Electron Transport Chain Subunits Research Articles

BHLHE40-mediated transcriptional activation of GRIN2D in gastric cancer is involved in metabolic reprogramming.

Gastric cancer (GC) is the third leading cause of death in developed countries. The reprogramming of energy metabolism represents a hallmark of cancer, particularly amplified dependence on aerobic glycolysis. Here, we aimed to illustrate the functional role of glutamate ionotropic receptor N-methyl-D-aspartate type subunit 2D (GRIN2D) in the regulation of glycolysis in GC and the mechanisms involved. Differentially expressed genes were analyzed using the GEO and GEPIA databases, followed by prognostic value prediction using the Kaplan-Meier Plotter database. The effect of GRIN2D knockdown on the malignant behavior and glycolysis of GC cells was explored. GRIN2D expression was upregulated in GC cells and promoted the malignant behavior of GC cells by activating glycolysis. Class E basic helix-loop-helix protein 40 (BHLHE40) was overexpressed in GC cells and mediated transcriptional activation of GRIN2D. The anti-tumor effects of BHLHE40 knockdown on GC cells in vitro and in vivo were reversed by GRIN2D overexpression. Knockdown of GRIN2D or BHLHE40 downregulated the expression of mRNA of electron transport chain subunits and phosphorylation of p38 MARK and inhibited calcium efflux in GC cells. Overexpression of GRIN2D promoted calcium efflux, phosphorylation of p38 MARK protein, and proliferation of GES1 cells. Altogether, the findings derived from this study suggest that BHLHE40 knockdown suppresses the growth, mobility, and glycolysis of GC cells by inhibiting GRIN2D transcription and disrupting the BHLHE40/GRIN2D axis may be an attractive therapeutic strategy for GC.

Inherent metabolic preferences differentially regulate the sensitivity of Th1 and Th2 cells to ribosome-inhibiting antibiotics.

Mitochondrial translation is essential to maintain mitochondrial function and energy production. Mutations in genes associated with mitochondrial translation cause several developmental disorders, and immune dysfunction is observed in many such patients. Besides genetic mutations, several antibiotics targeting bacterial ribosomes are well-established to inhibit mitochondrial translation. However, the effect of such antibiotics on different immune cells is not fully understood. Here, we addressed the differential effect of mitochondrial translation inhibition on different subsets of helper T cells (Th) of mice and humans. Inhibition of mitochondrial translation reduced the levels of mitochondrially encoded electron transport chain subunits without affecting their nuclear-encoded counterparts. As a result, mitochondrial oxygen consumption reduced dramatically, but mitochondrial mass was unaffected. Most importantly, we show that inhibition of mitochondrial translation induced apoptosis, specifically in Th2 cells. This increase in apoptosis was associated with higher expression of Bim and Puma, two activators of the intrinsic pathway of apoptosis. We propose that this difference in the sensitivity of Th1 and Th2 cells to mitochondrial translation inhibition reflects the intrinsic metabolic demands of these subtypes. Though Th1 and Th2 cells exhibit similar levels of oxidative phosphorylation, Th1 cells exhibit higher levels of aerobic glycolysis than Th2 cells. Moreover, Th1 cells are more sensitive to the inhibition of glycolysis, while higher concentrations of glycolysis inhibitor 2-deoxyglucose are required to induce cell death in the Th2 lineage. These observations reveal that selection of metabolic pathways for substrate utilization during differentiation of Th1 and Th2 lineages is a fundamental process conserved across species.

ATF4 and mTOR regulate metabolic reprogramming in TGF-β-treated lung fibroblasts.

Idiopathic pulmonary fibrosis is a fatal disease characterized by the TGF-β-dependent activation of lung fibroblasts, leading to excessive deposition of collagen proteins and progressive replacement of healthy lung with scar tissue. We and others have shown that fibroblast activation is supported by metabolic reprogramming, including the upregulation of the de novo synthesis of glycine, the most abundant amino acid found in collagen protein. How fibroblast metabolic reprogramming is regulated downstream of TGF-β is incompletely understood. We and others have shown that TGF-β-mediated activation of the Mechanistic Target of Rapamycin Complex 1 (mTORC1) and downstream upregulation of Activating Transcription Factor 4 (ATF4) promote increased expression of the enzymes required for de novo glycine synthesis; however, whether mTOR and ATF4 regulate other metabolic pathways in lung fibroblasts has not been explored. Here, we used RNA sequencing to determine how both ATF4 and mTOR regulate gene expression in human lung fibroblasts following TGF-β. We found that ATF4 primarily regulates enzymes and transporters involved in amino acid homeostasis as well as aminoacyl-tRNA synthetases. mTOR inhibition resulted not only in the loss of ATF4 target gene expression, but also in the reduced expression of glycolytic enzymes and mitochondrial electron transport chain subunits. Analysis of TGF-β-induced changes in cellular metabolite levels confirmed that ATF4 regulates amino acid homeostasis in lung fibroblasts while mTOR also regulates glycolytic and TCA cycle metabolites. We further analyzed publicly available single cell RNAseq data sets and found increased expression of ATF4 and mTOR metabolic targets in pathologic fibroblast populations from the lungs of IPF patients. Our results provide insight into the mechanisms of metabolic reprogramming in lung fibroblasts and highlight novel ATF4 and mTOR-dependent pathways that may be targeted to inhibit fibrotic processes.

Mono-(2-ethylhexyl) phthalate induces trophoblast hypoxia and mitochondrial dysfunction through HIF-1α-miR-210-3p axis in HTR-8/SVneo cell line

The exposure to the ubiquitous phthalate metabolite mono-(2-ethylhexyl) phthalate (MEHP) is connected to dysregulated trophoblast function and placenta health; however, the underlying mechanisms preluding this scenario remain to be elucidated. In this study, we explored the hypoxemic effects of MEHP on a human placental first-trimester trophoblast cell line (HTR-8/Svneo). MEHP-treated trophoblast cells displayed significantly increased levels of oxidative stress and hypoxia-inducible factor-1 alpha (HIF-1α) attributed by the induction of hypoxia. Further, HIF-1α exhibited higher DNA binding activity and upregulated gene expression of its downstream target vascular endothelial growth factor A (VEGFA). The hypoxia-induced microRNA miR-210-3p was also significantly increased upon MEHP treatment followed by disrupted mitochondrial ATP generation and membrane potential. This was identified to possibly be facilitated by lowered mitochondrial DNA copy number and inhibited expression of electron transport chain subunits, such as mitochondrial complex-IV. These results suggest potential adverse effects of MEHP exposure in a trophoblast cell line mediated by HIF-1α and the epigenetic modulator miR-210-3p. Chronic placental hypoxia and oxidative stress have long been implicated in the pathogenesis of pregnancy complications such as preeclampsia. As we’ve revealed genetic and epigenetic factors underscoring a potential mechanism induced by MEHP, this brings to light another significant implication of phthalate exposure on maternal and fetal health.

AGO2 Protects Against Diabetic Cardiomyopathy by Activating Mitochondrial Gene Translation.

Diabetes is associated with cardiovascular complications. microRNAs translocate into subcellular organelles to modify genes involved in diabetic cardiomyopathy. However, functional properties of subcellular AGO2 (Argonaute2), a core member of miRNA machinery, remain elusive. We elucidated the function and mechanism of subcellular localized AGO2 on mouse models for diabetes and diabetic cardiomyopathy. Recombinant adeno-associated virus type 9 was used to deliver AGO2 to mice through the tail vein. Cardiac structure and functions were assessed by echocardiography and catheter manometer system. AGO2 was decreased in mitochondria of diabetic cardiomyocytes. Overexpression of mitochondrial AGO2 attenuated diabetes-induced cardiac dysfunction. AGO2 recruited TUFM, a mitochondria translation elongation factor, to activate translation of electron transport chain subunits and decrease reactive oxygen species. Malonylation, a posttranslational modification of AGO2, reduced the importing of AGO2 into mitochondria in diabetic cardiomyopathy. AGO2 malonylation was regulated by a cytoplasmic-localized short isoform of SIRT3 through a previously unknown demalonylase function. Our findings reveal that the SIRT3-AGO2-CYTB axis links glucotoxicity to cardiac electron transport chain imbalance, providing new mechanistic insights and the basis to develop mitochondria targeting therapies for diabetic cardiomyopathy.

Metabolic reprogramming in inflammatory microglia indicates a potential way of targeting inflammation in Alzheimer's disease

Microglia activation drives the pro-inflammatory activity in the early stages of Alzheimer's disease (AD). However, the mechanistic basis is elusive, and the hypothesis of targeting microglia to prevent AD onset is little explored. Here, we demonstrated that upon LPS exposure, microglia shift towards an energetic phenotype characterised by high glycolysis and high mitochondrial respiration with dysfunction. Although the activity of electron transport chain (ETC) complexes is boosted by LPS, this is mostly devoted to the generation of reactive oxygen species. We showed that by inhibiting succinate dehydrogenase (SDH) with dimethyl malonate (DMM), it is possible to modulate the LPS-induced metabolic rewiring, facilitating an anti-inflammatory phenotype. DMM improves mitochondrial function in a direct way and by reducing LPS-induced mitochondrial biogenesis. Moreover, the block of SDH with DMM inhibits the recruitment of hypoxia inducible-factor 1 α (HIF-1α), which mediates the induction of glycolysis and cytokine expression. Similar bioenergetic alterations were observed in the microglia isolated from AD mice (3xTg-AD), which present high levels of circulating LPS and brain toll-like receptor4 (TLR4). Moreover, this well-established model of AD was used to show a potential effect of SDH inhibition in vivo as DMM administration abrogated brain inflammation and modulated the microglia metabolic alterations of 3xTg-AD mice. The RNA-sequencing analysis from a public dataset confirmed the consistent transcription of genes encoding for ETC subunits in the microglia of AD mice (5xFAD). In conclusion, TLR4 activation promotes metabolic changes and the pro-inflammatory activity in microglia, and SDH might represent a promising therapeutic target to prevent AD development.

Adult skeletal muscle peroxisome proliferator-activated receptor γ -related coactivator 1 is involved in maintaining mitochondrial content.

The peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) family of transcriptional coactivators are regulators of mitochondrial oxidative capacity and content in skeletal muscle. Many of these conclusions are based primarily on gain-of-function studies using muscle-specific overexpression of PGC1s. We have previously reported that genetic deletion of both PGC-1α and PGC-1β in adult skeletal muscle resulted in a significant reduction in oxidative capacity with no effect on mitochondrial content. However, the contribution of PGC-1-related coactivator (PRC), the third PGC-1 family member, in regulating skeletal muscle mitochondria is unknown. Therefore, we generated an inducible skeletal muscle-specific PRC knockout mouse (iMS-PRC-KO) to assess the contribution of PRC in skeletal muscle mitochondrial function. We measured mRNA expression of electron transport chain (ETC) subunits as well as markers of mitochondrial content in the iMS-PRC-KO animals and observed an increase in ETC gene expression and mitochondrial content. Furthermore, the increase in ETC gene expression and mitochondrial content was associated with increased expression of PGC-1α and PGC-1β. We therefore generated an adult-inducible PGC-1 knockout mouse in which all PGC-1 family members are deleted (iMS-PGC-1TKO). The iMS-PGC-1TKO animals exhibited a reduction in ETC mRNA expression and mitochondrial content. These data suggest that in the absence of PRC alone, compensation occurs by increasing PGC-1α and PGC-1β to maintain mitochondrial content. Moreover, the removal of all three PGC-1s in skeletal muscle results in a reduction in both ETC mRNA expression and mitochondrial content. Taken together, these results suggest that PRC plays a role in maintaining baseline mitochondrial content in skeletal muscle.

Constitutive Expression of Hif2α Confers Acute O2 Sensitivity to Carotid Body Glomus Cells.

Acute oxygen (O2) sensing and adaptation to hypoxia are essential for physiological homeostasis. The prototypical acute O2 sensing organ is the carotid body, which contains chemosensory glomus cells expressing O2-sensitive K+ channels. Inhibition of these channels during hypoxia leads to cell depolarization, transmitter release, and activation of afferent sensory fibers terminating in the brain stem respiratory and autonomic centers. Focusing on recent data, here we discuss the special sensitivity of glomus cell mitochondria to changes in O2 tension due to Hif2α-dependent expression of several atypical mitochondrial electron transport chain subunits and enzymes. These are responsible for an accelerated oxidative metabolism and the strict dependence of mitochondrial complex IV activity on O2 availability. We report that ablation of Epas1 (the gene coding Hif2α) causes a selective downregulation of the atypical mitochondrial genes and a strong inhibition of glomus cell acute responsiveness to hypoxia. Our observations indicate that Hif2α expression is required for the characteristic metabolic profile of glomus cells and provide a mechanistic explanation for the acute O2 regulation of breathing.

Mitochondrial dynamics, elimination and biogenesis during post-ischemic recovery in ischemia-resistant and ischemia-vulnerable gerbil hippocampal regions

Transient ischemic attacks (TIA) result from a temporary blockage in blood circulation in the brain. As TIAs cause disabilities and often precede full-scale strokes, the effects of TIA are investigated to develop neuroprotective therapies. We analyzed changes in mitochondrial network dynamics, mitophagy and biogenesis in sections of gerbil hippocampus characterized by a different neuronal survival rate after 5-minute ischemia-reperfusion (I/R) insult.Our research revealed a significantly greater mtDNA/nDNA ratio in CA2–3, DG hippocampal regions (5.8 ± 1.4 vs 3.6 ± 0.8 in CA1) that corresponded to a neuronal resistance to I/R. During reperfusion, an increase of pro-fission (phospho-Ser616-Drp1/Drp1) and pro-fusion proteins (1.6 ± 0.5 and 1.4 ± 0.3 for Mfn2 and Opa1, respectively) was observed in CA2–3, DG. Selective autophagy markers, PINK1 and SQSTM1/p62, were elevated 24-96 h after I/R and accompanied by significant elevation of transcription factors proteins PGC-1α and Nrf1 (1.2 ± 0.4, 1.78 ± 0.6, respectively) and increased respiratory chain proteins (e.g., 1.5 ± 0.3 for complex IV at I/R 96 h).Contrastingly, decreased enzymatic activity of citrate synthase, reduced Hsp60 protein level and electron transport chain subunits (0.88 ± 0.03, 0.74 ± 0.1 and 0.71 ± 0.1 for complex IV at I/R 96 h, respectively) were observed in I/R-vulnerable CA1. The phospho-Ser616-Drp1/Drp1 was increased while Mfn2 and total Opa1 reduced to 0.88 ± 0.1 and 0.77 ± 0.17, respectively. General autophagy, measured as LC3-II/I ratio, was activated 3 h after reperfusion reaching 2.37 ± 0.9 of control.This study demonstrated that enhanced mitochondrial fusion, followed by late and selective mitophagy and mitochondrial biogenesis might together contribute to reduced susceptibility to TIA.

Abstract 15761: SMYD1 Regulates OPA1-Mediated Cristae Remodeling and Supercomplex Formation to Protect From Ischemic Injury

SMYD1 is a lysine methyltransferase, which has been shown to methylate lysine 4 on histone H3, an established mark of gene activation. SMYD1 is only expressed in skeletal and cardiac muscle and was originally shown to play a significant role in regulating cardiac development. In the adult myocardium, using inducible, cardiomyocyte-specific Smyd1 knockout mice, loss of SMYD1 leads to massive downregulation of mitochondrial bioenergetics and overt heart failure. However, the effects of SMYD1 gain-of-function in the heart and its molecular function in the cardiomyocyte in response to ischemic stress remains unknown. Here we demonstrate that SMYD1a, the mouse ortholog of human SMYD1, positively regulates cardiac energetics and protects the heart from ischemic injury. To delineate how SMYD1a controls energy efficiency and metabolism in the cardiomyocyte, we generated a novel mouse model capable of inducible cardiomyocyte-specific SMYD1a overexpression. When subjected to ischemic injury these transgenic mice display reduced infarct size and cardiomyocyte death concomitant with enhanced mitochondrial respiratory efficiency. In addition, our molecular analysis revealed that the cardiac tissue in these animals is protected from ischemic injury through SMYD1a’s synergistic regulation of two key mitochondrial pathways. First, through its histone methyltransferase activity, SMYD1a maintains metabolic homeostasis by preserving basal expression of PGC-1α and its downstream targets including electron transport chain subunits. Second, SMYD1a regulates expression of OPA1, a key regulator of cristae morphology which drives the formation of electron transport chain supercomplexes to enhance mitochondrial respiration and ATP production. This work highlights SMYD1a as the only known epigenetic regulator of cristae morphology and identifies a novel molecular pathway by which cardiomyocytes dynamically regulate energy efficiency to protect from ischemic injury.

Dihydromyricetin ameliorates social isolation-induced anxiety by modulating mitochondrial function, antioxidant enzymes, and BDNF

Stress has been implicated in the etiology of neurological and psychological illnesses. Chronic social isolation (SI) is a psychological stressor that provokes neurobehavioral changes associated with psychiatric disorders, including anxiety disorders. Mitochondria dysfunction and oxidative stress are hallmarks of anxiety pathogenesis. Here we demonstrate the effects of SI-induced stress on mitochondrial function, antioxidative enzymes, autophagy, and brain derivative neurotrophic factor (BDNF). SI induced a reduction in electron transport chain subunits C–I, C-II, and C-VI and an increase in hydrogen peroxide. Treatment with dihydromyricetin (DHM), extracted from Ampelopsis grossedentata, counteracted these changes. A dramatic increase in several primary mitochondrial antioxidative enzymes such as superoxide dismutase 2 (SOD2), heme oxygenase-1 (HO-1), peroxiredoxin-3 (PRDX3), and glutathione peroxidase 4 (GPX4) was observed after SI and a repeated episode of SI. Both SI and repeated SI induced a reduction in sequestosome 1 (SQSTM1/p62). However, only repeated SI modulated autophagy primary protein beclin-1 (Bcl-1). In addition, SI and repeated SI modulated the BDNF-TrkB signaling pathway and the phosphorylation of the downstream extracellular signal-regulated MAP kinase1/2 (p-Erk p42 and p-Erk p44) cascade. DHM treatment ameliorated these changes. Collectively, we demonstrated that DHM treatment counteracted the effects of SI and repeated SI on antioxidative enzymes, autophagy, and the BDNF-TrkB signaling pathway. These findings highlight the molecular mechanisms that partially explain the anxiolytic effects of DHM.

Mitochondrial microproteins link metabolic cues to respiratory chain biogenesis.

Electron transport chain (ETC) biogenesis is tightly coupled to energy levels and availability of ETC subunits. Complex III (CIII), controlling ubiquinol:ubiquinone ratio in ETC, is an attractive node for modulating ETC levels during metabolic stress. Here, we report the discovery of mammalian Co-ordinator of mitochondrial CYTB (COM) complexes that regulate the stepwise CIII biogenesis in response to nutrient and nuclear-encoded ETC subunit availability. The COMA complex, consisting of UQCC1/2 and membrane anchor C16ORF91, facilitates translation of CIII enzymatic core subunit CYTB. Subsequently, microproteins SMIM4 and BRAWNIN together with COMA subunits form the COMB complex to stabilize nascent CYTB. Finally, UQCC3-containing COMC facilitates CYTB hemylation and association with downstream CIII subunits. Furthermore, when nuclear CIII subunits are limiting, COMB is required to chaperone nascent CYTB to prevent OXPHOS collapse. Our studies highlight CYTB synthesis as a key regulatory node of ETC biogenesis and uncover the roles of microproteins in maintaining mitochondrial homeostasis.

Mitochondrial Ca2+ Uptake Drives Endothelial Injury By Radiation Therapy.

Background:Radiation therapy strongly increases the risk of atherosclerotic vascular disease, such as carotid stenosis. Radiation induces DNA damage, in particular in mitochondria, but the upstream and downstream signaling events are poorly understood. The objective of this study was to define such mechanisms.Methods:Endothelial-specific MCU (mitochondrial Ca2+ uniporter) knockout and C57Bl6/J mice with or without a preinfusion of a mitoTEMPO (mitochondrial reactive oxygen species [ROS] scavenger) were exposed to a single dose of cranial irradiation. 24, and 240 hours postirradiation, vascular reactivity, endothelial function, and mitochondrial integrity were assessed ex vivo and in vitro.Results:In cultured human endothelial cells, irradiation with 4 Gy increased cytosolic Ca2+ transients and the mitochondrial Ca2+ concentration ([Ca2+]mt) and activated MCU. These outcomes correlated with increases in mitochondrial ROS (mtROS), loss of NO production, and sustained damage to mitochondrial but not nuclear DNA. Moreover, irradiation impaired activity of the ETC (electron transport chain) and the transcription of ETC subunits encoded by mitochondrial DNA (mtDNA). Knockdown or pharmacological inhibition of MCU blocked irradiation-induced mtROS production, mtDNA damage, loss of NO production, and impairment of ETC activity. Similarly, the pretreatment with mitoTEMPO, a scavenger of mtROS, reduced irradiation-induced Ca2+ entry, and preserved both the integrity of the mtDNA and the production of NO, suggesting a feed-forward loop involving [Ca2+]m and mtROS. Enhancement of DNA repair in mitochondria, but not in the nucleus, was sufficient to block prolonged mtROS elevations and maintain NO production. Consistent with the findings from cultured cells, in C57BL/6J mice, head and neck irradiation decreased endothelium-dependent vasodilation, and mtDNA integrity in the carotid artery after irradiation. These effects were prevented by endothelial knockout of MCU or infusion with mitoTEMPO.Conclusions:Irradiation-induced damage to mtDNA is driven by MCU-dependent Ca2+ influx and the generation of mtROS. Such damage leads to reduced transcription of mitochondrial genes and activity of the ETC, promoting sustained mtROS production that induces endothelial dysfunction. Our findings suggest that targeting MCU and mtROS might be sufficient to mitigate irradiation-induced vascular disease.

Abstract 5807: MDM2 mitochondrial translocation mediates metabolic reprogramming towards OXPHOS in TKI-resistant oncogene-addicted cancer

Abstract There is mounting evidence of oxidative phosphorylation (OXPHOS) dependency in cancers resistant to tyrosine kinase inhibitors (TKI), but the cause of this metabolic switch remains elusive. Mitochondria-bound MDM2 (mtMDM2), the fraction of MDM2 oncoprotein which is actively imported into the mitochondria, has been found to dysregulate electron transport chain (ETC) complex I function and OXPHOS. AKT is known to be a regulator of MDM2 protein stability. Given that AKT phosphorylation is commonly detected in oncogene-addicted tumours, we investigated the role of mtMDM2 and AKT in promoting TKI resistance through reprogrammed cellular metabolism. EGFR+ NSCLC [HCC827 and HCC827-GR (gefitinib-resistant)] and BRAF+ melanoma [A375 and A375-VR (vemurafenib-resistant)] cells were established via repetitive pulsed strategy. Translocation of MDM2 was examined through subcellular fractionation and Western Blotting. To investigate the role of MDM2 in mediating OXPHOS, genetic knockdown of MDM2 was performed via siRNA followed by measurement of oxygen consumption rate (Seahorse Analyzer); mRNA expression of mtDNA-encoded ETC subunits by real-time PCR (qPCR). ChIP-qPCR analysis was performed to examine the binding affinity of MDM2 and TFAM (mitochondrial transcription factor A) to mitochondrial DNA (mtDNA). Mitochondrial translocation of MDM2 was predominant in TKI-sensitive cells; MDM2 silencing upregulated OXPHOS and induced mRNA expression of mtDNA-encoded ETC complex I subunits, suggesting that mtMDM2 inhibits OXPHOS. ChIP analysis revealed competitive binding of MDM2 and TFAM at the LSP region of mtDNA. Conversely, MDM2 was primarily localized the cytoplasm in TKI-resistant cells and was associated with increased OXPHOS in TKI-resistant cells. Mechanistically, both pAKT and pMDM2 (Ser166/Ser186) were higher in TKI-resistant cells. Collectively, we describe a novel observation that OXPHOS upregulation underpinning TKI resistance in cancer cells could be mediated by AKT upregulation and MDM2 localisation. Citation Format: Jie Qing Eu, Li Ren Kong, Jayshree Hirpara, Boon Cher Goh, Andrea LA Wong, Shazib Pervaiz. MDM2 mitochondrial translocation mediates metabolic reprogramming towards OXPHOS in TKI-resistant oncogene-addicted cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5807.

Molecular hallmarks of heterochronic parabiosis at single-cell resolution.

The ability to slow or reverse biological ageing would have major implications for mitigating disease risk and maintaining vitality1. Although an increasing number of interventions show promise for rejuvenation2, their effectiveness on disparate cell types across the body and the molecular pathways susceptible to rejuvenation remain largely unexplored. Here we performed single-cell RNA sequencing on 20 organs to reveal cell-type-specific responses to young and aged blood in heterochronic parabiosis. Adipose mesenchymal stromal cells, haematopoietic stem cells and hepatocytes are among those cell types that are especially responsive. On the pathway level, young blood invokes new gene sets in addition to reversing established ageing patterns, with the global rescue of genes encoding electron transport chain subunits pinpointing a prominent role of mitochondrial function in parabiosis-mediated rejuvenation. We observed an almost universal loss of gene expression with age that is largely mimicked by parabiosis: aged blood reduces global gene expression, and young blood restores it in select cell types. Together, these data lay the groundwork for a systemic understanding of the interplay between blood-borne factors and cellular integrity.

Abstract 11214: Ibrutinib Decreases Mitochondrial Oxidative Phosphorylation and Metabolic Gene Expression in Atrial-Like Engineered Heart Tissue

Introduction: Ibrutinib is a Bruton kinase inhibitor which treats many hematological malignancies, but also precipitates atrial fibrillation (AF). The precise mechanism(s) remain to be elucidated. Hypothesis: We hypothesized ibrutinib treatment would decrease mitochondrial function and metabolic gene expression in human inducible pluripotent stem cell-derived atrial cardiomyocytes grown as atrial-like engineered heart tissues (aEHTs). Methods: aEHTs were treated with vehicle or 1 μM ibrutinib daily, for 72 hours. Oxidative phosphorylation was assessed (n=5/group) using an Oroboros Oxygraph. Gene expression was evaluated by RNAseq (n=3/group). Results: Oxidative phosphorylation in the presence of complex I and complex II substrates, and maximal oxidative capacity, was decreased in ibrutinib-treated aEHTs compared to vehicle (Panel A). Subunit expression of complex I (NDUFA6, NDUFA9, NDUFS1, NDUFS2, NDUFS4, ND3, ND4, ND5, ND4L), complex II (SDHA, SDHB, SDHD), complex III (UQCR10, UQCRB, UQCRC1, UQCRC2, UQCRFS1), and complex IV (CO2, COX10, COX7A2, CYCS) was decreased. Ingenuity Pathway Analysis of genes differentially expressed identified TCA Cycle and Glycolysis I (Panel B), pathways that provide reducing equivalents to the electron transport chain, as pathways that were decreased by ibrutinib. These changes were accompanied by decreased AMPK Signaling, including key metabolic regulators (PRKAA2, PRKAB2, PRKAG2, SIRT1, and PPARGC1A), and Calcium Signaling. Tumor protein (TP53), nuclear protein 1 (NUPR1), and erb-B2 receptor tyrosine kinase (ERBB2) were identified as top upstream regulators. Conclusions: Ibrutinib treatment decreased oxidative phosphorylation and gene expression of electron transport chain subunits and key modulators of atrial metabolism. Together, these data identify metabolic pathways that are altered by ibrutinib in aEHTs and metabolic regulators that could be targeted for AF therapeutic intervention.

Mitochondrial redox regulation and myocardial ischemia-reperfusion injury.

Mitochondrial reactive oxygen species (ROS) have emerged as an important mechanism of disease and redox signaling in the cellular system. Under basal or pathological conditions, electron leakage for ROS production is primarily mediated by complexes I and III of the electron transport chain (ETC) and by the proton motive force (PMF), consisting of a membrane potential (ΔΨ) and a proton gradient (ΔpH). Several factors control redox status in mitochondria, including ROS, the PMF, oxidative posttranslational modifications (OPTM) of the ETC subunits, SOD2, and cytochrome c heme lyase (HCCS). In the mitochondrial PMF, increased ΔpH-supported backpressure due to diminishing electron transport and chemiosmosis promotes a more reductive mitochondrial physiological setting. OPTM by protein cysteine sulfonation in complex I and complex III has been shown to affect enzymatic catalysis, the proton gradient, redox status, and enzyme-mediated ROS production. Pathological conditions associated with oxidative or nitrosative stress, such as myocardial ischemia and reperfusion (I/R), increase mitochondrial ROS production and redox dysfunction via oxidative injury to complexes I and III, intensely enhancing protein cysteine sulfonation and impairing heme integrity. The physiological conditions of reductive stress induced by gains in SOD2 function normalize I/R-mediated ROS overproduction and redox dysfunction. Further insight into the cellular mechanisms by which HCCS, biogenesis of c-type cytochrome, and OPTM regulate PMF and ROS production in mitochondria will enrich our understanding of redox signal transduction and identify new therapeutic targets for cardiovascular diseases in which oxidative stress perturbs normal redox signaling.

Adaptive optimization of the OXPHOS assembly line partially compensates lrpprc-dependent mitochondrial translation defects in mice

Mouse models of genetic mitochondrial disorders are generally used to understand specific molecular defects and their biochemical consequences, but rarely to map compensatory changes allowing survival. Here we took advantage of the extraordinary mitochondrial resilience of hepatic Lrpprc knockout mice to explore this question using native proteomics profiling and lipidomics. In these mice, low levels of the mtRNA binding protein LRPPRC induce a global mitochondrial translation defect and a severe reduction (>80%) in the assembly and activity of the electron transport chain (ETC) complex IV (CIV). Yet, animals show no signs of overt liver failure and capacity of the ETC is preserved. Beyond stimulation of mitochondrial biogenesis, results show that the abundance of mitoribosomes per unit of mitochondria is increased and proteostatic mechanisms are induced in presence of low LRPPRC levels to preserve a balance in the availability of mitochondrial- vs nuclear-encoded ETC subunits. At the level of individual organelles, a stabilization of residual CIV in supercomplexes (SCs) is observed, pointing to a role of these supramolecular arrangements in preserving ETC function. While the SC assembly factor COX7A2L could not contribute to the stabilization of CIV, important changes in membrane glycerophospholipid (GPL), most notably an increase in SC-stabilizing cardiolipins species (CLs), were observed along with an increased abundance of other supramolecular assemblies known to be stabilized by, and/or participate in CL metabolism. Together these data reveal a complex in vivo network of molecular adjustments involved in preserving mitochondrial integrity in energy consuming organs facing OXPHOS defects, which could be therapeutically exploited.

Berberine elevates cardiolipin in heart of offspring from mouse dams with high fat diet-induced gestational diabetes mellitus

Berberine (BBR) is an isoquinoline alkaloid from plants known to improve cardiac mitochondrial function in gestational diabetes mellitus (GDM) offspring but the mechanism is poorly understood. We examined the role of the mitochondrial phospholipid cardiolipin (CL) in mediating this cardiac improvement. C57BL/6 female mice were fed either a Lean-inducing low-fat diet or a GDM-inducing high-fat diet for 6 weeks prior to breeding. Lean and GDM-exposed male offspring were randomly assigned a low-fat, high-fat, or high-fat diet containing BBR at weaning for 12 weeks. The content of CL was elevated in the heart of GDM offspring fed a high fat diet containing BBR. The increase in total cardiac CL was due to significant increases in the most abundant and functionally important CL species, tetralinoleoyl-CL and this correlated with an increase in the expression of the CL remodeling enzyme tafazzin. Additionally, BBR treatment increased expression of cardiac enzymes involved in fatty acid uptake and oxidation and electron transport chain subunits in high fat diet fed GDM offspring. Thus, dietary BBR protection from cardiac dysfunction in GDM exposed offspring involves improvement in mitochondrial function mediated through increased synthesis of CL.

MOMC-2. Glioblastoma Metabolic Symbiosis: When Lactate Takes The Lead

Glioblastoma (GBM) is a common and devastating brain tumor, associated with a low median survival, despite standard therapeutic management. Among its major features, GBMs are highly angiogenic and exhibit paradoxically an elevated glycolysis. Most of differentiated cells convert glucose into pyruvate that enters into the Krebs cycle to maximize energy production in the presence of oxygen. For cancer cells, glucose uptake and catabolism are increased regardless of oxygen level. However, their energy needs are important – mainly for rapid growth – that it requires a much faster production flow. It is at this step that lactate dehydrogenase (LDH) are involved: LDHA converts efficiently pyruvate into lactate and generates NAD+ to maintain glycolysis. Thus, the lactate formed is exported into the extracellular compartment inducing an unfavourable acidification of the microenvironment. Moreover, LDHB, another LDH isoform, metabolizes lactate into pyruvate for generating energy in mitochondria. Though LDHA has already been studied in many cancers including GBM, the simultaneous role of LDH enzymes have not yet been investigated in GBM development.Hypoxia-driven LDHA expression and lactate production increased cell invasion. Infusing 13C-lactate in starved cells rescued TCA cycle. Then, we showed that, under hypoxia, double sgLDHA/B cell growth and invasion was dramatically decreased in comparison to control cells, mainly caused by an increase in apoptosis. Moreover, double impairment of LDHA and B significantly reduced tumor growth and cell invasion, and induces a massive increase in mouse survival. Tracing experiments with 13C-Glucose coupled with RNA sequencing revealed how metabolism adapts to these contraints, by modifying electron transport chain subunit expressions or by increasing lipid droplet formation.Considered for a long time as a metabolic waste, lactate is shown here to play a critical role in GBM cell symbiosis. This study highlighted GBM adaptability through the LDH isoforms and their involvement in GBM development.