bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2022‒01‒09
33 papers selected by
Kelsey Fisher-Wellman, East Carolina University



  1. Front Chem. 2021 ;9 775226
      Clinically, the prognosis of tumor therapy is fundamentally affected by multidrug resistance (MDR), which is primarily a result of enhanced drug efflux mediated by channels in the membrane that reduce drug accumulation in tumor cells. How to restore the sensitivity of tumor cells to chemotherapy is an ongoing and pressing clinical issue. There is a prevailing view that tumor cells turn to glycolysis for energy supply due to hypoxia. However, studies have shown that mitochondria also play crucial roles, such as providing intermediates for biosynthesis through the tricarboxylic acid (TCA) cycle and a plenty of ATP to fuel cells through the complete breakdown of organic matter by oxidative phosphorylation (OXPHOS). High OXPHOS have been found in some tumors, particularly in cancer stem cells (CSCs), which possess increased mitochondria mass and may be depends on OXPHOS for energy supply. Therefore, they are sensitive to inhibitors of mitochondrial metabolism. In view of this, we should consider mitochondrial metabolism when developing drugs to overcome MDR, where mitochondrial RNA polymerase (POLRMT) would be the focus, as it is responsible for mitochondrial gene expression. Inhibition of POLRMT could disrupt mitochondrial metabolism at its source, causing an energy crisis and ultimately eradicating tumor cells. In addition, it may restore the energy supply of MDR cells to glycolysis and re-sensitize them to conventional chemotherapy. Furthermore, we discuss the rationale and strategies for designing new therapeutic molecules for MDR cancers by targeting POLRMT.
    Keywords:  OxPhos; POLRMT; RNA polymerase; cancer stem cell; multidrug resisitance
    DOI:  https://doi.org/10.3389/fchem.2021.775226
  2. ChemMedChem. 2022 Jan 04.
      Metformin and other biguanides represent a new class of inhibitors of mitochondrial complex I that show promising anti-tumor effects, but stronger inhibition of mitochondrial complex I is generally associated with upregulation of glycolysis and higher risk of lactic acidosis. Here we reported a novel biguanide derivative N-cystaminylbiguanide (MC001) that inhibited mitochondrial complex I with higher potency while induced similar lactate production compared with metformin. Furthermore, MC001 was found to efficiently inhibit a panel of colorectal cancer (CRC) cells in vitro, and suppress tumor growth in HCT116 xenograft nude mice model while did not enhance lactate production compared with metformin, exhibiting superior safety profile to other potent biguanides such as phenformin. Mechanistically, MC001 efficiently inhibited mitochondrial complex I, activated AMPK and repressed mTOR, leading to cell cycle arrest and apoptosis. Notably, MC001 inhibited both oxidative phosphorylation (OXPHOS) and glycolysis. We thus propose that MC001 warrants further investigation in cancer treatment.
    Keywords:  Biguanide; Glycolysis; Lactate production; Mitochondrial complex I; Oxidative phosphorylation
    DOI:  https://doi.org/10.1002/cmdc.202100674
  3. Cell Rep. 2022 Jan 04. pii: S2211-1247(21)01701-0. [Epub ahead of print]38(1): 110197
      AMP-activated protein kinase (AMPK) regulates the balance between cellular anabolism and catabolism dependent on energy resources to maintain proliferation and survival. Small-compound AMPK activators show anti-cancer activity in preclinical models. Using the direct AMPK activator GSK621, we show that the unfolded protein response (UPR) is activated by AMPK in acute myeloid leukemia (AML) cells. Mechanistically, the UPR effector protein kinase RNA-like ER kinase (PERK) represses oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and pyrimidine biosynthesis and primes the mitochondrial membrane to apoptotic signals in an AMPK-dependent manner. Accordingly, in vitro and in vivo studies reveal synergy between the direct AMPK activator GSK621 and the Bcl-2 inhibitor venetoclax. Thus, selective AMPK-activating compounds kill AML cells by rewiring mitochondrial metabolism that primes mitochondria to apoptosis by BH3 mimetics, holding therapeutic promise in AML.
    Keywords:  AML; AMPK; GSK621; PERK; mitochondrial apoptosis; unfolded protein response; venetoclax
    DOI:  https://doi.org/10.1016/j.celrep.2021.110197
  4. FEBS J. 2022 Jan 05.
      Nek4 is a serine/threonine kinase which has been implicated in primary cilia stabilization, DNA damage response, autophagy and epithelial-to-mesenchymal transition. The role of Nek4 in cancer cell survival and chemotherapy resistance has also been shown. However, the precise mechanisms by which Nek4 operates remain to be elucidated. Here, we show that Nek4 overexpression activates mitochondrial respiration coupled to ATP production, which is paralleled by increased mitochondrial membrane potential, and resistance to mitochondrial DNA damage. Congruently, Nek4 depletion reduced mitochondrial respiration and mtDNA integrity. Nek4 deficiency caused mitochondrial elongation, probably via reduced activity of the fission protein DRP1. In Nek4 overexpressing cells the increase in mitochondrial fission was concomitant to enhanced phosphorylation of DRP1 and Erk1/2 proteins, and the effects on mitochondrial respiration were abolished in the presence of a DRP1 inhibitor. This study shows Nek4 as a novel regulator of mitochondrial function that may explain the joint appearance of high mitochondrial respiration and mitochondrial fragmentation.
    Keywords:  DRP1; Nek4; fission; mitochondrial function
    DOI:  https://doi.org/10.1111/febs.16343
  5. J Biol Chem. 2021 Dec 29. pii: S0021-9258(21)01364-8. [Epub ahead of print] 101554
      The mitochondrial pyruvate carrier (MPC) is an inner mitochondrial membrane complex that plays a critical role in intermediary metabolism. Inhibition of the MPC, especially in liver, may have efficacy for treating type 2 diabetes mellitus. Herein, we examined the anti-diabetic effects of zaprinast and 7ACC2, small molecules which have been previously reported to act as MPC inhibitors. Both compounds activated a bioluminescence resonance energy transfer (BRET)-based MPC reporter assay (reporter sensitive to pyruvate; RESPYR) and potently inhibited pyruvate-mediated respiration in isolated mitochondria. Furthermore, zaprinast and 7ACC2 acutely improved glucose tolerance in diet-induced obese mice in vivo. Although some findings were suggestive of improved insulin sensitivity, hyperinsulinemic-euglycemic clamp studies did not detect enhanced insulin action in response to 7ACC2 treatment. Rather, our data suggest acute glucose-lowering effects of MPC inhibition may be due to suppressed hepatic gluconeogenesis. Finally, we used RESPYR to screen a chemical library (Pharmakon 1600) of drugs and identified 35 potentially novel MPC modulators. Using available evidence, we generated a pharmacophore model to prioritize which hits to pursue. Our analysis revealed carsalam and six quinolone antibiotics, as well as 7ACC1, share a common pharmacophore with 7ACC2. We validated that these compounds are novel inhibitors of the MPC and suppress hepatocyte glucose production, and demonstrated that one quinolone (nalidixic acid) improved glucose tolerance in obese mice. In conclusion, these data demonstrate the feasibility of therapeutic targeting of the MPC for treating diabetes and provide scaffolds that can be used to develop potent and novel classes of MPC inhibitors.
    Keywords:  diabetes; gluconeogenesis; metabolic disease; mitochondrial metabolism; pyruvate
    DOI:  https://doi.org/10.1016/j.jbc.2021.101554
  6. Clin Chem. 2022 Jan 05. pii: hvab268. [Epub ahead of print]
      BACKGROUND: Many studies have demonstrated the high efficacy of cell-free nuclear DNA in cancer diagnostics. Compared to nuclear DNA, mitochondrial DNA (mtDNA) exhibits distinct characteristics, including multiple copies per cell and higher mutation frequency. However, the potential applicability of cell-free mtDNA (cf-mtDNA) in plasma and urine remains poorly investigated.METHODS: Here, we comprehensively analyzed the fragmentomic and mutational characteristics of cf-mtDNA in urine and plasma samples from controls and cancer patients using next-generation sequencing.
    RESULTS: Compared to plasma cf-mtDNA, urine cf-mtDNA exhibited increased copy numbers and wider spread in fragment size distributions. Based on 2 independent animal models, urine cf-mtDNA originated predominantly from local shedding and transrenal excretion. Further analysis indicated an enhanced fragmentation of urine cf-mtDNA in renal cell carcinoma (RCC) and colorectal cancer (CRC) patients. Using the mtDNA sequence of peripheral blood mononuclear cells for reference, the mutant fragments were shorter than wild-type fragments in urine cf-mtDNA. Size selection of short urine cf-mtDNA fragments (<150 bp) significantly enhanced the somatic mutation detection. Our data revealed remarkably different base proportions of fragment ends between urine and plasma cf-mtDNA that also were associated with fragment size. Moreover, both RCC and CRC patients exhibited significantly higher T-end and lower A-end proportions in urine cf-mtDNA than controls. By integrating the fragmentomic and mutational features of urine cf-mtDNA, our nomogram model exhibited a robust efficacy for cancer diagnosis.
    CONCLUSIONS: Our proof-of-concept findings revealed aberrant fragmentation and mutation profiles of urine cf-mtDNA in cancer patients that have diagnostic potential.
    Keywords:  cancer; cell-free mtDNA; fragmentomics; liquid biopsy; next-generation sequencing; urine
    DOI:  https://doi.org/10.1093/clinchem/hvab268
  7. Front Pharmacol. 2021 ;12 734078
      T cell activation and differentiation is associated with metabolic reprogramming to cope with the increased bioenergetic demand and to provide metabolic intermediates for the biosynthesis of building blocks. Antigen receptor stimulation not only promotes the metabolic switch of lymphocytes but also triggers the uptake of calcium (Ca2+) from the cytosol into the mitochondrial matrix. Whether mitochondrial Ca2+ influx through the mitochondrial Ca2+ uniporter (MCU) controls T cell metabolism and effector function remained, however, enigmatic. Using mice with T cell-specific deletion of MCU, we here show that genetic inactivation of mitochondrial Ca2+ uptake increased cytosolic Ca2+ levels following antigen receptor stimulation and store-operated Ca2+ entry (SOCE). However, ablation of MCU and the elevation of cytosolic Ca2+ did not affect mitochondrial respiration, differentiation and effector function of inflammatory and regulatory T cell subsets in vitro and in animal models of T cell-mediated autoimmunity and viral infection. These data suggest that MCU-mediated mitochondrial Ca2+ uptake is largely dispensable for murine T cell function. Our study has also important technical implications. Previous studies relied mostly on pharmacological inhibition or transient knockdown of mitochondrial Ca2+ uptake, but our results using mice with genetic deletion of MCU did not recapitulate these findings. The discrepancy of our study to previous reports hint at compensatory mechanisms in MCU-deficient mice and/or off-target effects of current MCU inhibitors.
    Keywords:  calcium (Ca2+); immunometabolism; mitochondria; mitochondrial Ca2+ handling; mitochondrial calcium uniporter (MCU); oxidative phosphorylation; store-operated Ca2+ entry
    DOI:  https://doi.org/10.3389/fphar.2021.734078
  8. J Clin Invest. 2022 Jan 06. pii: e153157. [Epub ahead of print]
      The bone marrow (BM) microenvironment regulates acute myeloid leukemia (AML) initiation, proliferation and chemotherapy resistance. Following cancer cell death, a growing body of evidence suggests an important role for remaining apoptotic debris in regulating the immunologic response to, and growth of, solid tumors. Here we investigated the role of macrophage LC3-associated phagocytosis (LAP) within the BM microenvironment of AML. Depletion of BM macrophages increased AML growth in-vivo. We showed that LAP is the predominate method of BM macrophage phagocytosis of dead and dying cells in the AML microenvironment. Targeted inhibition of LAP led to accumulation of apoptotic cells (AC) and apoptotic bodies (AB) resulting in accelerated leukemia growth. Mechanistically, LAP of AMLderived-AB by BM macrophages, resulted in STING pathway activation. We identified that AML derived mitochondrial damage associated molecular patterns were processed by BM macrophages via LAP. Moreover, depletion of mitochondrial DNA (mtDNA) in AML derived-AB showed that it is this mtDNA which was responsible for the induction of STING signalling in BM macrophages. Phenotypically we found that STING activation suppressed AML growth through a mechanism related to increased phagocytosis. In summary, we report that macrophage LAP of apoptotic debris in the AML BM microenvironment suppressed tumor growth.
    Keywords:  Autophagy; Hematology; Leukemias; Mitochondria; Oncology
    DOI:  https://doi.org/10.1172/JCI153157
  9. J Proteome Res. 2022 Jan 05.
      The outer mitochondrial membrane protein SLC25A46 has been recently identified as a novel genetic cause of a wide spectrum of neurological diseases. The aim of the present work was to elucidate the physiological role of SLC25A46 through the identification of its interactome with immunoprecipitation and proteomic analysis in whole cell extracts from the cerebellum, cerebrum, heart, and thymus of transgenic mice expressing ubiquitously SLC25A46-FLAG. Our analysis identified 371 novel putative interactors of SLC25A46 and confirmed 17 known ones. A total of 79 co-immunoprecipitated proteins were common in two or more tissues, mainly participating in mitochondrial activities such as oxidative phosphorylation (OXPHOS) and ATP production, active transport of ions or molecules, and the metabolism. Tissue-specific co-immunoprecipitated proteins were enriched for synapse annotated proteins in the cerebellum and cerebrum for metabolic processes in the heart and for nuclear processes and proteasome in the thymus. Our proteomic approach confirmed known mitochondrial interactors of SLC25A46 including MICOS complex subunits and also OPA1 and VDACs, while we identified novel interactors including the ADP/ATP translocases SLC25A4 and SLC25A5, subunits of the OXPHOS complexes and F1Fo-ATP synthase, and components of the mitochondria-ER contact sites. Our results show that SLC25A46 interacts with a large number of proteins and protein complexes involved in the mitochondria architecture, energy production, and flux and also in inter-organellar contacts.
    Keywords:  LC−MS/MS; SLC25A46; interactome; mitochondria; neurological diseases; quantitative proteomics; transgenic mice
    DOI:  https://doi.org/10.1021/acs.jproteome.1c00728
  10. Cancer Commun (Lond). 2022 Jan 04.
      BACKGROUND: Mitochondria are dynamic organelles that constantly change their morphology through fission and fusion processes. Recently, abnormally increased mitochondrial fission has been observed in several types of cancer. However, the functional roles of increased mitochondrial fission in lipid metabolism reprogramming in cancer cells remain unclear. This study aimed to explore the role of increased mitochondrial fission in lipid metabolism in hepatocellular carcinoma (HCC) cells.METHODS: Lipid metabolism was determined by evaluating the changes in the expressions of core lipid metabolic enzymes and intracellular lipid content. The rate of fatty acid oxidation was evaluated by [3 H]-labelled oleic acid. The mitochondrial morphology in HCC cells was evaluated by fluorescent staining. The expression of protein was determined by real-time PCR, iimmunohistochemistry and Western blotting.
    RESULTS: Activation of mitochondrial fission significantly promoted de novo fatty acid synthesis in HCC cells through upregulating the expression of lipogenic genes fatty acid synthase (FASN), acetyl-CoA carboxylase1 (ACC1), and elongation of very long chain fatty acid protein 6 (ELOVL6), while suppressed fatty acid oxidation by downregulating carnitine palmitoyl transferase 1A (CPT1A) and acyl-CoA oxidase 1 (ACOX1). Consistently, suppressed mitochondrial fission exhibited the opposite effects. Moreover, in vitro and in vivo studies revealed that mitochondrial fission-induced lipid metabolism reprogramming significantly promoted the proliferation and metastasis of HCC cells. Mechanistically, mitochondrial fission increased the acetylation level of sterol regulatory element-binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α) by suppressing nicotinamide adenine dinucleotide (NAD+)/Sirtuin 1 (SIRT1) signaling. The elevated SREBP1 then upregulated the expression of FASN, ACC1 and ELOVL6 in HCC cells, while PGC-1α/PPARα suppressed the expression of CPT1A and ACOX1.
    CONCLUSIONS: Increased mitochondrial fission plays a crucial role in the reprogramming of lipid metabolism in HCC cells, which provides strong evidence for the use of this process as a drug target in the treatment of this malignancy.
    Keywords:  Sirtuin 1; fatty acid oxidation; hepatocellular carcinoma; lipogenesis; metabolic reprogramming; mitochondrial fission
    DOI:  https://doi.org/10.1002/cac2.12247
  11. STAR Protoc. 2021 Dec 17. 2(4): 101021
      Drosophila flight muscles are highly enriched with mitochondria and have emerged as a powerful genetic system for studying how oxidative phosphorylation (OXPHOS) complexes are assembled. Here, we describe a series of protocols for analyzing the integrity of OXPHOS complexes in Drosophila via blue native polyacrylamide gel electrophoresis (BN PAGE). We have also included protocols for the additional steps that are typically performed after OXPHOS complexes are separated by BN PAGE, such as Coomassie staining, silver staining, and in-gel OXPHOS activities. For complete details on the use and execution of this protocol, please refer to Murari et al. (2020).
    Keywords:  Cell Biology; Genetics; Metabolism; Model Organisms; Protein Biochemistry
    DOI:  https://doi.org/10.1016/j.xpro.2021.101021
  12. Life Sci. 2022 Jan 03. pii: S0024-3205(21)01261-3. [Epub ahead of print] 120274
      AIMS: The purpose of this study was to evaluate the heterogeneities of glutamine metabolism in EGFR-TKI-resistant lung cancer cells and its potential as a therapeutic target.MAIN METHODS: Cell proliferation and cell cycle assays was performed by IncuCyte real-time analysis and flow cytometry, respectively. Tumor growth was assessed in xenografts implanted with HCC827 GR. An isotopologue analysis was conducted by LC-MS/MS using 13C-(U)-glutamine labeling to determine the amounts of metabolites. Cellular ATP and mitochondrial oxidative phosphorylation were determined by XFp analysis.
    KEY FINDINGS: We found that the cell growth of the two acquired EGFR-TKI-resistant lung cancer cells lines (HCC827 GR and H292 ER) depends on glutamine. In HCC827 GR, glutamine deficiency caused reduced GSH synthesis and, subsequently, enhanced ROS generation relative to their parental cells, HCC827. On the other hand, in H292 ER, glutamine mainly acted as a carbon source for TCA-cycle intermediates, and its depletion led to reduced mitochondrial ATP production. CB-839, a specific GLS inhibitor, inhibited the latter's conversion of glutamine to glutamate and exerted enhanced anti-proliferating effects on the two acquired EGFR-TKI-resistant lung cancer cell lines versus their parental cell lines. Moreover, oral administration of CB-839 significantly suppressed HCC827 GR tumor growth in the xenograft model.
    SIGNIFICANCE: These findings suggest that glutamine dependency in acquired EGFR-TKI-resistant lung cancer is heterogeneous and that inhibition of glutamine metabolism by CB-839 may serve as a therapeutic tool for acquired EGFR-TKI-resistant lung cancer.
    Keywords:  Acquired EGFR-TKI-resistant lung cancer; CB-839; CB-839 (Telaglenastat; Glutamine metabolism; PubChem CID: 71577426)
    DOI:  https://doi.org/10.1016/j.lfs.2021.120274
  13. Int J Biol Sci. 2022 ;18(1): 374-385
      Anti-cancer chemo-drugs can cause a rapid elevation of intracellular reactive oxygen species (ROS) levels. An imbalance in ROS production and elimination systems leads to cancer cell resistance to chemotherapy. This study aimed to evaluate the mechanism and effect of ROS on multidrug resistance in various human chemoresistant cancer cells by detecting the changes in the amount of ROS, the expression of ROS-related and glycolysis-related genes, and cell death. We found that ROS was decreased while oxidative phosphorylation was increased in chemoresistant cells. We verified that the chemoresistance of cancer cells was achieved in two ways. First, chemoresistant cells preferred oxidative phosphorylation instead of anaerobic glycolysis for energy generation, which increased ATPase activity and produced much more ATP to provide energy. Second, ROS-scavenging systems were enhanced in chemoresistant cancer cells, which in turn decreased ROS amount and thus inhibited chemo-induced cell death. Our in vitro and in vivo photodynamic therapy further demonstrated that elevated ROS production efficiently inhibited chemo-drug resistance and promoted chemoresistant cell death. Taken together, targeting ROS systems has a great potential to treat cancer patients with chemoresistance.
    Keywords:  Chemoresistance; ROS; glycolysis; malignant tumor; oxidative phosphorylation; photodynamic therapy
    DOI:  https://doi.org/10.7150/ijbs.66602
  14. Cancer Lett. 2021 Dec 31. pii: S0304-3835(21)00655-8. [Epub ahead of print]529 19-36
      Cancer cells are typically characterized by abnormal quality control of mitochondria, production of reactive oxygen species (ROS), dysregulation of the cell redox state, and the Warburg effect. Mutation or depletion of PTEN-induced kinase 1 (PINK1) or Parkin leads to mitophagy defects and accumulation of malfunctioning mitochondria, and is often detected in a variety of tumors. However, PINK1's role in the progression of gastric cancer (GC) remains unclear, with its main effect being on mitochondrial turnover, metabolic reprogramming, and tumor microenvironment (TME) alteration. To address these issues, we first assessed the expression levels of PINK1, mitophagy-associated molecules, ROS, HIF-1α, glycolysis-associated genes, and macrophage signatures in GC tissues and matched tumor-adjacent normal samples. In addition, GC cell lines (AGS and MKN-45) and xenograft mouse models were used to determine the mechanism by which PINK1 regulates mitophagy, metabolic reprogramming, tumor-associated macrophage (TAM) polarization, and GC progression. We found that PINK1 loss correlated with advanced stage GC and poorer overall survival. GC tissues with lower PINK1 levels showed compromised mitophagy signaling and enhanced glycolytic enzyme expression. In vitro experiments demonstrated that PINK1 deficiency promoted GC cell proliferation and migration through the inhibition of mitophagy, production of mitochondrial ROS, stabilization of HIF-1α, and facilitation of the Warburg effect under both normoxic and hypoxic conditions. Moreover, PINK1 deficiency in GC cells promoted TAM polarization toward the M2-like phenotype. Reintroduction of PINK1 or inhibition of HIF-1α effectively repressed PINK1 deficiency-mediated effects on GC cell growth, metabolic shift, and TAM polarization. Thus, mitophagy defects caused by PINK1 loss conferred a metabolic switch through accumulation of mtROS and stabilization of HIF-1α, thereby facilitating the M2 polarization of TAM to remodel an immunosuppressive microenvironment in GC. Our results clarify the mechanism between PINK1 and GC progression and may provide a novel strategy for the treatment of GC.
    Keywords:  Metabolic reprogramming; Mitochondria; ROS,HIF-1α; Tumor-associated macrophage
    DOI:  https://doi.org/10.1016/j.canlet.2021.12.032
  15. Mol Metab. 2021 Dec 31. pii: S2212-8778(21)00287-8. [Epub ahead of print] 101429
      STIM1 is a single-pass transmembrane endoplasmic/sarcoplasmic reticulum (E/SR) protein recognized for its role in store operated Ca2+ entry (SOCE), an ancient and ubiquitous signaling pathway. Whereas STIM1 is indispensable during development, its biological and metabolic functions in mature muscle were unclear. Shown here, STIM1 is abundant in adult skeletal muscle, upregulated by exercise, and present at SR-mitochondria interfaces. Among its multifaceted roles, STIM1 regulates Ca2+ signaling, mitochondrial Ca2+ loading, energy metabolism and protein homeostasis. Thus, inducible tissue-specific deletion of STIM1 (iSTIM1 KO) in adult muscle leads to diminished lean mass, reduced exercise capacity, and perturbed fuel selection in settings of energetic stress, without affecting whole-body glucose tolerance. Proteomics and phospho-proteomics analyses of iSTIM1 KO muscles revealed molecular signatures of low-grade E/SR stress and broad activation of processes and signaling networks involved in proteostasis. The findings provide insight into the pathophysiology of muscle diseases linked to disturbances in STIM1-dependent calcium handling.
    DOI:  https://doi.org/10.1016/j.molmet.2021.101429
  16. J Cancer. 2022 ;13(1): 304-312
      Kidney cancers including clear cell carcinoma (RCC) are identified with very vulnerable mitochondria DNA (mtDNA) and frequent epigenetic aberrations. Bone metastasis from RCC is prevalent and destructive. Bone marrow contains a quite hypoxic microenvironment that usually insitigate 50% of hypermethylation events in conferring a selective advantage for tumor growth. We hypothesized that hypermethylation of mtDNA in RCC cells would significantly contribute to bone metastatic tumor progression. Methylation-specific polymerase chain reaction assay (MSP) was adopted to measure the methylation status of D-loop region of mtDNA in 15 pairs of bone metastatic and primary RCC as well as tumor adjescent normal kidney tissues. mtDNA copy number was examined by the real-time quantitative polymerase chain reaction (qPCR). Western blotting analysis was used to measure the accumulation of several DNA methyltransferases (DNMTs) in the mitochondria and nucleus fractions of bone metastatic RCC cells. mRNA expression of mitochondria encoded genes was examined by RT-PCR. Reactive oxygen species (ROS), mitochondrial membrane potential and ATP content were measured using in vitro cells treated with de-methylation drug 5-Azacytidine (5-Aza). Non-invasive bioluminescent imaging was performed to monitor tumor occurrence in skeleton in mice. Our results showed that the D-loop region in bone metastatic tumor cells was markedly hypermethylated than those in primary RCC tumor cells, that is associated with a decreased mtDNA copy number and accumulation of DNMT1 in the mitochondria. The bone-tropism tumor colonization and progression of RCC cells was significantly suppressed by demethylating the D-loop region of mtDNA and reducing the intracellular level of ROS and ATP by 5-Aza treatment. In conclusion, our study provided a direct association between hypermethylation of mtDNA in RCC with bone metastastic tumor growth.
    Keywords:  5-Azacytidine; Bone metastasis; clear cell carcinoma; hypermethylation; mitochondria DNA
    DOI:  https://doi.org/10.7150/jca.62278
  17. ACS Biomater Sci Eng. 2022 Jan 03.
      Mitochondria play an essential role in cellular metabolism and generate energy in cells. To support these functions, several proteins are encoded in the mitochondrial DNA (mtDNA). The mutation of mtDNA causes mitochondrial dysfunction and ultimately results in a variety of inherited diseases. To date, gene delivery systems targeting mitochondria have been developed to ameliorate mtDNA mutations. However, applications of these strategies in mitochondrial gene therapy are still being explored and optimized. Thus, from this perspective, we herein highlight recent mitochondria-targeting strategies for gene therapy and discuss future directions for effective mitochondria-targeted gene delivery.
    Keywords:  gene therapy; mitochondria; mitochondria-targeting peptides; organelle targeting
    DOI:  https://doi.org/10.1021/acsbiomaterials.1c01114
  18. Theranostics. 2022 ;12(2): 859-874
      Rationale: Caloric restriction improves the efficacy of anti-cancer therapy. This effect is largely dependent on the increase of the extracellular ATP concentration in the tumor microenvironment (TME). Pathways for ATP release triggered by nutrient deprivation are largely unknown. Methods: The extracellular ATP (eATP) concentration was in vivo measured in the tumor microenvironment of B16F10-inoculated C57Bl/6 mice with the pmeLuc probe. Alternatively, the pmeLuc-TG-mouse was used. Caloric restriction was in vivo induced with hydroxycitrate (HC). B16F10 melanoma cells or CT26 colon carcinoma cells were in vitro exposed to serum starvation to mimic nutrient deprivation. Energy metabolism was monitored by Seahorse. Microparticle release was measured by ultracentrifugation and by Nanosight. Results: Nutrient deprivation increases eATP release despite the dramatic inhibition of intracellular energy synthesis. Under these conditions oxidative phosphorylation was dramatically impaired, mitochondria fragmented and glycolysis and lactic acid release were enhanced. Nutrient deprivation stimulated a P2X7-dependent release of ATP-loaded, mitochondria-containing, microparticles as well as of naked mitochondria. Conclusions: Nutrient deprivation promotes a striking accumulation of eATP paralleled by a large release of ATP-laden microparticles and of naked mitochondria. This is likely to be a main mechanism driving the accumulation of eATP into the TME.
    Keywords:  P2X7.; extracellular ATP; microparticles; nutrient deprivation; tumor microenvironment
    DOI:  https://doi.org/10.7150/thno.66274
  19. Cancer Sci. 2022 Jan 02.
      T cells survival, proliferation and anti-tumor response are tightly linked to their mitochondrial health. Complement C1q binding protein (C1QBP) promotes the mitochondrial fitness through regulation of mitochondrial metabolism and morphology. However, whether C1QBP regulates T cells survival, proliferation and anti-tumor immune function remains unclear. Our data demonstrated that C1QBP knocking down induced the accumulation of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential to impair T cells mitochondrial fitness. At the same time, C1QBP insufficiency led to the less recruitment of the anti-apoptotic proteins including Bcl-2 and Bcl-XL to repress caspase-3 activation and PARP cleavage, which consequently accelerated T cells apoptotic process. On the other hand, C1QBP knocking-down rendered T cells with the relatively weaker proliferation due to the inhibition of AKT/mTOR signaling pathway. In order to investigate the exact role of C1QBP in anti-tumor response, C1QBP+/- and C1QBP+/+ mice were given the subcutaneous injection of murine MC38 cells, respectively. We found that C1QBP deficiency attenuated T cells tumor infiltration and aggravated these tumor-infiltrating T lymphocytes (TILs) exhaustion. Moreover, we further clarified the potential function of C1QBP in the chimeric antigen receptor (CAR)-T cells immunotherapy. Our data showed that C1QBP+/- CAR-T cells exhibited relatively weaker anti-tumor response than the corresponding C1QBP+/+ CAR-T cells. Given that C1QBP knocking down impairs T cells anti-apoptotic capacity, proliferation as well as anti-tumor immune function, development of the strategy to potentiation of T cells mitochondrial fitness through C1QBP would have a promise to optimize their efficacy of the related immunotherapy.
    Keywords:  Anti-tumor immune function; C1QBP; Mitochondrial fitness; Proliferation; T cells survival
    DOI:  https://doi.org/10.1111/cas.15261
  20. Biochem Biophys Rep. 2022 Mar;29 101192
      The human antimicrobial peptide LL-37 permeabilizes the plasma membrane of host cells, but LL-37-induced direct effects on mitochondrial membrane permeability and function has not been reported. Here, we demonstrate that LL-37 is rapidly (within 20 min) internalized by human osteoblast-like MG63 cells, and that the peptide co-localizes with MitoTracker arguing for accumulation in mitochondria. Subcellular fractionation and Western blot disclose that stimulation with LL-37 (8 μM) for 2 h triggers release of the mitochondrial protein apoptosis-inducing factor (AIF) to the cytosol, whereas LL-37 causes no release of cytochrome C oxidase subunit IV of the inner mitochondrial membrane, suggesting that LL-37 affects mitochondrial membrane permeability in a specific manner. Next, we investigated release of AIF and cytochrome C from isolated mitochondria by measuring immunoreactivity by dot blot. The media of mitochondria treated with LL-37 (8 μM) for 2 h contained 50% more AIF and three times more cytochrome C than that of control mitochondria, showing that LL-37 promotes release of both AIF and cytochrome C. Moreover, in vesicles reflecting mitochondrial membrane lipid composition, LL-37 stimulates membrane permeabilization and release of tracer molecules. We conclude that LL-37 is rapidly internalized by MG63 cells and accumulates in mitochondria, and that the peptide triggers release of pro-apoptotic AIF and directly affects mitochondrial membrane structural properties.
    Keywords:  Apoptosis; Cathelicidin; Innate immunity; Mitochondria; Mitochondria model membranes
    DOI:  https://doi.org/10.1016/j.bbrep.2021.101192
  21. J Med Chem. 2022 Jan 04.
      Inhibition of oxidative phosphorylation (OXPHOS) is a promising therapeutic strategy for select cancers that are dependent on aerobic metabolism. Here, we report the discovery, optimization, and structure-activity relationship (SAR) study of a series of novel OXPHOS inhibitors. The hit compound, benzene-1,4-disulfonamide 1, was discovered in a phenotypic screen selective for cytotoxicity in a galactose-containing medium. Our multi-parameter optimization campaign led to the discovery of 65 (DX3-235), showing nanomolar inhibition of complex I function and adenosine triphosphate (ATP) production in a galactose-containing medium resulting in significant cytotoxicity. Importantly, 64 (DX3-234), a close analogue of 65, is well tolerated in mice and shows significant single agent efficacy in a Pan02 syngeneic pancreatic cancer model, suggesting that highly potent and selective OXPHOS inhibitors can be useful for the treatment of pancreatic cancer.
    DOI:  https://doi.org/10.1021/acs.jmedchem.1c01509
  22. Cell Metab. 2022 Jan 04. pii: S1550-4131(21)00620-3. [Epub ahead of print]34(1): 90-105.e7
      HER2+ breast cancer patients are presented with either synchronous (S-BM), latent (Lat), or metachronous (M-BM) brain metastases. However, the basis for disparate metastatic fitness among disseminated tumor cells of similar oncotype within a distal organ remains unknown. Here, employing brain metastatic models, we show that metabolic diversity and plasticity within brain-tropic cells determine metastatic fitness. Lactate secreted by aggressive metastatic cells or lactate supplementation to mice bearing Lat cells limits innate immunosurveillance and triggers overt metastasis. Attenuating lactate metabolism in S-BM impedes metastasis, while M-BM adapt and survive as residual disease. In contrast to S-BM, Lat and M-BM survive in equilibrium with innate immunosurveillance, oxidize glutamine, and maintain cellular redox homeostasis through the anionic amino acid transporter xCT. Moreover, xCT expression is significantly higher in matched M-BM brain metastatic samples compared to primary tumors from HER2+ breast cancer patients. Inhibiting xCT function attenuates residual disease and recurrence in these preclinical models.
    Keywords:  HER2; breast cancer brain metastasis; immune surveillance; late recurrences; metabolism; metastasis; metastatic dormancy; metastatic latency; redox homeostasis; relapse
    DOI:  https://doi.org/10.1016/j.cmet.2021.12.001
  23. Mol Cell Proteomics. 2021 Dec 30. pii: S1535-9476(21)00163-8. [Epub ahead of print] 100191
      Mitophagy, the selective degradation of mitochondria by autophagy, affects defective mitochondria following damage or stress. At the onset of mitophagy, parkin ubiquitylates proteins on the mitochondrial outer membrane (MOM). While the role of parkin at the onset of mitophagy is well understood, less is known about its activity during later stages of the process. Here we used HeLa cells expressing catalytically active or inactive parkin to perform temporal analysis of the proteome, ubiquitylome and phosphoproteome during 18 hours after induction of mitophagy by mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP). Abundance profiles of proteins downregulated in parkin-dependent manner revealed a stepwise, "outside-in" directed degradation of mitochondrial subcompartments. While ubiquitylation of MOM proteins was enriched among early parkin-dependent targets, numerous mitochondrial inner membrane, matrix and cytosolic proteins were also found ubiquitylated at later stages of mitophagy. Phosphoproteome analysis revealed a possible cross-talk between phosphorylation and ubiquitylation during mitophagy on key parkin targets, such as VDAC1/2.
    Keywords:  Mitochondria; Mitophagy; Parkin; Quantitative proteomics; Ubiquitin
    DOI:  https://doi.org/10.1016/j.mcpro.2021.100191
  24. EMBO Rep. 2022 Jan 07. e48754
      Mitochondria are unavoidably subject to organellar stress resulting from exposure to a range of reactive molecular species. Consequently, cells operate a poorly understood quality control programme of mitophagy to facilitate elimination of dysfunctional mitochondria. Here, we used a model stressor, deferiprone (DFP), to investigate the molecular basis for stress-induced mitophagy. We show that mitochondrial fission 1 protein (Fis1) is required for DFP-induced mitophagy and that Fis1 is SUMOylated at K149, an amino acid residue critical for Fis1 mitochondrial localization. We find that DFP treatment leads to the stabilization of the SUMO protease SENP3, which is mediated by downregulation of the E3 ubiquitin (Ub) ligase CHIP. SENP3 is responsible for Fis1 deSUMOylation and depletion of SENP3 abolishes DFP-induced mitophagy. Furthermore, preventing Fis1 SUMOylation by conservative K149R mutation enhances Fis1 mitochondrial localization. Critically, expressing a Fis1 K149R mutant restores DFP-induced mitophagy in SENP3-depleted cells. Thus, we propose a model in which SENP3-mediated deSUMOylation facilitates Fis1 mitochondrial localization to underpin stress-induced mitophagy.
    Keywords:  Fis1; SENP3; SUMO; mitophagy; organellar stress
    DOI:  https://doi.org/10.15252/embr.201948754
  25. Mol Biol Cell. 2022 Jan 05. mbcE21120610T
      Positioning organelles at the right place and time is critical for their function and inheritance. In budding yeast, mitochondrial and nuclear positioning require the anchoring of mitochondria and dynein to the cell cortex by clusters of Num1. We have previously shown that mitochondria drive the assembly of cortical Num1 clusters, which then serve as anchoring sites for mitochondria and dynein. When mitochondrial inheritance is inhibited, mitochondrial-driven assembly of Num1 in buds is disrupted and defects in dynein-mediated spindle positioning are observed. Using a structure-function approach to dissect the mechanism of mitochondria-dependent dynein anchoring, we found the EF hand-like motif (EFLM) of Num1 and its ability to bind calcium are required to bias dynein anchoring on mitochondria-associated Num1 clusters. Consistently, when the EFLM is disrupted, we no longer observe defects in dynein activity following inhibition of mitochondrial inheritance. Thus, the Num1 EFLM functions to bias dynein anchoring and activity in nuclear inheritance subsequent to mitochondrial inheritance. We hypothesize that this hierarchical integration of organelle positioning pathways by the Num1 EFLM contributes to the regulated order of organelle inheritance during the cell cycle.
    DOI:  https://doi.org/10.1091/mbc.E21-12-0610-T
  26. J Thorac Dis. 2021 Nov;13(11): 6427-6438
      Background: Lung cancer is the leading cause of cancer-related death globally, with many of these patients also suffering from diabetes. Previous studies have shown that diabetes may contribute to cancer progression through hyperglycemia. However, the underlying mechanism remains largely unknown. This study aimed to investigate the role of succinate dehydrogenase 5 (SDH5), an enzyme required for assembling respiratory complex II in lung cancer patients with diabetes.Methods: The expression levels of SDH5 in patient plasma and tissue were determined by RT-qPCR. Western blotting, immunofluorescence (IF), and immunohistology (IHC) were used to examine protein expression. Migration and invasion assays were performed using Transwell assays. Reactive oxygen species (ROS) production was detected by flow cytometry. Bioluminescent imaging (BLI) was used to detect tumor metastasis in a lung orthotopic mouse model.
    Results: In samples from non-small cell lung cancer (NSCLC) patients with diabetes, SDH5 mRNA levels were significantly lower in both plasma and tissue among later stage patients. TCGA data showed that low SDH5 expression was correlated with a higher expression of genes involved in glycolysis and metastasis. In vitro, high glucose conditions alone induced epithelial-to-mesenchymal transition (EMT) in cells, an effect that was further reinforced by SDH5 depletion. Additionally, depleting SDH5 promoted glucose consumption and lactate production. The underlying mechanism indicates that depleting SDH5 stabilizes hypoxia-inducible factor 1-alpha (HIF-1α), which is dependent on ROS production. In vivo, SDH5-deficient tumor-bearing mice had multiple organ metastases, which is consistent with the in vitro findings.
    Conclusions: Our findings suggest that SDH5 deficiency activates HIF-1α to promote EMT under high glucose conditions and represents a predictive marker for NSCLC patients with diabetes.
    Keywords:  Non-small cell lung cancer (NSCLC); epithelial-to-mesenchymal transition (EMT); high glucose; hypoxia-inducible factor 1-alpha (HIF-1α); succinate dehydrogenase 5 (SDH5)
    DOI:  https://doi.org/10.21037/jtd-21-1769
  27. Leukemia. 2022 Jan 08.
      Mitochondria can function as signaling organelles, and part of this output leads to epigenetic remodeling. The full extent of this far-reaching interplay remains undefined. Here, we show that MYC transcriptionally activates IDH2 and increases alpha-ketoglutarate (αKG) levels. This regulatory step induces the activity of αKG-dependent DNA hydroxylases and RNA demethylases, thus reducing global DNA and RNA methylation. MYC, in a IDH2-dependent manner, also promotes the nuclear accumulation of TET1-TET2-TET3, FTO and ALKBH5. Notably, this subcellular movement correlated with the ability of MYC, in an IDH2-dependent manner, and, unexpectedly, of αKG to directly induce O-GlcNAcylation. Concordantly, modulation of the activity of OGT and OGA, enzymes that control the cycling of this non-canonical mono-glycosylation, largely recapitulated the effects of the MYC-IDH2-αKG axis on the subcellular movement of DNA and RNA demethylases. Together, we uncovered a hitherto unsuspected crosstalk between MYC, αKG and O-GlcNAcylation which could influence the epigenome and epitranscriptome homeostasis.
    DOI:  https://doi.org/10.1038/s41375-021-01489-7
  28. J Phys Chem Lett. 2022 Jan 05. 387-392
      Fo subcomplex of ATP synthase is a membrane-embedded rotary motor that converts proton motive force into mechanical energy. Despite a rapid increase in the number of high-resolution structures, the mechanism of tight coupling between proton transport and motion of the rotary c-ring remains elusive. Here, using extensive all-atom free energy simulations, we show how the motor's directionality naturally arises from the interplay between intraprotein interactions and energetics of protonation of the c-ring. Notably, our calculations reveal that the strictly conserved arginine in the a-subunit (R176) serves as a jack-of-all-trades: it dictates the direction of rotation, controls the protonation state of the proton-release site, and separates the two proton-access half-channels. Therefore, arginine is necessary to avoid slippage between the proton flux and the mechanical output and guarantees highly efficient energy conversion. We also provide mechanistic explanations for the reported defective mutations of R176, reconciling the structural information on the Fo motor with previous functional and single-molecule data.
    DOI:  https://doi.org/10.1021/acs.jpclett.1c03358
  29. Mol Cancer Res. 2022 Jan 06. pii: molcanres.0374.2021. [Epub ahead of print]
      There is a continued need to identify novel therapeutic targets to prevent the mortality associated with prostate cancer. In this context, Mitochondrial Rho GTPase 2 (MIRO2) mRNA was upregulated in metastatic prostate cancer compared to localized tumors, and higher MIRO2 levels were correlated with poor patient survival. Using human cell lines that represent androgen-independent or -sensitive prostate cancer, we showed that MIRO2 depletion impaired cell growth, colony formation and tumor growth in mice. Network analysis of MIRO2's binding partners identified metabolism and cellular responses to extracellular stimuli as top over-represented pathways. The top hit on our screen, General Control Non-derepressible 1 (GCN1), was overexpressed in prostate cancer, and interacted with MIRO2 in prostate cancer cell lines and in primary prostate cancer cells. Functional analysis of MIRO2 mutations present in prostate cancer patients led to the identification of MIRO2 159L, which increased GCN1 binding. Importantly, MIRO2 was necessary for efficient GCN1-mediated GCN2 kinase signaling and induction of the transcription factor ATF4 levels. Further, MIRO2's effect on regulating prostate cancer cell growth was mediated by ATF4. Finally, levels of activated GCN2 and ATF4 were correlated with MIRO2 expression in prostate cancer xenografts. Both MIRO2 and activated GCN2 levels were higher in hypoxic areas of prostate cancer xenografts. Overall, we propose that targeting the MIRO2-GCN1 axis may be a valuable strategy to halt prostate cancer growth. Implications: MIRO2/GCN1/GCN2 constitute a novel mitochondrial signaling pathway that controls androgen-independent and androgen-sensitive prostate cancer cell growth.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-21-0374
  30. Anal Chem. 2022 Jan 03.
      Data-dependent acquisition (DDA) methods are the current standard for quantitative proteomics in many biological systems. However, DDA preferentially measures highly abundant proteins and generates data that is plagued with missing values, requiring extensive imputation. Here, we demonstrate that library-free BoxCarDIA acquisition, combining MS1-level BoxCar acquisition with MS2-level data-independent acquisition (DIA) analysis, outperforms conventional DDA and other library-free DIA (directDIA) approaches. Using a combination of low- (HeLa cells) and high- (Arabidopsis thaliana cell culture) dynamic range sample types, we demonstrate that BoxCarDIA can achieve a 40% increase in protein quantification over DDA without offline fractionation or an increase in mass-spectrometer acquisition time. Further, we provide empirical evidence for substantial gains in dynamic range sampling that translates to deeper quantification of low-abundance protein classes under-represented in DDA and directDIA data. Unlike both DDA and directDIA, our new BoxCarDIA method does not require full MS1 scans while offering reproducible protein quantification between replicate injections and providing more robust biological inferences. Overall, our results advance the BoxCarDIA technique and establish it as the new method of choice for label-free quantitative proteomics across diverse sample types.
    DOI:  https://doi.org/10.1021/acs.analchem.1c03338
  31. Theranostics. 2022 ;12(2): 976-998
      Rationale: We found that a subset of signal transducer and activator of transcription 3 (STAT3) translocated into mitochondria in phagocytes, including macrophages isolated from individuals with sepsis. However, the role of mitochondrial STAT3 in macrophages remains unclear. Method: To investigate the function of mitochondrial STAT3 in vivo, we generated inducible mitochondrial STAT3 knock-in mice. A cytokine array analysis, a CBA analysis, flow cytometry, immunofluorescence staining and quantification and metabolic analyses in vivo were subsequently performed in an LPS-induced sepsis model. Single-cell RNA sequencing, a microarray analysis, metabolic assays, mass spectrometry and ChIP assays were utilized to gain insight into the mechanisms of mitochondrial STAT3 in metabolic reprogramming in LPS-induced sepsis. Results: We found that mitochondrial STAT3 induced NF-κB nuclear localization and exacerbated LPS-induced sepsis in parallel with a metabolic switch from mainly using glucose to an increased reliance on fatty acid oxidation (FAO). Moreover, mitochondrial STAT3 abrogated carnitine palmitoyl transferase 1a (CPT1a) ubiquitination and degradation in LPS-treated macrophages. Meanwhile, an interaction between CPT1a and ubiquitin-specific peptidase 50 (USP50) was observed. In contrast, knocking down USP50 decreased CPT1a expression and FAO mediated by mitochondrial STAT3. The ChIP assays revealed that NF-κB bound the USP50 promoter. Curcumin alleviated LPS-mediated sepsis by suppressing the activities of mitochondrial STAT3 and NF-κB. Conclusion: Our findings reveal that mitochondrial STAT3 could trigger FAO by inducing CPT1a stabilization mediated by USP50 in macrophages, at least partially.
    Keywords:  CPT1a stabilization; FAO; USP50; mitochondrial STAT3
    DOI:  https://doi.org/10.7150/thno.63751
  32. Mol Cell. 2022 Jan 06. pii: S1097-2765(21)01077-7. [Epub ahead of print]82(1): 60-74.e5
      Acetyl-CoA is a key intermediate situated at the intersection of many metabolic pathways. The reliance of histone acetylation on acetyl-CoA enables the coordination of gene expression with metabolic state. Abundant acetyl-CoA has been linked to the activation of genes involved in cell growth or tumorigenesis through histone acetylation. However, the role of histone acetylation in transcription under low levels of acetyl-CoA remains poorly understood. Here, we use a yeast starvation model to observe the dramatic alteration in the global occupancy of histone acetylation following carbon starvation; the location of histone acetylation marks shifts from growth-promoting genes to gluconeogenic and fat metabolism genes. This reallocation is mediated by both the histone deacetylase Rpd3p and the acetyltransferase Gcn5p, a component of the SAGA transcriptional coactivator. Our findings reveal an unexpected switch in the specificity of histone acetylation to promote pathways that generate acetyl-CoA for oxidation when acetyl-CoA is limiting.
    Keywords:  Gcn5p; Rpd3p; SAGA; acetyl-CoA; environmental stress response; fat metabolism; gluconeogenesis; glucose starvation; histone acetylation; transcription
    DOI:  https://doi.org/10.1016/j.molcel.2021.12.015
  33. Acta Physiol (Oxf). 2022 Jan 05. e13772
      AIM: Assessments of mitochondrial respiration and mitochondrial content are common in skeletal muscle research and exercise science. However, many sources of technical and biological variation render these analyses susceptible to error. This study aimed to better quantify the reliability of the experimental design and/or techniques employed, therefore assist researcher in obtaining more reliable data.METHODS: We examined the repeatability of maximal mitochondrial oxidative phosphorylation in permeabilized muscle fibres via high-resolution respirometry, and of citrate synthase activity (a biomarker for mitochondrial content) in a microplate with spectrophotometery.
    RESULTS: For mitochondrial respiration using permeabilised skeletal muscle fibres, the variability was reduced by using three chambers and removing outliers compared to two chambers (CV reduced from 12.7% to 11.0%), and the minimal change that can be detected with 10 participants reduced from 17% to 13% according to modelling. For citrate synthase activity, the within-plate CV (3.5%) increased when the assay was repeated after 4 hours (CV = 10.2%) and 4 weeks (CV = 30.5%). The readings were correlated, but significantly different after 4 hours and 4 weeks.
    CONCLUSION: This research provides evidence for important technical considerations when measuring mitochondrial respiration and content using citrate synthase activity as a biomarker. When assessing mitochondrial respiration in human skeletal muscle, the technical variability of high-resolution respirometry can be reduced by increasing technical repeats and excluding outliers, practices which are not currently common. When analysing citrate synthase activity, our results highlight the importance of analysing all samples from the same study at the same time.
    Keywords:  Exercise; Human skeletal muscle; Mitochondrial content; Mitochondrial respiration
    DOI:  https://doi.org/10.1111/apha.13772