bims-midhyp Biomed News
on Mitochondrial dysfunction and hypoxia
Issue of 2023‒12‒17
fourteen papers selected by
Alia Abukiwan, University of Heidelberg

  1. Respir Res. 2023 Dec 13. 24(1): 310
      BACKGROUND: Hypoxic pulmonary hypertension (HPH) is a common type of pulmonary hypertension and characterized by pulmonary vascular remodeling and constriction. A large number of studies have shown that pulmonary vascular endothelial cells (PVECs) dysfunction plays an important role in the initiation and development stages of HPH, but the mechanism of PVECs dysfunction after hypoxia remains unclear. In this study, we explored the exact mechanism of PVECs dysfunction after hypoxia.METHODS: In vitro, we used primary cultured PVECs hypoxia model to mimic HPH injury. We detected the expressions of mitochondrial biogenesis markers, mitochondrial transcription factor A (TFAM) level inside mitochondria, mitochondrial quantity and function, and the components expressions of translocase of outer mitochondrial membrane (TOM) at 24 h after hypoxia. To explore the effects of Tom70 on mitochondrial biogenesis and functions of PVECs after hypoxia, Tom70 overexpression adenovirus was constructed, and the expressions of mitochondrial biogenesis markers, TFAM level inside mitochondria, mitochondrial quantity and function, and the functions of PVECs were detected. And in vivo, we used cre-dependent overexpression adenovirus of Tom70 in the Cdh5-CreERT2 mouse model of HPH to verify the role of upregulating PVECs Tom70 in improving HPH.
    RESULTS: Hypoxia obviously increased the expressions of mitochondrial biogenesis markers for PGC-1α, NRF-1 and TFAM, but reduced the content of TFAM in mitochondria and the quantity and functions of mitochondria. In addition, only Tom70 expression among the TOM components was significantly decreased after hypoxia, and up-regulation of Tom70 significantly increased the content of TFAM in mitochondria of PVECs by transporting TFAM into mitochondria after hypoxia, enhanced the quantity and functions of mitochondria, improved the functions of PVECs, and ultimately alleviated HPH.
    CONCLUSION: The findings of present study demonstrated that hypoxia induced the decreased expression of Tom70 in PVECs, reduced the mitochondrial biogenesis-associated TFAM protein transporting into mitochondria, inhibited mitochondrial biogenesis, caused PVECs injury, and prompted the formation of HPH. However, up-regulation of Tom70 abolished the hypoxia-induced injurious effects on PVECs and alleviated HPH.
    Keywords:  Hypoxic pulmonary hypertension; Mitochondrial biogenesis; Pulmonary vascular endothelial cells; Translocase of outer mitochondrial membrane
  2. Cancer Metab. 2023 Dec 08. 11(1): 25
      BACKGROUND: A hypoxic environment often persists within solid tumors, including hepatocellular carcinoma (HCC). Hypoxia-inducible factor-1α (HIF-1α) can accelerate cancer malignancy by inducing hypoxia-dependent expression of various genes. Tumor hypoxia can also induce metabolic reprogramming of fatty acid (FA) metabolism, through which HIF-1α plays an essential role in diminishing fatty acid β-oxidation (FAO) in hypoxic cancer cells.METHODS: We aimed to investigate potential new drug therapy options for targeting hypoxic cancer cells within HCC tumors, specifically through combining HIF-1α inhibition with palmitic acid (PA) + L-carnitine (LC) treatment to effectively induce apoptosis in hypoxic HCC cells. To test this hypothesis, in vitro and in vivo studies were performed.
    RESULTS: We first demonstrated that hypoxia-dependent apoptosis was induced by an overload of PA in two HCC cell lines (HepG2 and Hep3B) via excessive production of reactive oxygen species (ROS). Moreover, this observed PA-induced apoptosis was enhanced by HIF-1α knockdown (KD) in these cells under hypoxia. In addition, the combination of PA with FAO activator LC increased FAO activity and led to stronger cell death than PA alone in hypoxic HIF-1α KD cells, specifically through further ROS generation. To clarify the mechanism of hypoxia-induced FA metabolism reprogramming, expression levels of the genes encoding FAO enzymes CPT1A, ACSL1, MCAD, and LCAD, FA transporter CD36, and FA esterification enzymes DGAT and APGAT were analyzed using HIF-1α KD and scramble control (SC) cells. The results suggested that HIF-1α could repress mRNA expression of the FAO-related enzymes and CD36, while it upregulated FA esterification gene expression. This suggested a central role for HIF-1α in hypoxia-induced reprogramming of FA metabolism in HCC cells. Using a nude mouse model, PA administration was found to induce apoptosis from ROS overproduction in HIF-1α KD tumors compared with SC tumors. Additional LC treatment synergistically enhanced the PA-induced apoptosis in HIF-1α KD tumors. Finally, in vivo therapy composed of HIF-1α inhibitor YC-1 with PA + LC could induce ROS-mediated apoptosis in HepG2 tumors without significant toxicity.
    CONCLUSIONS: A combination therapy of YC-1 with PA + LC may be a unique anti-tumor therapy for targeting hypoxic HCC cells, specifically by ROS overproduction leading to forced FAO activation.
    Keywords:  Fatty acid oxidation; HCC; HIF-1α; Hypoxia; L-carnitine; Palmitic acids; ROS; YC-1
  3. Bio Protoc. 2023 Dec 05. 13(23): e4892
      Human mitochondrial DNA (mtDNA) encodes several components of oxidative phosphorylation responsible for the bulk of cellular energy production. The mtDNA is transcribed by a dedicated human mitochondrial RNA polymerase (POLRMT) that is structurally distinct from its nuclear counterparts, instead closely resembling the single-subunit viral RNA polymerases (e.g., T7 RNA polymerase). The initiation of transcription by POLRMT is aided by two initiation factors: transcription factor A, mitochondrial (TFAM), and transcription factor B2, mitochondrial (TFB2M). Although many details of human mitochondrial transcription initiation have been elucidated with in vitro biochemical and structural studies, much remains to be addressed relating to the mechanism and regulation of transcription. Studies of such mechanisms require reliable, high-yield, and high-purity methods for protein production, and this protocol provides the level of detail and troubleshooting tips that are necessary for a novice to generate meaningful amounts of proteins for experimental work. The current protocol describes how to purify recombinant POLRMT, TFAM, and TFB2M from Escherichia coli using techniques such as affinity column chromatography (Ni2+ and heparin), how to remove the solubility tags with TEV protease and recover untagged proteins of interest, and how to overcome commonly encountered challenges in obtaining high yield of each protein. Key features • This protocol builds upon purification methods developed by Patel lab (Ramachandran et al., 2017) and others with greater detail than previously published works. • The protocol requires several days to complete as various steps are designed to be performed overnight. • The recombinantly purified proteins have been successfully used for in vitro transcription experiments, allowing for finer control of experimental components in a minimalistic system.
    Keywords:  Bacterial protein expression; Heparin; Maltose binding protein (MBP) fusion protein purification; Ni-NTA; POLRMT; Protein purification; TEV protease; TFAM; TFB2M
  4. Elife. 2023 Dec 11. pii: RP89232. [Epub ahead of print]12
      Based on studies with a fluorescent reporter dye, Mito Thermo Yellow (MTY), and the genetically encoded gTEMP ratiometric fluorescent temperature indicator targeted to mitochondria, the temperature of active mitochondria in four mammalian and one insect cell line was estimated to be up to 15°C above that of the external environment to which the cells were exposed. High mitochondrial temperature was maintained in the face of a variety of metabolic stresses, including substrate starvation or modification, decreased ATP demand due to inhibition of cytosolic protein synthesis, inhibition of the mitochondrial adenine nucleotide transporter and, if an auxiliary pathway for electron transfer was available via the alternative oxidase, even respiratory poisons acting downstream of oxidative phosphorylation (OXPHOS) complex I. We propose that the high temperature of active mitochondria is an inescapable consequence of the biochemistry of OXPHOS and is homeostatically maintained as a primary feature of mitochondrial metabolism.
    Keywords:  D. melanogaster; OXPHOS; biochemistry; bioenergetics; cell biology; chemical biology; human; mitochondria; mouse; organelle; temperature; thermogenesis
  5. Proc Natl Acad Sci U S A. 2023 Dec 19. 120(51): e2303713120
      The mitochondrial permeability transition pore (mPTP) is a channel in the inner mitochondrial membrane whose sustained opening in response to elevated mitochondrial matrix Ca2+ concentrations triggers necrotic cell death. The molecular identity of mPTP is unknown. One proposed candidate is the mitochondrial ATP synthase, whose canonical function is to generate most ATP in multicellular organisms. Here, we present mitochondrial, cellular, and in vivo evidence that, rather than serving as mPTP, the mitochondrial ATP synthase inhibits this pore. Our studies confirm previous work showing persistence of mPTP in HAP1 cell lines lacking an assembled mitochondrial ATP synthase. Unexpectedly, however, we observe that Ca2+-induced pore opening is markedly sensitized by loss of the mitochondrial ATP synthase. Further, mPTP opening in cells lacking the mitochondrial ATP synthase is desensitized by pharmacological inhibition and genetic depletion of the mitochondrial cis-trans prolyl isomerase cyclophilin D as in wild-type cells, indicating that cyclophilin D can modulate mPTP through substrates other than subunits in the assembled mitochondrial ATP synthase. Mitoplast patch clamping studies showed that mPTP channel conductance was unaffected by loss of the mitochondrial ATP synthase but still blocked by cyclophilin D inhibition. Cardiac mitochondria from mice whose heart muscle cells we engineered deficient in the mitochondrial ATP synthase also demonstrate sensitization of Ca2+-induced mPTP opening and desensitization by cyclophilin D inhibition. Further, these mice exhibit strikingly larger myocardial infarctions when challenged with ischemia/reperfusion in vivo. We conclude that the mitochondrial ATP synthase does not function as mPTP and instead negatively regulates this pore.
    Keywords:  mitochondrial ATP synthase; mitochondrial permeability transition pore; necrosis
  6. Nat Commun. 2023 Dec 12. 14(1): 8248
      The Mitochondrial Complex I Assembly (MCIA) complex is essential for the biogenesis of respiratory Complex I (CI), the first enzyme in the respiratory chain, which has been linked to Alzheimer's disease (AD) pathogenesis. However, how MCIA facilitates CI assembly, and how it is linked with AD pathogenesis, is poorly understood. Here we report the structural basis of the complex formation between the MCIA subunits ECSIT and ACAD9. ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9, releasing the FAD cofactor and converting ACAD9 from a fatty acid β-oxidation (FAO) enzyme to a CI assembly factor. We provide evidence that ECSIT phosphorylation downregulates its association with ACAD9 and is reduced in neuronal cells upon exposure to amyloid-β (Aβ) oligomers. These findings advance our understanding of the MCIA complex assembly and suggest a possible role for ECSIT in the reprogramming of bioenergetic pathways linked to Aβ toxicity, a hallmark of AD.
  7. Elife. 2023 Dec 14. pii: e89297. [Epub ahead of print]12
      Retrograde monosynaptic tracing using glycoprotein-deleted rabies virus is an important component of the toolkit for investigation of neural circuit structure and connectivity. It allows for the identification of first-order presynaptic connections to cell populations of interest across both the central and peripheral nervous system, helping to decipher the complex connectivity patterns of neural networks that give rise to brain function. Despite its utility, the factors that influence the probability of transsynaptic rabies spread are not well understood. While it is well established that expression levels of rabies glycoprotein used to trans-complement G-deleted rabies can result in large changes in numbers of inputs labeled per starter cell (convergence index [CI]), it is not known how typical values of CI relate to the proportions of synaptic contacts or input neurons labeled. And it is not known whether inputs to different cell types, or synaptic contacts that are more proximal or distal to the cell body, are labeled with different probabilities. Here, we use a new rabies virus construct that allows for the simultaneous labeling of pre- and postsynaptic specializations to quantify the proportion of synaptic contacts labeled in mouse primary visual cortex. We demonstrate that with typical conditions about 40% of first-order presynaptic excitatory synapses to cortical excitatory and inhibitory neurons are labeled. We show that using matched tracing conditions there are similar proportions of labeled contacts onto L4 excitatory pyramidal, somatostatin (Sst) inhibitory, and vasoactive intestinal peptide (Vip) starter cell types. Furthermore, we find no difference in the proportions of labeled excitatory contacts onto postsynaptic sites at different subcellular locations.
    Keywords:  cerebral cortex; interneuron; mouse; neural circuit; neuroscience; rabies virus; synapse
  8. Curr Opin Struct Biol. 2023 Dec 09. pii: S0959-440X(23)00214-2. [Epub ahead of print]84 102740
      During protein synthesis, the growing nascent peptide chain moves inside the polypeptide exit tunnel of the ribosome from the peptidyl transferase center towards the exit port where it emerges into the cytoplasm. The ribosome defines the unique energy landscape of the pioneering round of protein folding. The spatial confinement and the interactions of the nascent peptide with the tunnel walls facilitate formation of secondary structures, such as α-helices. The vectorial nature of protein folding inside the tunnel favors local intra- and inter-molecular interactions, thereby inducing cotranslational folding intermediates that do not form upon protein refolding in solution. Tertiary structures start to fold in the lower part of the tunnel, where interactions with the ribosome destabilize native protein folds. The present review summarizes the recent progress in understanding the driving forces of nascent protein folding inside the tunnel and at the surface of the ribosome.
  9. Int J Mol Sci. 2023 Nov 28. pii: 16887. [Epub ahead of print]24(23):
      The tumor microenvironment (TME) is characterized by an acidic pH and low oxygen concentrations. Hypoxia induces neoplastic cell evasion of the immune surveillance, rapid DNA repair, metabolic reprogramming, and metastasis, mainly as a response to the hypoxic inducible factors (HIFs). Likewise, cancer cells increase matrix metalloproteinases' (MMPs) expression in response to TME conditions, allowing them to migrate from the primary tumor to different tissues. Since HIFs and MMPs are augmented in the hypoxic TME, it is easy to consider that HIFs participate directly in their expression regulation. However, not all MMPs have a hypoxia response element (HRE)-HIF binding site. Moreover, different transcription factors and signaling pathways activated in hypoxia conditions through HIFs or in a HIF-independent manner participate in MMPs' transcription. The present review focuses on MMPs' expression in normal and hypoxic conditions, considering HIFs and a HIF-independent transcription control. In addition, since the hypoxic TME causes resistance to anticancer conventional therapy, treatment approaches using MMPs as a target alone, or in combination with other therapies, are also discussed.
    Keywords:  HIFs; cancer treatment; hypoxia; matrix metalloproteinases; nanotechnology; transcription factors; tumor microenvironment
  10. Redox Biol. 2023 Dec 05. pii: S2213-2317(23)00385-3. [Epub ahead of print]69 102984
      BACKGROUND: Acute kidney injury (AKI), often experienced at the intensive care units, is associated with high morbidity/mortality where ischemia-reperfusion injury is a main causative factor. Succinate accumulation during ischemia contributes to the excessive generation of reactive oxygen species at reperfusion. Inhibition of succinate dehydrogenase has been associated with protective outcome in cardiac ischemia-reperfusion after 24h, but the effects on kidney and mitochondrial functions are less well studied.AIM: To investigate the therapeutic potential of succinate dehydrogenase inhibition, by using dimethyl malonate (DMM), on kidney and mitochondria functions in a mouse model of AKI.
    METHODS: Male C57BL/6J mice were pre-treated with DMM or placebo, i.p. 30min prior to bilateral renal ischemia (20min). After 3-days of reperfusion, glomerular filtration rate (GFR) was calculated from plasma clearance of FITC-inulin. Kidney mitochondria was isolated and mass specific and intrinsic mitochondrial function were evaluated by high resolution respirometry. Kidney sections were stained (i.e., hematoxylin-eosin and TUNEL) and analyzed for histopathological evaluation of injuries and apotosis, respectively. NADPH oxidase activity in kidney and human proximal tubular cell-line (HK2) were measured luminometrically.
    RESULTS: DMM treatment improved GFR (p < 0.05) and reduced levels of blood urea nitrogen (p < 0.01) compared to untreated animals, which was associated with lower degree of ischemia-reperfusion-induced tubular injuries (P < 0.001) and apoptosis (P < 0.01). These therapeutic renal effects were linked with improved mitochondrial function, both mass-specific and intrinsic. Finally, DMM treatment prevented ischemia-reperfusion-induced NADPH oxidase activity in the kidney (p < 0.001), which was showed also in HK2 cells exposed to hypoxia and reoxygenation (P < 0.01).
    CONCLUSION: Inhibition of succinate dehydrogenase with DMM, in conjunction with the ischemia-reperfusion phase, significantly improved both renal and mitochondrial functions. These findings may have clinical implications for future therapeutic strategies to prevent development of AKI and associated adverse complications, especially in high risk hospitalized patients.
    Keywords:  Dimethyl malonate; Glomerular filtration rate; Ischemia-reperfusion; Kidney; Reverse electron transfer
  11. Cell Signal. 2024 Jan;pii: S0898-6568(23)00370-4. [Epub ahead of print]113 110955
      Diabetic retinopathy (DR) is a leading cause of blindness, and ferroptosis may be an essential component of the pathological process of DR. In this study, we aimed to screen five hub genes (TLR4, CAV1, HMOX1, TP53, and IL-1B) using bioinformatics analysis and experimentally verify their expression and effects on ferroptosis and cell function. The online Gene Expression Omnibus microarray expression profiling datasets GSE60436 and GSE1025485 were selected for investigation. Ferroptosis-related genes that might be differentially expressed in DR were identified. Then, Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and protein-protein interaction (PPI) network analyses were conducted to characterize the differentially-expressed ferroptosis-related genes. After tissue-specific analyses and external dataset validation of hub genes, the mRNA and protein levels of hub genes in retinal microvascular endothelial cells (HRMECs) symbiotic with high glucose were verified using real-time quantitative PCR (qRT-PCR) and immunocytochemistry (ICC). Finally, hub genes were knocked down using siRNA, and changes in ferroptosis and cell function were observed. Based on the differential expression analysis, 19 ferroptosis-related genes were identified. GO and KEGG enrichment analyses showed that ferroptosis-related genes were significantly enriched in reactive oxygen species metabolic processes, necrotic cell death, hypoxia responses, iron ion responses, positive regulation of cell migration involved in sprouting angiogenesis, NF-kappa B signaling pathway, ferroptosis, fluid shear stress, and atherosclerosis. Subsequently, PPI network analysis and critical module construction were used to identify five hub genes. Based on bioinformatics analysis of mRNA microarrays, qRT-PCR confirmed higher mRNA expression of five genes in the DR model, and immunocytochemistry confirmed their higher protein expression. Finally, siRNA interference was used to verify the effects of five genes on ferroptosis and cell function. Based on bioinformatics analysis, five potential genes related to ferroptosis were identified, and their upregulation may affect the onset or progression of DR. This study sheds new light on the pathogenesis of DR.
    Keywords:  Bioinformatics analysis; Diabetic retinopathy; Ferroptosis; HRMECs; Oxidative stress
  12. Curr Opin Pulm Med. 2023 Dec 14.
      PURPOSE OF REVIEW: Despite the advent of effective and mechanistically diverse treatments for pulmonary arterial hypertension (PAH) and their positive impacts on the functional capacities and outcomes for PAH patients, the much larger population of patients with pulmonary hypertension (PH) in chronic lung diseases like chronic obstructive pulmonary disease (PH-COPD) remain without effective therapies.RECENT FINDINGS: In this review, we will highlight advances in the understanding of PH-COPD pathobiology, the clinical impact comorbid PH has on COPD outcomes, and detail the spectrum of disease and clinical phenotypes that encompass the heterogenous disease manifestations of PH-COPD. Finally, we will examine recent studies exploring the effects of potential treatments for PH-COPD and highlight sub-populations and treatment options that warrant further study.
    SUMMARY: As the PAH population-base ages and comorbid diseases become more frequently diagnosed in PAH patients, the need to clearly delineate subpopulations for clinical applications of PH therapies and research becomes even more urgent. Through an improved understanding of the clinical phenotypes of PH-COPD and the overlap with certain subpopulations of PAH, a framework for future research and potential for therapeutic impact is highlighted.
  13. Am J Transl Res. 2023 ;15(11): 6425-6436
      BACKGROUND: Despite a crucial role of miR-155 in human cancers, its function in heart failure (HF) is still under investigation. This study was designed to explore its association with HF.METHODS: The abdominal transverse aortic constriction (TAC) was adopted for establishment of mouse HF models. qRT-PCR and WB were adopted to detect the changes of miR-155, HIF-1α, Cle-caspase-3, BCL2 and Bax levels in myocardial cells and heart tissues. The changes of cardiac function were checked by ultrasound. Additionally, luciferase reporter gene was adopted for interaction analysis of miR-155 with HIF-1α, and in situ end labelling method was used for detecting myocardial apoptosis.
    RESULTS: MiR-155 in myocardial tissue of HF mice was significantly down regulated. In HF mice injected with agomiR-155, the up-regulation of miR-155 strongly improved cardiac function, and also significantly lowered the protein levels of apoptosis-associated markers, C-caspase-3 and Bax, but up regulated Bcl-2. Additionally, HIF-1α was identified as the direct target of miR-155. As expected, over-expression of HIF-1α greatly reversed the effects of agomiR-155 on cardiac function and the expression of apoptosis-associated markers in heart tissues of HF mice.
    CONCLUSION: MiR-155 overexpression can suppress myocardial cell apoptosis through HIF-1α, and strongly alleviate the cardiac function damage in HF mice, indicating the potential of miR-155/HIF-1α axis to be a target for the diagnosis and therapy of HF.
    Keywords:  Apoptosis; HIF-1α; heart failure; miR-155
  14. Biochem Pharmacol. 2023 Dec 08. pii: S0006-2952(23)00565-8. [Epub ahead of print] 115972
      Photorhabdus luminescens is a gram-negative bioluminescent bacterium known as an intestinal bacterium that coexists in the digestive tract of insect-pathogenic nematodes. As part of our ongoing exploration to identify bioactive compounds from diverse natural resources, the chemical analysis of the cultures of P. luminescens KACC 12254 via LC/MS and TLC-based analyses enabled the isolation and identification of a major fluorescent compound. Its chemical structure was elucidated as 1,8-dihydroxy-3-methoxyanthraquinone (DMA) using HR-ESI-MS and NMR analysis. In this study, we conducted comprehensive investigations utilizing human colorectal cancer HCT116 cells, human umbilical cord vascular endothelial cells (HUVECs), and zebrafish embryos to assess the potential benefits of DMA in suppressing tumor angiogenesis. Our results convincingly demonstrate that DMA effectively suppresses the stability of hypoxia-inducible factor-1α (HIF-1α) protein and its target genes without inducing any cytotoxic effects. Furthermore, DMA demonstrates the ability to inhibit HIF-1α transcriptional activation and mitigate the production of reactive oxygen species (ROS). In our in vitro experiments, DMA exhibits notable inhibitory effects on VEGF-mediated tube formation, migration, and invasion in HUVECs. Additionally, in vivo investigations using zebrafish embryos confirm the antiangiogenic properties of DMA. Notably, DMA does not exhibit any adverse developmental or cardiotoxic effects in the in vivo setting. Moreover, we observe DMA's capability to restrain tumor growth through the downregulation of PI3K/AKT and c-RAF/ERK pathway. Collectively, these compelling findings underscore DMA's potential as a promising therapeutic candidate for targeted intervention against HIF-1α and angiogenesis in cancer treatment.
    Keywords:  Angiogenesis; Anthraquinone; Bioluminescent bacterium; HIF-1α; Photorhabdus luminescens; VEGF