Changes In Mitochondrial Respiration Research Articles

Mitochondrial Dysfunction in Sporadic Amyotrophic Lateral Sclerosis Patients: Insights from High-Resolution Respirometry.

Amyotrophic lateral sclerosis is a severe neurodegenerative disease whose exact cause is still unclear. Currently, research attention is turning to the mitochondrion as a critical organelle of energy metabolism. Current knowledge is sufficient to confirm the involvement of the mitochondria in the pathophysiology of the disease, since the mitochondria are involved in many processes in the cell; however, the exact mechanism of involvement is still unclear. We used peripheral blood mononuclear cells isolated from whole fresh blood from patients with amyotrophic lateral sclerosis for measurement and matched an age- and sex-matched set of healthy subjects. The group of patients consisted of patients examined and diagnosed at the neurological clinic of the University Hospital Martin. The set of controls consisted of healthy individuals who were actively searched, and controls were selected on the basis of age and sex. The group consisted of 26 patients with sporadic forms of ALS (13 women, 13 men), diagnosed based on the definitive criteria of El Escorial. The average age of patients was 54 years, and the average age of healthy controls was 56 years. We used a high-resolution O2K respirometry method, Oxygraph-2k, to measure mitochondrial respiration. Basal respiration was lower in patients by 29.48%, pyruvate-stimulated respiration (respiratory chain complex I) was lower by 29.26%, and maximal respiratory capacity was lower by 28.15%. The decrease in succinate-stimulated respiration (respiratory chain complex II) was 26.91%. Our data confirm changes in mitochondrial respiration in ALS patients, manifested by the reduced function of complex I and complex II of the respiratory chain. These defects are severe enough to confirm this disease's hypothesized mitochondrial damage. Therefore, research interest in the future should be directed towards a deeper understanding of the involvement of mitochondria and respiratory complexes in the pathophysiology of the disease. This understanding could develop new biomarkers in diagnostics and subsequent therapeutic interventions.

Oxidative stress in the remaining muscle after volumetric muscle loss contributes to impaired mitochondrial bioenergetics and is attenuated by SS-31

Volumetric muscle loss (VML) injury results in the non-recoverable loss of muscle mass and function. Our previous studies indicate mitochondrial dysfunction in the remaining muscle fibers after VML injury, marked by changes in mitochondrial respiration, membrane potential, and enzyme activities. In particular, mitochondrial membrane potential is hyperpolarized during the first 14-days post-injury, a condition that could lead to the production of reactive oxygen species (ROS). When ROS emissions exceed the antioxidant buffering capacity, cellular damage can ensue. The primary objective of this study was to determine ROS production, ROS emission, and antioxidant buffering capacity of remaining muscle fibers after VML injury in the first month after injury. The secondary objective of this study was to determine if a mitochondrial antioxidant (SS-31) could mitigate mitochondrial dysfunction after VML injury. We hypothesized that VML injury will alter the redox status of the remaining muscle. Study 1: Male C57BL/6J mice (n=40) were randomized to Uninjured or VML-injured cohorts. ROS (H2O2) emission/production and endogenous antioxidant buffering capacity were analyzed fluorometrically during live muscle fiber respiration (Oroboros O2k) at 3-, 7-, 14-, 21-, and 30-days post-injury. Study 2: Male C57BL/6J mice (n=32) were randomized into two groups: VML and VML+SS-31. SS-31 is a mitochondrial-targeted peptide with ROS-scavenging properties. VML+SS-31 received SS-31 (8mg/kg/day) for 14 days, and the VML received equal volume saline injections. At 14- and 28-days post-VML, mitochondrial respiration and electron conductance, ROS emission, and antioxidant buffering capacity were assessed in permeabilized gastrocnemius muscle fibers from the muscle remaining after the initial injury. Group differences were detected through ANOVA and unpaired t-test. ROS emission and antioxidant buffering capacity were different between VML-injured and Uninjured mice through 14-days post-injury, specifically at 3-, 7-, and 14-days, mitochondrial ROS emission was significantly higher (+28%, p<0.001), and antioxidant buffering capacity was less (-28%, p=0.002) in VML mice compared to Uninjured mice. Two weeks of SS-31 treatment lead to greater muscle fiber respiration and electron conductance (p≤0.008) at both 14- and 28-days post-VML. Importantly, at 14-days post-injury, mitochondrial ROS emission was 71% lower in VML+SS-31 than VML. At both 14- and 28-days post-injury, antioxidant buffering capacity was 12% greater in VML+SS-31 (p≤0.003) compared to VML. Oxidative stress is a feature of early VML pathophysiology marked by greater ROS emission and less antioxidant buffering capacity. Attenuating oxidative stress during the first two-weeks post-VML injury improves mitochondrial respiration. W81XWH-20-1-0885 and R01AR078903 to SG and JC. 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.

Flexibility underlies differences in mitochondrial respiratory performance between migratory and non-migratory White-crowned Sparrows (Zonotrichia leucophrys)

Migration is one of the most energy-demanding behaviors observed in birds. Mitochondria are the primary source of energy used to support these long-distance movements, yet how mitochondria meet the energetic demands of migration is scarcely studied. We quantified changes in mitochondrial respiratory performance in the White-crowned Sparrow (Zonotrichia leucophrys), which has a migratory and non-migratory subspecies. We hypothesized that the long-distance migratory Gambel’s subspecies (Z. l. gambelii) would show higher mitochondrial respiratory performance compared to the non-migratory Nuttall’s subspecies (Z. l. nuttalli). We sampled Gambel’s individuals during spring pre-migration, active fall migration, and a period with no migration or breeding (winter). We sampled Nuttall’s individuals during periods coinciding with fall migration and the winter period of Gambel’s annual cycle. Overall, Gambel’s individuals had higher citrate synthase, a proxy for mitochondrial volume, than Nuttall’s individuals. This was most pronounced prior to and during migration. We found that both OXPHOS capacity (state 3) and basal respiration (state 4) of mitochondria exhibit high seasonal flexibility within Gambel’s individuals, with values highest during active migration. These values in Nuttall’s individuals were most similar to Gambel’s individuals in winter. Our observations indicate that seasonal changes in mitochondrial respiration play a vital role in migration energetics.

A transcriptomic analysis of correlation between mitochondrial function and energy metabolism remodeling in mice with myocardial fibrosis following myocardial infarction

To investigate the changes of mitochondrial respiratory function during myocardial fibrosis in mice with myocardial infarction (MI) and its correlation with the increase of glycolytic flux. Forty C57BL/6N mice were randomized into two equal groups to receive sham operation or ligation of the left anterior descending coronary artery to induce acute MI. At 28 days after the operation, 5 mice from each group were euthanized and left ventricular tissue samples were collected for transcriptomic sequencing. FPKM method was used to calculate gene expression levels to identify the differentially expressed genes (DEGs) in MI mice, which were analyzed using GO and KEGG databases to determine the pathways affecting the disease process. Heat maps were drawn to show the differential expressions of the pathways and the related genes in the enrichment analysis. In primary cultures of neonatal mouse cardiac fibroblasts (CFs), the changes in mitochondrial respiration and glycolysis levels in response to treatment with the pro-fibrotic agonist TGF-β1 were analyzed using Seahorse experiment. The mouse models of MI showed significantly increased diastolic and systolic left ventricular diameter (P < 0.05) and decreased left ventricular ejection fraction (P < 0.0001). A total of 124 up-regulated and 106 down-regulated DEGs were identified in the myocardial tissues of MI mice, and GO and KEGG enrichment analysis showed that these DEGs were significantly enriched in fatty acid metabolism, organelles and other metabolic pathways and in the mitochondria. Heat maps revealed fatty acid beta oxidation, mitochondrial dysfunction and increased glycolysis levels in MI mice. In the primary culture of CFs, treatment with TGF-β1 significantly reduced the basal and maximum respiratory levels and increased the basal and maximum glycolysis levels (P < 0.0001). During myocardial fibrosis, energy metabolism remodeling occurs in the CFs, manifested by lowered mitochondrial function and increased energy generation through glycolysis.

Cardiac mitochondrial metabolism during pregnancy and the postpartum period.

The goal of the present study was to characterize changes in mitochondrial respiration in the maternal heart during pregnancy and after birth. Timed pregnancy studies were performed in 12-wk-old female FVB/NJ mice, and cardiac mitochondria were isolated from the following groups of mice: nonpregnant (NP), midpregnancy (MP), late pregnancy (LP), and 1-wk postbirth (PB). Similar to our previous studies, we observed increased heart size during all stages of pregnancy (e.g., MP and LP) and postbirth (e.g., PB) compared with NP mice. Differential cardiac gene and protein expression analyses revealed changes in several mitochondrial transcripts at LP and PB, including several mitochondrial complex subunits and members of the Slc family, important for mitochondrial substrate transport. Respirometry revealed that pyruvate- and glutamate-supported state 3 respiration was significantly higher in PB vs. LP mitochondria, with respiratory control ratio (RCR) values higher in PB mitochondria. In addition, we found that PB mitochondria respired more avidly when given 3-hydroxybutyrate (3-OHB) than mitochondria from NP, MP, and LP hearts, with no differences in RCR. These increases in respiration in PB hearts occurred independent of changes in mitochondrial yield but were associated with higher abundance of 3-hydroxybutyrate dehydrogenase 1. Collectively, these findings suggest that, after birth, maternal cardiac mitochondria have an increased capacity to use 3-OHB, pyruvate, and glutamate as energy sources; however, increases in mitochondrial efficiency in the postpartum heart appear limited to carbohydrate and amino acid metabolism.NEW & NOTEWORTHY Few studies have detailed the physiological adaptations that occur in the maternal heart. We and others have shown that pregnancy-induced cardiac growth is associated with significant changes in cardiac metabolism. Here, we examined mitochondrial respiration and substrate preference in isolated mitochondria from the maternal heart. We show that following birth, cardiac mitochondria are "primed" to respire on carbohydrate, amino acid, and ketone bodies. However, heightened respiratory efficiency is observed only with carbohydrate and amino acid sources. These results suggest that significant changes in mitochondrial respiration occur in the maternal heart in the postpartum period.

Abstract 5579: MGAloss-of-function regulates mitochondrial oxidative metabolism-induced cancer stem cell phenotype during lymphomagenesis with therapeutic potential

Abstract As an antagonist of MYC oncogene, Max gene associated (MGA) gene are implicated in multiple cancers, proposing the potential role of tumor suppressor. Using multi-omics data of The Cancer Genome Atlas (TCGA, n=10,295) and lymphoma datasets from our group (n=702), we defined genetic features associated with MGA and its related MYC/MAX/MXD network members across 36 cancers. Pan-cancer, MGA mutation, predominant as nonsense or frameshift, was observed in 4.39% of all samples. MGA expression was significantly downregulated in mutated samples than that of wild type samples. Moreover, MGA mutation was positively associated with inferior clinical outcomes in diffuse large B-cell lymphoma (P=0.001 and P&amp;lt;0.001 in 2 independent datasets), peripheral T-cell lymphoma (P=0.044) and natural killer/T-cell lymphoma (P=0.012), encouraging in-depth study on the important role of MGA in lymphomagenesis. Conditional knockout of MGA in hematopoietic system using Vav-iCre (Mgafl/fl, Vav-iCre mice) led to reduction in the numbers of peripheral blood lymphocyte due to blockade differentiation and enhanced stemness as evidenced by flowcytometry and RNA-sequencing (RNA-seq). To investigate the molecular mechanism, we performed pulldown assay in NK-92 cells molecular silencing of MGA or scramble control, and subsequently subjected for mass spectrum analysis. A total of 97 proteins were identified to interaction with MGA and mainly enriched to nucleosome (26), cytosolic ribosome (16), immune response (13), and glycolysis (6). Molecular silencing of MGA, localizing in the nucleolus, interfered with ribosomal RNA processing, enhanced “onco-ribosome” biogenesis, and contributed to aberrant translational program characterized by mitochondrial oxidative metabolism. Changes in mitochondrial respiration and membrane potential are key to maintaining stemness in cancer cells. Considering that stemness is important for tumorigenesis, recurrence, metastasis, and drug resistance, we exploited high-throughput drug screening platform and found that targeted inhibition of Exportin 1 (XPO1) significantly suppressed the viability of tumor cells molecular silencing MGA, which were resistant to chemotherapeutic agents such as gemcitabine, etoposide, etc. Mechanically, molecular silencing MGA induced abnormalities of nucleocytoplasmic localization in 739 proteins involving in mitochondrial respiratory chain complex I assembly, oxidative phosphorylation, and mitochondrial translation, 50% (332/739) of which were rescued by treatment with XPO1 inhibitor Selinexor. In summary, MGA initiated lymphoma cell stemness via onco-ribosome mediated activation of mitochondrial oxidative metabolism. Selinexor targeted the aberrant nucleocytoplasmic localization and served as a promising therapeutic agent. Citation Format: Jie Xiong, Wen-Fang Wang, Wei-Li Zhao. MGAloss-of-function regulates mitochondrial oxidative metabolism-induced cancer stem cell phenotype during lymphomagenesis with therapeutic potential [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 5579.

The PINK1/Parkin pathway of mitophagy exerts a protective effect during prion disease.

The PINK1/Parkin pathway of mitophagy has been implicated in the pathogenesis of Parkinson's disease. In prion diseases, a transmissible neurodegenerative disease caused by the misfolded and infectious prion protein (PrPSc), expression of both PINK1 and Parkin are elevated, suggesting that PINK1/Parkin mediated mitophagy may also play a role in prion pathogenesis. Using mice in which expression of either PINK1 (PINK1KO) or Parkin (ParkinKO) has been ablated, we analyzed the potential role of PINK1 and Parkin in prion pathogenesis. Prion infected PINK1KO and ParkinKO mice succumbed to disease more rapidly (153 and 150 days, respectively) than wild-type control C57Bl/6 mice (161 days). Faster incubation times in PINK1KO and ParkinKO mice did not correlate with altered prion pathology in the brain, altered expression of proteins associated with mitochondrial dynamics, or prion-related changes in mitochondrial respiration. However, the expression level of mitochondrial respiration Complex I, a major site for the formation of reactive oxygen species (ROS), was higher in prion infected PINK1KO and ParkinKO mice when compared to prion infected control mice. Our results demonstrate a protective role for PINK1/Parkin mitophagy during prion disease, likely by helping to minimize ROS formation via Complex I, leading to slower prion disease progression.

Age-related changes of skeletal muscle metabolic response to contraction are also sex-dependent.

Mitochondria adapt to increased energy demands during muscle contraction by acutely altering metabolite fluxes and substrate oxidation. With age, an impaired mitochondrial metabolic response may contribute to reduced exercise tolerance and decreased skeletal muscle mass, specific force, increased overall fatty depositions in the skeletal muscle, frailty and depressed energy maintenance. We hypothesized that elevated energy stress in mitochondria with age alters the capacity of mitochondria to utilize different substrates following muscle contraction. To test this hypothesis, we used in vivo electrical stimulation to simulate high-intensity intervals (HII) or low intensity steady-state (LISS) exercise in young (5-7months) and aged (27-29months) male and female mice to characterize effects of age and sex on mitochondrial substrate utilization in skeletal muscle following contraction. Mitochondrial respiration using glutamate decreased in aged males following HII and glutamate oxidation was inhibited following HII in both the contracted and non-stimulated muscle of aged female muscle. Analyses of the muscle metabolome of female mice indicated that changes in metabolic pathways induced by HII and LISS contractions in young muscle are absent in aged muscle. To test improved mitochondrial function on substrate utilization following HII, we treated aged females with elamipretide (ELAM), a mitochondrially-targeted peptide shown to improve mitochondrial bioenergetics and restore redox status in aged muscle. ELAM removed inhibition of glutamate oxidation and showed increased metabolic pathway changes following HII, suggesting rescuing redox status and improving bioenergetic function in mitochondria from aged muscle increases glutamate utilization and enhances the metabolic response to muscle contraction in aged muscle. KEY POINTS: Acute local contraction of gastrocnemius can systemically alter mitochondrial respiration in non-stimulated muscle. Age-related changes in mitochondrial respiration using glutamate or palmitoyl carnitine following contraction are sex-dependent. Respiration using glutamate after high-intensity contraction is inhibited in aged female muscle. Metabolite level and pathway changes following muscle contraction decrease with age in female mice. Treatment with the mitochondrially-targeted peptide elamipretide can partially rescue metabolite response to muscle contraction.

Metabolic Disruption Induced by mTOR Signaling Pathway Inhibition in Regulatory T-Cell Expansion for Clinical Application.

Regulatory T cell (Treg) therapy is considered an alternative approach to induce tolerance in transplantation. If successful, this therapy may have implications on immunosuppression minimization/withdrawal to reduce drug-induced toxicity in patients. The aim of this study was to assess the efficacy of the mTORC1/C2 inhibitor, AZD8055, in the manufacturing of clinically competent Treg cells and compare the effects with those induced by rapamycin (RAPA), another mTOR inhibitor commonly used in Treg expansion protocols. Primary human Treg cells were isolated from leukapheresis product. Cell viability, expansion rates, suppressive function, autophagy, mitochondrial unfolded protein response (mitoUPR), and cell metabolic profile were assessed. We observed a stronger inhibition of the mTORC2 signaling pathway and downstream events triggered by Interleukin 2 (IL2)-receptor in AZD8055-treated cells compared with those treated with RAPA. AZD8055 induced progressive metabolic changes in mitochondrial respiration and glycolytic pathways that disrupted the long-term expansion and suppressive function of Tregs. Unlike RAPA, AZD8055 treatment impaired autophagy and enhanced the mitoUPR cell stress response pathway. A distinct pattern of mTOR inhibition by AZD, compared with RAPA, induced mitochondrial stress response and dysfunction, impaired autophagy, and disrupted cellular bioenergetics, resulting in the loss of proliferative potential and suppressive function of Treg cells.

The bioenergetic "CK Clamp" technique detects substrate-specific changes in mitochondrial respiration and membrane potential during early VML injury pathology.

Volumetric muscle loss (VML) injuries are characterized by non-recoverable loss of tissue resulting in contractile and metabolic dysfunction. The characterization of metabolic dysfunction in volumetric muscle loss-injured muscle has been interpreted from permeabilized myofiber respiration experiments involving saturating ADP levels and non-physiologic ATP:ADP concentration ratios. The extent to which this testing condition obscures the analysis of mitochondrial (dys) function after volumetric muscle loss injury is unclear. An alternative approach is described that leverages the enzymatic reaction of creatine kinase and phosphocreatine to assess mitochondrial respiration and membrane potential at clamped physiologic ATP:ADP ratios, "CK Clamp." The objective of this study was to validate the CK Clamp in volumetric muscle loss-injured muscle and to detect differences that may exist between volumetric muscle loss-injured and uninjured muscles at 1, 3, 5, 7, 10, and 14days post-injury. Volumetric muscle loss-injured muscle maintains bioenergetic features of the CK Clamp approach, i.e., mitochondrial respiration rate (JO2) titters down and mitochondrial membrane potential is more polarized with increasing ATP:ADP ratios. Pyruvate/malate/succinate-supported JO2 was significantly less in volumetric muscle loss-injured muscle at all timepoints compared to uninjured controls (-26% to -84%, p < 0.001) and electron conductance was less at day 1 (-60%), 5 (-52%), 7 (-35%), 10 (-59%), and 14 (-41%) (p < 0.001). Palmitoyl-carnitine/malate-supported JO2 and electron conductance were less affected following volumetric muscle loss injury. volumetric muscle loss-injury also corresponded with a more polarized mitochondrial membrane potential across the clamped ATP:ADP ratios at day 1 and 10 (pyruvate and palmitoyl-carnitine, respectively) (+5%, p < 0.001). This study supports previous characterizations of metabolic dysfunction and validates the CK Clamp as a tool to investigate bioenergetics in traumatically-injured muscle.

OR09-5 mTOR Signaling is Associated with Regulation of Mitochondrial Respiration in Thyroid Cancer

Abstract Background Thyroid cancers (TC) are divided into BRAF-like and RAS-like tumors based on their molecular signature. The mTOR (mammalian target of rapamycin) signaling pathway is commonly activated in RAS-like tumors. Oncogene-driven signaling pathways are involved in the regulation of intracellular metabolism, including glycolysis and oxidative phosphorylation (OXPHOS). There is inadequate information on the role of mTOR signaling in the regulation of TC metabolism. The purpose of this study was to determine the association between mTOR signaling and mitochondrial respiration in TC. Methods We conducted an in vitro study using 4 TC cell lines (2 BRAF-like, 2 RAS-like) characterized by variable activation of mTOR signaling pathway, as documented by immunoblotting to quantify a ratio of p-mTOR/mTOR. OXPHOS was measured through oxygen consumption rate (OCR) of TC cells by the Seahorse XF Cell Mito Stress Test Assay. Standard MTT assay was performed to evaluate cell proliferation. A specific mTOR inhibitor (rapamycin) as well as an mTOR activator (leucine) were used for functional studies. The association between OCR and p-mTOR/mTOR was calculated by Pearson correlation coefficient (r). The one-way Anova or Kruskal-Wallis test were used for between group comparisons, as appropriate. P-values of ≤ 0.05 were considered statistically significant. Results The baseline activation of mTOR signaling in TC cells correlated with baseline (r=0.75, p=0.04) and maximum OCR (r=0.91, p=0.01). In addition, the RAS-like cell lines with the highest activation of mTOR showed considerable response after inhibition of mTOR with rapamycin by inhibiting baseline OCR (FTC133: 6320 ± 1531 vs 3368 ± 651 pmol/min/mg, p=0.01; THJ29T: 4484 ± 514 vs 3067 ± 620 pmol/min/mg, p=0.047), while the TC cell lines with minimal baseline mTOR activation did not show change in mTOR phosphorylation status nor basal OCR after rapamycin treatment (TPC1: 1817 ± 201 vs 1974 ± 336 pmol/min/mg, p&amp;gt;0.99, BCPAP: 1051 ± 162 vs 1130 ± 228 pmol/min/mg, p&amp;gt;0.99). Treatment with rapamycin significantly reduced cell proliferation rates in all examined cells. On the other hand, treatment with the mTOR activator leucine increased both mTOR phosphorylation and basal OCR in TC cell lines characterized by minimal baseline mTOR activation (TPC1: 1778 ± 280 vs 2372 ± 249 pmol/min/mg, p=0.004, BCPAP: 1581 ± 603 vs 2064 ± 560 pmol/min/mg, p=0.04), but did not significantly increase OCR further in cell lines with high baseline mTOR activation and OXPHOS rate (FTC133: 4512 ± 683 vs 5355 ± 870 pmol/min/mg, p=0.27; THJ29T: 3881 ± 880 vs 4764 ± 1273 pmol/min/mg, p=0.14). Conclusion mTOR signaling is associated with the regulation of mitochondrial respiration as its inhibition decreases OXPHOS rate, while its activation results in increased mitochondrial respiration. The change in mitochondrial respiration might be one of the mechanisms of the growth inhibition caused by medications targeting mTOR signaling in TC. Presentation: Saturday, June 11, 2022 12:30 p.m. - 12:45 p.m.

CoCl2-Mimicked Endothelial Cell Hypoxia Induces Nucleotide Depletion and Functional Impairment That Is Reversed by Nucleotide Precursors.

Chronic hypoxia drives vascular dysfunction by various mechanisms, including changes in mitochondrial respiration. Although endothelial cells (ECs) rely predominantly on glycolysis, hypoxia is known to alter oxidative phosphorylation, promote oxidative stress and induce dysfunction in ECs. Our work aimed to analyze the effects of prolonged treatment with hypoxia-mimetic agent CoCl2 on intracellular nucleotide concentration, extracellular nucleotide breakdown, mitochondrial function, and nitric oxide (NO) production in microvascular ECs. Moreover, we investigated how nucleotide precursor supplementation and adenosine deaminase inhibition protected against CoCl2-mediated disturbances. Mouse (H5V) and human (HMEC-1) microvascular ECs were exposed to CoCl2-mimicked hypoxia for 24 h in the presence of nucleotide precursors: adenine and ribose, and adenosine deaminase inhibitor, 2′deoxycoformycin. CoCl2 treatment decreased NO production by ECs, depleted intracellular ATP concentration, and increased extracellular nucleotide and adenosine catabolism in both H5V and HMEC-1 cell lines. Diminished intracellular ATP level was the effect of disturbed mitochondrial phosphorylation, while nucleotide precursors effectively restored the ATP pool via the salvage pathway and improved endothelial function under CoCl2 treatment. Endothelial protective effects of adenine and ribose were further enhanced by adenosine deaminase inhibition, that increased adenosine concentration. This work points to a novel strategy for protection of hypoxic ECs by replenishing the adenine nucleotide pool and promoting adenosine signaling.

148-OR: Partial Knockout of Skeletal Muscle Dynamin-Related Protein 1 Improves Insulin Sensitivity in Diet-Induced Insulin Resistant Mice, but Not in Normal Control Mice

Dynamin-related protein 1 (Drp1) is the key regulator of mitochondrial fission. We and others have reported excessive Drp1 activity in skeletal muscle from both animals and humans with insulin resistance, which is strongly correlated to impaired skeletal muscle insulin sensitivity. To examine whether Drp1 directly regulates skeletal muscle insulin sensitivity and whole-body glucose homeostasis, we generated tamoxifen-inducible skeletal muscle-specific heterozygous Drp1 partial knockout mice (mDrp1+/-) . Male mDrp1+/- and wildtype (WT) mice were fed with either a high-fat diet (HFD) or low-fat diet (LFD) for four weeks, received tamoxifen injections for five consecutive days, and remained on their respective diet for another four weeks. Improvements in whole-body glucose tolerance and insulin sensitivity were observed in HFD-fed mDrp1+/- mice (P&amp;lt;0.05) , but not in LFD-fed mDrp1+/- mice, when compared to their respective WT controls. Further, HFD-induced impairment in skeletal muscle insulin-stimulated Akt Ser473 phosphorylation (P&amp;lt;0.05) was mitigated in HFD-fed mDrp1+/- mice. In contrast, insulin-stimulated Akt Ser473 phosphorylation was reduced in LFD-fed mDrp1+/- mice compared to LFD-fed WT mice (P&amp;lt;0.05) . mDrp1+/- mice exhibited more fused and interconnected mitochondrial networks in skeletal muscle than WT mice. The examination of mitochondrial function revealed no change in mitochondrial respiration but reductions in mitochondrial Complex I and II and pyruvate dehydrogenase derived H2O2 production in HFD-fed mDrp1+/- mice compared to HFD-fed WT mice (P&amp;lt;0.05) . In conclusion, our data suggest that partial knockout of skeletal muscle Drp1 improves skeletal muscle insulin sensitivity and whole-body glucose homeostasis in diet-induced insulin-resistant mice but not in normal control mice. These improvements are, at least in part, due to reduced mitochondrial-derived H2O2 production. Disclosure B.A.Kugler: None. N.Lin: None. P.D.Nguyen: None. A.Ali: None. A.Sesay: None. B.Kalemba: None. H.Sesaki: None. K.Zou: None. Funding National Institutes of Health (R15DK131512) Diabetes Action Research and Education Foundation Grant (#505)

The role of mitochondrial fission in cardiovascular health and disease.

Mitochondria are organelles involved in the regulation of various important cellular processes, ranging from ATP generation to immune activation. A healthy mitochondrial network is essential for cardiovascular function and adaptation to pathological stressors. Mitochondria undergo fission or fusion in response to various environmental cues, and these dynamic changes are vital for mitochondrial function and health. In particular, mitochondrial fission is closely coordinated with the cell cycle and is linked to changes in mitochondrial respiration and membrane permeability. Another key function of fission is the segregation of damaged mitochondrial components for degradation by mitochondrial autophagy (mitophagy). Mitochondrial fission is induced by the large GTPase dynamin-related protein 1 (DRP1) and is subject to sophisticated regulation. Activation requires various post-translational modifications of DRP1, actin polymerization and the involvement of other organelles such as the endoplasmic reticulum, Golgi apparatus and lysosomes. A decrease in mitochondrial fusion can also shift the balance towards mitochondrial fission. Although mitochondrial fission is necessary for cellular homeostasis, this process is often aberrantly activated in cardiovascular disease. Indeed, strong evidence exists that abnormal mitochondrial fission directly contributes to disease development. In this Review, we compare the physiological and pathophysiological roles of mitochondrial fission and discuss the therapeutic potential of preventing excessive mitochondrial fission in the heart and vasculature.

The Effects of In Utero Fetal Hypoxia and Creatine Treatment on Mitochondrial Function in the Late Gestation Fetal Sheep Brain.

Near-term acute hypoxia in utero can result in significant fetal brain injury, with some brain regions more vulnerable than others. As mitochondrial dysfunction is an underlying feature of the injury cascade following hypoxia, this study is aimed at characterizing mitochondrial function at a region-specific level in the near-term fetal brain after a period of acute hypoxia. We hypothesized that regional differences in mitochondrial function would be evident, and that prophylactic creatine treatment would mitigate mitochondrial dysfunction following hypoxia; thereby reducing fetal brain injury. Pregnant Border-Leicester/Merino ewes with singleton fetuses were surgically instrumented at 118 days of gestation (dGa; term is ~145 dGA). A continuous infusion of either creatine (n = 15; 6 mg/kg/h) or isovolumetric saline (n = 16; 1.5 ml/kg/h) was administered to the fetuses from 121 dGa. After 10 days of infusion, a subset of fetuses (8 saline-, 7 creatine-treated) were subjected to 10 minutes of umbilical cord occlusion (UCO) to induce a mild global fetal hypoxia. At 72 hours after UCO, the fetal brain was collected for high-resolution mitochondrial respirometry and molecular and histological analyses. The results show that the transient UCO-induced acute hypoxia impaired mitochondrial function in the hippocampus and the periventricular white matter and increased the incidence of cell death in the hippocampus. Creatine treatment did not rectify the changes in mitochondrial respiration associated with hypoxia, but there was a negative relationship between cell death and creatine content following treatment. Irrespective of UCO, creatine increased the proportion of cytochrome c bound to the inner mitochondrial membrane, upregulated the mRNA expression of the antiapoptotic gene Bcl2, and of PCG1-α, a driver of mitogenesis, in the hippocampus. We conclude that creatine treatment prior to brief, acute hypoxia does not fundamentally modify mitochondrial respiratory function, but may improve mitochondrial structural integrity and potentially increase mitogenesis and activity of antiapoptotic pathways.

Abstract 14116: Exercise and Specialized Proresolving Lipid Mediators Stimulate Mitochondrial Metabolism in Macrophages

Exercise may prevent the development and progression of chronic inflammatory diseases through alterations in immune cell function. We find that macrophages isolated from mice subjected to 4 weeks of voluntary wheel exercise display enhanced basal and maximal mitochondrial respiration. We have previously shown that exercise also increases the production of specialized proresolving mediators (SPMs), macrophage phagocytosis, and resolution of acute inflammation; however, whether exercise-induced SPM formation directly alters macrophage metabolism remains unknown. SPMs stimulate resolution through actions on specific G-protein coupled receptors (GPCRs) in macrophages ( e.g. , RvD1=&gt; Fpr2). Therefore, we treated macrophages for 1 h with SPMs that act through distinct GPCRs ( e.g. , RvD1, RvD2, RvE1, and MaR1) and found enhanced basal (vehicle vs 1nM SPM; 6.11±0.1 vs RvD1 7.76±0.6 vs RvD2 9.48±0.9 vs RvE1 7.20±0.5 vs MaR1 8.73±0.9 pmol/min/μg protein; P&lt;0.05; n =3), maximal (vehicle vs 0.1nM SPM; 9.06±1.1 vs RvD1 14.37±1.1 vs RvD2 16.54±2.7 vs RvE1 12.32±1.5 vs MaR1 13.28±1.3 pmol/min/μg protein; P&lt;0.05; n =3), and ATP-linked respiration (vehicle vs 1nM SPM; 2.83±0.2 vs RvD1 4.50±0.3 vs RvD2 4.39±0.2 vs RvE1 4.71±0.3 vs MaR1 4.20±0.2 pmol/min/μg protein; P&lt;0.05; n =3). Stimulation of mitochondrial metabolism with RvD1 was receptor mediated as Fpr2 -deficient macrophages displayed no significant changes in mitochondrial respiration following treatment (vehicle vs 1nM RvD1; basal 6.74±0.6 vs 6.52±0.2; maximal 11.54±1.2 vs 13.26±1.0; ATP-linked 5.84±0.8 vs 5.33±0.2 pmol/min/μg protein). Collectively, these data suggest that exercise-induced production of SPMs stimulates mitochondrial metabolism in macrophages, which could underlie their common proresolving properties and the exercise-induced amelioration of chronic inflammatory diseases.

Monitoring Mitochondrial Function in Aedes albopictus C6/36 Cell Line during Dengue Virus Infection.

Simple SummaryDengue is an important and growing public health problem. To date, no specific therapeutic or effective prophylactic measures exist. Therefore, vector control remains the primary approach to prevent dengue virus (DENV) infection in humans. Recent findings highlight that viruses regulate mitochondrial function and dynamics to facilitate viral proliferation. In this study, we report that DENV infection modulates mitochondrial physiology in C6/36 mosquito cells. Our results revealed that DENV alters redox metabolism and mitochondrial membrane potential without any significant change in cellular ATP pool or viability. In addition, we observed preservation of the respiratory control ratio and translocation of mitofusins to mitochondria. These results suggest that mitochondrial fusion could be required for the maintenance of mitochondrial function in C6/36 mosquito cells infected with DENV.Aedes aegypti and Aedes albopictus mosquitoes are responsible for dengue virus (DENV) transmission in tropical and subtropical areas worldwide, where an estimated 3 billion people live at risk of DENV exposure. DENV-infected individuals show symptoms ranging from sub-clinical or mild to hemorrhagic fever. Infected mosquitoes do not show detectable signs of disease, even though the virus maintains a lifelong persistent infection. The interactions between viruses and host mitochondria are crucial for virus replication and pathogenicity. DENV infection in vertebrate cells modulates mitochondrial function and dynamics to facilitate viral proliferation. Here, we describe that DENV also regulates mitochondrial function and morphology in infected C6/36 mosquito cells (derived from Aedes albopictus). Our results showed that DENV infection increased ROS (reactive oxygen species) production, modulated mitochondrial transmembrane potential and induced changes in mitochondrial respiration. Furthermore, we offer the first evidence that DENV causes translocation of mitofusins to mitochondria in the C6/36 mosquito cell line. Another protein Drp-1 (Dynamin-related protein 1) did not localize to mitochondria in DENV-infected cells. This observation therefore ruled out the possibility that the abovementioned alterations in mitochondrial function are associated with mitochondrial fission. In summary, this report provides some key insights into the virus–mitochondria crosstalk in DENV infected mosquito cells.

Intermittent Hypoxia-Hyperoxia and Oxidative Stress in Developing Human Airway Smooth Muscle.

Premature infants are frequently and intermittently administered supplemental oxygen during hypoxic episodes, resulting in cycles of intermittent hypoxia and hyperoxia. The relatively hypoxic in utero environment is important for lung development while hyperoxia during the neonatal period is recognized as detrimental towards the development of diseases such as bronchopulmonary dysplasia and bronchial asthma. Understanding early mechanisms that link hypoxic, hyperoxic, and intermittent hypoxic-hyperoxic exposures to altered airway structure and function are key to developing advanced therapeutic approaches in the clinic. Changes in oxygen availability can be detrimental to cellular function and contribute to oxidative damage. Here, we sought to determine the effect of oxygen on mitochondria in human fetal airway smooth muscle cells exposed to either 5% O2, 21% O2, 40% O2, or cycles of 5% and 40% O2 (intermittent hypoxia-hyperoxia). Reactive oxygen species production, altered mitochondrial morphology, and changes in mitochondrial respiration were assessed in the context of the antioxidant N-acetylcysteine. Our findings show developing airway smooth muscle is differentially responsive to hypoxic, hyperoxic, or intermittent hypoxic-hyperoxic exposure in terms of mitochondrial structure and function. Cycling O2 decreased mitochondrial branching and branch length similar to hypoxia and hyperoxia in the presence of antioxidants. Additionally, hypoxia decreased overall mitochondrial respiration while the addition of antioxidants increased respiration in normoxic and O2-cycling conditions. These studies show the necessity of balancing oxidative damage and antioxidant defense systems in the developing airway.

Sample Preparation as a Critical Aspect of Blood Platelet Mitochondrial Respiration Measurements-The Impact of Platelet Activation on Mitochondrial Respiration.

Blood platelets are considered as promising candidates as easily-accessible biomarkers of mitochondrial functioning. However, their high sensitivity to various stimulus types may potentially affect mitochondrial respiration and lead to artefactual outcomes. Therefore, it is crucial to identify the factors associated with platelet preparation that may lead to changes in mitochondrial respiration. A combination of flow cytometry and advanced respirometry was used to examine the effect of blood anticoagulants, the media used to suspend isolated platelets, respiration buffers, storage time and ADP stimulation on platelet activation and platelet mitochondria respiration. Our results clearly show that all the mentioned factors can affect platelet mitochondrial respiration. Briefly, (i) the use of EDTA as anticoagulant led to a significant increase in the dissipative component of respiration (LEAK), (ii) the use of plasma for the suspension of isolated platelets with MiR05 as a respiration buffer allows high electron transfer capacity and low platelet activation, and (iii) ADP stimulation increases physiological coupling respiration (ROUTINE). Significant associations were observed between platelet activation markers and mitochondrial respiration at different preparation steps; however, the fact that these relationships were not always apparent suggests that the method of platelet preparation may have a greater impact on mitochondrial respiration than the platelet activation itself.

A single session of high‐intensity interval exercise in hypoxia did not change mitochondrial respiration or content in human skeletal muscle biopsies

Both high-intensity interval exercise (HIIE) and hypoxia have been suggested to affect mitochondria. However, it is not yet fully elucidated how a single session of HIIE in hypoxia affects mitochondrial respiration and content in human skeletal muscle. In this current study, we investigated the effects of a single session of HIIE combined with simulated hypoxia (3200 m, oxygen fraction of 14%) on mitochondrial respiration and content. We hypothesized that HIIE in hypoxia would induce substantial changes in mitochondrial respiration, but not in mitochondrial content. Ten healthy male participants (age, 28 ± 5 years; BMI, 26.0 ± 3.4) were recruited and completed three HIIE sessions (six 4-minute exercise bouts, separated by 2 minutes of resting) in a random order, separated by at least one week. Exercise sessions include an HIIE in hypoxia (88.4% of peak oxygen uptake (VO2peak) determined in hypoxia), an HIIE in normoxia matched for relative intensity (88.7% of VO2peak determined in normoxia), and an HIIE in normoxia matched for absolute intensity (74.1% of VO2peak determined in normoxia). Skeletal muscle samples were collected prior to, 3 hours, and 24 hours post-exercise to determine mitochondrial respiration and content. Hypoxia decreased VO2peak (by 20.2 ± 9.1%, p < 0.01), peak power output (PPO, by 9.4 ± 2.1%, p<0.01), and lactate threshold (LT, by 13.1 ± 3.0%, p<0.01). In the skeletal muscle biopsies, mitochondrial respiration, measured by Oroboros O2k with high-resolution respirometry, was similar among the three conditions prior to the HIIE interventions (p > 0.05) and remained unchanged both 3 and 24 hours after HIIE interventions performed in different conditions (p > 0.05). Citrate synthase activity, as a marker of mitochondrial content, did not differ prior to, or 3 and 24 hours after HIIE interventions among the three conditions (p > 0.05) . In conclusion, a single session of HIIE in hypoxia did not alter mitochondrial respiration and content. Gene expression and protein content of mitochondrial biogenesis markers are currently being analyzed.