neuroblastoma cells the interaction between Parkin and SLP-2 was investigated, and the Parkin domain responsible for the binding to SLP-2 was mapped. High resolution respirometry, immunofluorescence analysis and live imaging were used to analyze mitochondrial function.ResultsUsing a proximity ligation assay, we quantitatively assessed the Parkin-SLP-2 interaction in skin fibroblasts and hiPSC-derived neurons. When PD-associated PRKN mutations were present, we detected a significantly reduced interaction between the two proteins. We found a preferential binding of SLP-2 to the N-terminal part of Parkin, with a highest affinity for the RING0 domain. Computational modeling based on the crystal structure of Parkin protein predicted several potential binding sites for SLP-2 within the Parkin RING0 domain. Amongst these, three binding sites were observed to overlap with natural PD-causing missense mutations, which we demonstrated interfere substantially with the binding of Parkin to SLP-2. Finally, delivery of the isolated Parkin RING0 domain and a Parkin mini-peptide, conjugated to cell-permeant and mitochondrial transporters, rescued compromised mitochondrial function in Parkin-deficient neuroblastoma cells and hiPSC-derived neurons with endogenous, disease causing PRKN mutations.ConclusionsThese findings place further emphasis on the importance of the protein–protein interaction between Parkin and SLP-2 for the maintenance of optimal mitochondrial function. The possibility of restoring an abolished binding to SLP-2 by delivering the Parkin RING0 domain or the Parkin mini-peptide involved in this specific protein–protein interaction into cells might represent a novel organelle-specific therapeutic approach for correcting mitochondrial dysfunction in Parkin-linked PD.

Human papillomavirus-associated head and neck squamous cell carcinoma cells rely on glycolysis and display reduced oxidative phosphorylation.

Head and neck squamous cell carcinoma (HNSCC) constitutes a heterogeneous group of cancers. Human papilloma virus (HPV) is associated with a subtype of HNSCC with a better response to treatment and more favorable prognosis. Mitochondrial function and metabolism vary depending on cancer type and can be related to tumor aggressiveness. This study aims to characterize the metabolism of HPV-positive and HPV-negative HNSCC cell lines. Oxidative phosphorylation (OXPHOS) and glycolysis were assessed in intact cells, in four HNSCC cell lines using Seahorse XF Analyzer. OXPHOS was further studied in permeabilized cells using high-resolution respirometry in an Oroboros O2K. Metabolomic analysis was performed using mass spectroscopy. The HPV-negative cell lines were found to display a higher OXPHOS capacity and were also able to upregulate glycolysis when needed. The HPV-positive cell line had a higher basal glycolytic rate but lower spare OXPHOS capacity. These cells were also unable to increase respiration in response to succinate, unlike the HPV-negative cells. In the metabolomic analysis, the HPV-positive cells showed a higher kynurenine/tryptophan ratio. HPV-positive HNSCC preferred glycolysis to compensate for lower OXPHOS reserves, while the HPV-negative HNSCC displayed a more versatile metabolism, which might be related to increased tumor aggressiveness. The higher kynurenine/tryptophan ratio of HPV-positive HNSCC might be related to increased indoleamine 2,3-dioxygenase activity due to the carcinoma's viral origin. This study highlights important metabolic differences between HPV-positive and HPV-negative cancers and suggests that future metabolic targets for cancer treatment should be individualized based on specific tumor metabolism.

The Association of Skeletal Muscle Energetics With Recurrent Falls in Older Adults Within the Study of Muscle, Mobility and Aging.

Falls in the older population are a major public health concern. While many physiological and environmental factors have been associated with fall risk, muscle mitochondrial energetics has not yet been investigated. In this analysis, 835 Study of Muscle, Mobility and Aging (SOMMA) participants aged 70-94 were surveyed for number of falls (total), recurrent falls (2+), and fall-related injuries over the past 12 months at baseline and again after 1 year. Skeletal muscle energetics were assessed at baseline in vivo using 31P Magnetic Resonance Spectroscopy for the maximal rate of adenosine triphosphate recovery (ATPmax)after an acute bout of exercise, and ex vivo by High-Resolution Respirometry for the maximal rate of complex I and II supported oxygen consumption (MaxOXPHOS) in permeabilized muscle fibers from the vastus lateralis. At least 1 fall was reported in 28.7% of SOMMA participants in the first year of the study, with 12% of older adults reporting recurrent falls (2+). Individuals who experienced recurrent falls had a slower 400-m walk gait speed (1.0±0.2 vs 1.1±0.2, p < .001), reported fewer alcoholic drinks per week in the past year (2.4±4.3 vs 2.8±4.4, p = .054), and took a significantly greater number of medication in the 30 days before their baseline visit (5.6±4.4 vs 4.2±3.4, p < .05). A history of falls was reported in 63% of individuals who experienced recurrent falls in the first year of the study compared to 22.8% who experienced 1 or fewer falls. MaxOXPHOS was significantly lower in those who reported recurrent falls (p = .008) compared to those with 1 or fewer falls, but there was no significant difference in ATPmax (p = .369). Neither muscle energetics measure was significantly associated with total number of falls or injurious falls, but recurrent falls were significantly higher with lower MaxOXPHOS (risk ratio = 1.33, 95% confidence interval = 1.02-1.73, p = .033). However, covariates accounted for the increased risk. Mitochondrial energetics were largely unrelated to fall risk in older adults when accounting for variables, suggesting that the complex etiology of falls may not be related to a single "hallmark of aging" biological pathway.

13C-Metabolic flux analysis detected a hyperoxemia-induced reduction of tricarboxylic acid cycle metabolism in granulocytes during two models of porcine acute subdural hematoma and hemorrhagic shock.

Supplementation with increased inspired oxygen fractions has been suggested to alleviate the harmful effects of tissue hypoxia during hemorrhagic shock (HS) and traumatic brain injury. However, the utility of therapeutic hyperoxia in critical care is disputed to this day as controversial evidence is available regarding its efficacy. Furthermore, in contrast to its hypoxic counterpart, the effect of hyperoxia on the metabolism of circulating immune cells remains ambiguous. Both stimulating and detrimental effects are possible; the former by providing necessary oxygen supply, the latter by generation of excessive amounts of reactive oxygen species (ROS). To uncover the potential impact of increased oxygen fractions on circulating immune cells during intensive care, we have performed a 13C-metabolic flux analysis (MFA) on PBMCs and granulocytes isolated from two long-term, resuscitated models of combined acute subdural hematoma (ASDH) and HS in pigs with and without cardiovascular comorbidity. Swine underwent resuscitation after 2 h of ASDH and HS up to a maximum of 48 h after HS. Animals received normoxemia (PaO2 = 80 - 120 mmHg) or targeted hyperoxemia (PaO2 = 200 - 250 mmHg for 24 h after treatment initiation, thereafter PaO2 as in the control group). Blood was drawn at time points T1 = after instrumentation, T2 = 24 h post ASDH and HS, and T3 = 48h post ASDH and HS. PBMCs and granulocytes were isolated from whole blood to perform electron spin resonance spectroscopy, high resolution respirometry and 13C-MFA. For the latter, we utilized a parallel tracer approach with 1,2-13C2 glucose, U-13C glucose, and U-13C glutamine, which covered essential pathways of glucose and glutamine metabolism and supplied redundant data for robust Bayesian estimation. Gas chromatography-mass spectrometry further provided multiple fragments of metabolites which yielded additional labeling information. We obtained precise estimations of the fluxes, their joint credibility intervals, and their relations, and characterized common metabolic patterns with principal component analysis (PCA). 13C-MFA indicated a hyperoxia-mediated reduction in tricarboxylic acid (TCA) cycle activity in circulating granulocytes which encompassed fluxes of glutamine uptake, TCA cycle, and oxaloacetate/aspartate supply for biosynthetic processes. We further detected elevated superoxide levels in the swine strain characterized by a hypercholesterolemic phenotype. PCA revealed cell type-specific behavioral patterns of metabolic adaptation in response to ASDH and HS that acted irrespective of swine strains or treatment group. In a model of resuscitated porcine ASDH and HS, we saw that ventilation with increased inspiratory O2 concentrations (PaO2 = 200 - 250 mmHg for 24 h after treatment initiation) did not impact mitochondrial respiration of PBMCs or granulocytes. However, Bayesian 13C-MFA results indicated a reduction in TCA cycle activity in granulocytes compared to cells exposed to normoxemia in the same time period. This change in metabolism did not seem to affect granulocytes' ability to perform phagocytosis or produce superoxide radicals.