Mitochondrial Metabolic Function Research Articles

Identification of markers correlating with mitochondrial function in myocardial infarction by bioinformatics.

Myocardial infarction (MI), one of the most serious cardiovascular diseases, is also affected by altered mitochondrial metabolism and immune status, but their crosstalk is poorly understood. In this paper, we use bioinformatics to explore key targets associated with mitochondrial metabolic function in MI. The datasets (GSE775, GSE183272 and GSE236374) were from National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) in conjunction with mitochondrial gene data that were downloaded from the MitoCarta 3.0 database. Differentially expressed genes (DEGs) in the dataset were screened by ClusterGVis, Weighted Gene Co-Expression Network Analysis (WGCNA) and GEO2R, and functional enrichment was performed by Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genomes (KEGG). Then mitochondria-associated DEGs (MitoDEGs) were obtained. Protein-protein interaction (PPI) networks were constructed to identify central MitoDEGs that are strongly associated with MI. The Cytoscape and miRWalk databases were then used to predict the transcription factors and target miRNAs of the central MitoDEG, respectively. Finally, the mouse model has been established to demonstrate the expression of MitoDEGs and their association with cardiac function. MitoDEGs in MI were mainly involved in mitochondrial function and adenosine triphosphate (ATP) synthesis pathways. The 10 MI-related hub MitoDEGs were then obtained by eight different algorithms. Immunoassays showed a significant increase in monocyte macrophage and T cell infiltration. According to animal experiments, the expression trends of the four hub MitoDEGs (Aco2, Atp5a1, Ndufs3, and Ndufv1) were verified to be consistent with the bioinformatics results. Our study identified key genes (Aco2, Atp5a1, Ndufs3, and Ndufv1) associated with mitochondrial function in myocardial infarction.

ALDHHigh Breast Cancer Stem Cells Exhibit a Mesenchymal-Senescent Hybrid Phenotype, with Elevated Metabolic and Migratory Activities.

Cancer stem cells (CSCs) account for 0.01 to 2% of the total tumor mass; however, they play a key role in tumor progression, metastasis and resistance to current cancer therapies. The generation and maintenance of CSCs are usually linked to the epithelial-mesenchymal transition (EMT), a dynamic process involved in reprogramming cancer cells towards a more aggressive and motile phenotype with increased stemness potential. Cells that undergo an EMT process have shown to be more resistant to conventional chemo/radiotherapies. In this context, aldehyde dehydrogenase (ALDH) enzymes, known for their role in the cellular detoxification of aldehydes and enhancement of cell survival, are often upregulated in cancer cells, promoting their resistance to conventional cancer treatments. Indeed, high ALDH levels have become a hallmark biomarker of CSCs and are often used to isolate this sub-population from the more abundant cancer cell populations. Herein, we isolated human breast cancer epithelial cells with higher ALDH abundance (ALDHHigh) and compared them to those with low ALDH abundance (ALDHLow). ALDHHigh sub-populations exhibited more characteristic EMT biomarkers by adopting a more mesenchymal phenotype with increased stemness and enhanced migratory potential. Furthermore, ALDHHigh sub-populations displayed elevated senescent markers. Moreover, these cells also demonstrated higher levels of mitochondria DNA/mass, as well as greater mitochondrial and glycolytic metabolic function. Conversely, ALDHLow sub-populations showed a higher efficiency of mammosphere/colony formation and an increased proliferative capacity. Therefore, we demonstrated that these ALDH sub-populations have distinct characteristics, underscoring their role in EMT, the formation of tumors and the mechanisms of metastasis.

Short-chain chlorinated paraffins induce liver injury in mice through mitochondrial disorders and disruption of cholesterol-bile acid pathway

Short-chain chlorinated paraffins (SCCPs) are pervasive organic pollutants recognized for their persistence and bio-toxicity. This study investigated the hepatotoxic mechanisms of SCCPs at environmentally relevant concentration (0.7 μg/kg). The results showed that SCCPs exposure in mice resulted in dysregulated blood and liver lipids, marked by elevated cholesterol levels. Additionally, liver function was compromised, as indicated by increased levels of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase. Histopathological examination of liver tissue post-SCCPs exposure revealed hepatocyte enlargement, vacuolar degeneration, and mild ballooning degeneration. Mechanistically, SCCPs induced mitochondrial abnormalities, evidenced by heightened Hoechst 33258 fluorescence, and augmented reactive oxygen species and malondialdehyde levels in liver tissue. This was accompanied by a reduction in total antioxidant capacity, culminating in elevated apoptosis markers, including cytochrome C and caspase-3. Moreover, SCCPs perturbed hepatocellular energy metabolism, characterized by increased glycolysis, lactic acid, and fatty acid oxidation, alongside a disruption in the tricarboxylic acid cycle and a decline in mitochondrial energy metabolic function. Furthermore, SCCPs exposure downregulated the expression of genes involved in bile acid synthesis (cyp27a1, fxr, and shp), thereby precipitating the cholesterol-bile acid metabolism disorders and cholesterol accumulation. Collectively, these findings underscore that SCCPs, even at environmentally relevant levels, can induce lipid dysregulation, mitochondrial disorders and cholesterol deposition in the hepatocytes, contributing to liver damage. The study's insights contribute to a comprehension of SCCPs-induced hepatotoxicity and may inform potential preventative and treatment targets for hepatic damage associated with SCCPs exposure.

Glutaminase 1 plays critical roles in myelodysplastic syndrome and acute myeloid leukemia cells.

Myelodysplastic syndrome (MDS) features bone marrow failure and a heightened risk of evolving into acute myeloid leukemia (AML), increasing with age and reducing overall survival. Given the unfavorable outcomes of MDS, alternative treatments are necessary. Glutamine, the most abundant amino acid in the blood, is metabolized first by the enzyme glutaminase (GLS). To investigate whether GLS is involved in the progression of MDS. The efficacy of GLS inhibitors (CB839 or IPN60090) and BCL2 inhibitor venetoclax was also examined. We employed GLS inhibitors (CB839, IPN60090) and the BCL2 inhibitor venetoclax, prepared as detailed. MDS and AML cell lines were cultured under standard and modified (hypoxic, glutamine-free) conditions. Viability, proliferation, and caspase activity were assessed with commercial kits. RT-PCR quantified gene expression post-shRNA transfection. Mitochondrial potential, ATP levels, proteasome activity, and metabolic functions were evaluated using specific assays. Statistical analyses (t-tests, ANOVA) validated the findings. The glutamine-free medium inhibited the growth of MDS cells. GLS1 expression was higher in AML cells than in normal control samples (GSE15061), whereas GLS2 expression was not. Treatment of MDS and AML cells for 72 h was inhibited in a dose-dependent manner by GLS inhibitors. Co-treatment with the B-cell lymphoma 2 (BCL2) inhibitor venetoclax and GLS inhibitors increased potency. Cells transfected with GLS1 short hairpin RNA showed suppressed proliferation under hypoxic conditions and increased sensitivity to venetoclax. Targeting glutaminolysis and BCL2 inhibition enhances the therapeutic efficacy and has been proposed as a novel strategy for treating high-risk MDS and AML.

MSC-mediated mitochondrial transfer restores mitochondrial DNA and function in neural progenitor cells of Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is a debilitating mitochondrial disease associated with mutations in mitochondrial DNA (mtDNA). Unfortunately, the available treatment options for LHON patients are limited due to challenges in mitochondrial replacement. In our study, we reprogramming LHON urine cells into induced pluripotent stem cells (iPSCs) and differentiating them into neural progenitor cells (NPCs) and neurons for disease modeling. Our research revealed that LHON neurons exhibited significantly higher levels of mtDNA mutations and reduced mitochondrial function, confirming the disease phenotype. However, through co-culturing LHON iPSC-derived NPCs with mesenchymal stem cells (MSCs), we observed a remarkable rescue of mutant mtDNA and a significant improvement in mitochondrial metabolic function in LHON neurons. These findings suggest that co-culturing with MSCs can enhance mitochondrial function in LHON NPCs, even after their differentiation into neurons. This discovery holds promise as a potential therapeutic strategy for LHON patients.

Preparation of peptide-functionalized affinity materials for the highly specific capture and analysis of mitochondria

Mitochondria perform various metabolic processes that significantly affect cell differentiation, proliferation, signal transduction, and programmed cell death. The disruption of mitochondrial bioenergetic and metabolic functions is closely related to many disorders. The specific isolation and purification of intact, high-purity, and functional mitochondria are central to the understanding of their mechanism of action but remain challenging tasks. In this study, a mitochondrial penetrating peptide (MPP) with the sequence FrFKFrFK(Ac) was used as a mitochondrial recognition motif to construct a peptide-guided affinity separation material. The multiple aromatic phenylalanine (F) residues in this amphiphilic peptide can confer lipophilicity to the mitochondrial membrane, whereas the basic residues (D-arginine and lysine) render the MPP surface positively charged, thereby promoting the binding of negatively charged mitochondria. After the derivatization of the N terminal of MPP with an oligoglycine spacer, the peptide ligands were conjugated to matrix beads (MB) with surface aldehyde functional groups. Peptide functionalization was performed via a condensation reaction between the amino group in the peptide ligand and the aldehyde group on the beads. The generated Schiff bases were reduced, affording stable covalent bonds. The dense and stable functionalization of the beads with the mitochondria-targeting peptides was demonstrated using high performance liquid chromatography (HPLC), zeta potential assay, and scanning electron microscopy (SEM). The immobilization efficiency of the peptide ligands was 1.47 μmol/g, and the surface potential of MB@MPP was 11 mV. MB@MPP was used for the direct isolation of mitochondria after cell homogenization. As observed by SEM, mitochondria with a cross-sectional diameter of 500 nm were efficiently captured on the MB@MPP surface. Because the mitochondrial membrane potential is an important marker of mitochondrial function and the driving force behind the staining of mitochondria with Mito Tracker dyes, the specific binding and separation of fluorescent mitochondria from the cell samples revealed that the proposed MB@MPP-based isolation approach can keep mitochondria intact and retain their functions. Western blot assays were employed to characterize the protein markers of the mitochondria (citrate synthase (CS) and voltage-dependent anion channel protein (VDAC)) and cytoplasmic protein (vinculin), and examine the integrity and purity of the captured mitochondria. The results showed that the lysates released from MB@MPP had high CS and VDAC contents. By contrast, vinculin, which is highly abundant in whole-cell lysates, was barely detected in the lysates from MB@MPP. These results suggest that MB@MPP isolates mitochondria with high affinity, specificity, and antifouling ability by using the targeting peptide as the capture handle. A comparison with a commercial mitochondrial isolation kit demonstrated that MB@MPP can separate mitochondria with higher CS and VDAC abundance and purity. Given the superior separation performance of MB@MPP, the molecular profiles of the isolated mitochondria under stress were subjected to further analysis of their molecular profiles under stress. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established to detect tryptophan (Trp) and riboflavin in the mitochondria. Quantification was performed in multiple-reaction monitoring (MRM) mode. Owing to the high purity of the mitochondria, the Trp and riboflavin contents were determined to be 265 and 0.67 nmol/mg, respectively. The metabolic response of mitochondria to external stimuli was further examined using acadesine, an adenosine 5'-monophosphate (AMP)-activated protein kinase activator with a wide range of metabolic effects, to treat cells. After cell homogenization, MB@MPP was used to separate the mitochondria from the cell samples with and without acadesine treatment, followed by LC-MS/MS analysis. The quantification results demonstrated that acadesine induced a 14% upregulation of Trp content in the mitochondria. By contrast, the riboflavin content decreased to 0.48 nmol/mg, which is 72% of that in untreated mitochondria. The changes in Trp and riboflavin contents could influence their metabolic pathways and, thus, the levels of their metabolites, such as nicotinamide adenine dinucleotide, flavin mononucleotide, and flavin adenine dinucleotide, which are essential coenzymes in mitochondria. Peptide-functionalized affinity microbeads with high affinity and specificity for mitochondria are promising for the efficient isolation of high-quality mitochondria, and offer a useful tool for understanding the complicated functions and dynamics of this unique organelle.

Na/K-ATPase α1/Src Signaling Regulates Mitochondrial Metabolic Function and ROS Production in Human iPSC-derived Cardiomyocytes

Through an isoform specific mechanism, the Na/K-ATPase (NKA) α1 polypeptide-mediated regulation of Src kinase has emerged as a novel regulator of mitochondrial function in various cell types. Mitochondrial metabolism ensures adequate myocardial performance and adaptation to physiological demand. It is also a critical cellular determinant of cardiac repair and remodeling. To investigate the importance of the proposed NKA/Src regulatory signaling on cardiac mitochondrial metabolic function independently from the classic Na/K-ATPase enzymatic function, we used a gene targeting approach in cardiomyocytes. Human induced pluripotent stem cells (hiPSC) expressing a Src-signaling null mutant (A420P) form of NKA α1 were generated using CRISPR/Cas9-mediated genome editing. NKA isoform protein expression and enzymatic activity remained unchanged. However, baseline levels of Src and ERK1/2 phosphorylation were decreased by 55% and 80%, respectively in the A420P hiPSC (n=3, p<0.05). Both the WT and A420P hiPSC differentiated into cardiomyocytes (iCM) as assessed by morphology, spontaneous cell contraction, marker gene expression, and subcellular striations. Cell metabolism assessed by Seahorse extracellular flux analysis revealed significant reductions in basal and maximal rates of mitochondrial respiration (50%), spare respiratory capacity (50%), ATP production (60%), and coupling effciency (20%) (n=4, p<0.05). ROS production was monitored in live cells by measuring the oxidation of the CM-H2DCFDA dye using a fluorescence microscope. The slope of the ROS production rate curve was dramatically reduced in A420P iCM (80%, n=3, p<0.05). Protein carbonylation levels, analyzed by ELISA as markers of oxidative damage, were also decreased in A420P iCM (n=4, p<0.05). Taken together, these data provide genetic evidence for a role of NKA α1/Src in the tonic stimulation of basal mitochondrial metabolism and ROS production in human cardiac myocytes. This has potential significance in cardiometabolic diseases and suggests that targeting the NKA α1/Src signaling axis may offer a novel approach to address mitochondrial dysfunction. NIH Grant R15 HL145666, NIH Grant P20GM103434 to the West Virginia IDeA Network of Biomedical Research Excellence, and American Heart Association Postdoctoral Fellowship — 22POST917776 — to Marco T. Pessoa. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Potential utility of l-carnitine for preventing liver tumors derived from metabolic dysfunction-associated steatohepatitis.

Recent reports have unveiled the potential utility of l-carnitine to alleviate metabolic dysfunction-associated steatohepatitis (MASH) by enhancing mitochondrial metabolic function. However, its efficacy at preventing the development of HCC has not been assessed fully. l-carnitine (2g/d) was administered to 11 patients with MASH for 10 weeks, and blood liver function tests were performed. Five patients received a serial liver biopsy, and liver histology and hepatic gene expression were evaluated using this tissue. An atherogenic plus high-fat diet MASH mouse model received long-term l-carnitine administration, and liver histology and liver tumor development were evaluated. Ten-week l-carnitine administration significantly improved serum alanine transaminase and aspartate transaminase levels along with a histological improvement in the NAFLD activity score, while steatosis and fibrosis were not improved. Gene expression profiling revealed a significant improvement in the inflammation and profibrotic gene signature as well as the recovery of lipid metabolism. Long-term l-carnitine administration to atherogenic plus high-fat diet MASH mice substantially improved liver histology (inflammation, steatosis, and fibrosis) and significantly reduced the incidence of liver tumors. l-carnitine directly reduced the expression of the MASH-associated and stress-induced transcriptional factor early growth response 1. Early growth response 1 activated the promoter activity of neural precursor cell expressed, developmentally downregulated protein 9 (NEDD9), an oncogenic protein. Thus, l-carnitine reduced the activation of the NEDD9, focal adhesion kinase 1, and AKT oncogenic signaling pathway. Short-term l-carnitine administration ameliorated MASH through its anti-inflammatory effects. Long-term l-carnitine administration potentially improved the steatosis and fibrosis of MASH and may eventually reduce the risk of HCC.

Aberrant activated Notch1 promotes prostate enlargement driven by androgen signaling via disrupting mitochondrial function in mouse

The prostate is a vital accessory gonad in the mammalian male reproductive system. With the ever-increasing proportion of the population over 60 years of age worldwide, the incidence of prostate diseases, such as benign prostatic hyperplasia (BPH) and prostate cancer (PCa), is on the rise and is gradually becoming a significant medical problem globally. The notch signaling pathway is essential in regulating prostate early development. However, the potential regulatory mechanism of Notch signaling in prostatic enlargement and hyperplasia remains unclear. In this study, we proved that overactivation of Notch1 signaling in mouse prostatic epithelial cells (OEx) led to prostatic enlargement via enhancing proliferation and inhibiting apoptosis of prostatic epithelial cells. Further study showed that N1ICD/RBPJ directly up-regulated the androgen receptor (AR) and enhanced prostatic sensitivity to androgens. Hyper-proliferation was not found in orchidectomized OEx mice without androgen supply but was observed after Dihydrotestosterone (DHT) supplementation. Our data showed that the number of mitochondrion in prostatic epithelial cells of OEx mice was increased, but the mitochondrial function was impaired, and the essential activity of the mitochondrial respiratory electron transport chain was significantly weakened. Disordered mitochondrial number and metabolic function further resulted in excessive accumulation of reactive oxygen species (ROS). Importantly, anti-oxidant N-Acetyl-L-Cysteine (NAC) therapy could alleviate prostatic hyperplasia caused by the over-activation of Notch1 signaling. Furthermore, we observed the incremental Notch signaling activity in progenitor-like club cells in the scRNA-seq data set of human BPH patients. Moreover, the increased number of TROP2+ progenitors and Club cells was also confirmed in our OEx mice. In conclusion, our study revealed that over-activated Notch1 signaling induces prostatic enlargement by increasing androgen receptor sensitivity, disrupting cellular mitochondrial metabolism, increasing ROS, and a higher number of progenitor cells, all of which can be effectively rescued by NAC treatment.

The AMPK-related kinase NUAK1 controls cortical axons branching by locally modulating mitochondrial metabolic functions

The cellular mechanisms underlying axonal morphogenesis are essential to the formation of functional neuronal networks. We previously identified the autism-linked kinase NUAK1 as a central regulator of axon branching through the control of mitochondria trafficking. However, (1) the relationship between mitochondrial position, function and axon branching and (2) the downstream effectors whereby NUAK1 regulates axon branching remain unknown. Here, we report that mitochondria recruitment to synaptic boutons supports collateral branches stabilization rather than formation in mouse cortical neurons. NUAK1 deficiency significantly impairs mitochondrial metabolism and axonal ATP concentration, and upregulation of mitochondrial function is sufficient to rescue axonal branching in NUAK1 null neurons in vitro and in vivo. Finally, we found that NUAK1 regulates axon branching through the mitochondria-targeted microprotein BRAWNIN. Our results demonstrate that NUAK1 exerts a dual function during axon branching through its ability to control mitochondrial distribution and metabolic activity.

Catalpolaglycone disrupts mitochondrial thermogenesis by specifically binding to a conserved lysine residue of UCP2 on the proton leak tunnel

BackgroundCatalpol (CAT), a naturally occurring iridoid glycoside sourced from the root of Rehmannia glutinosa, affects mitochondrial metabolic functions. However, the mechanism of action of CAT against pyrexia and its plausible targets remain to be fully elucidated. PurposeThis study aimed to identify the specific targets of CAT for blocking mitochondrial thermogenesis and to unveil the unique biological mechanism of action of the orthogonal binding mode between the hemiacetal group and lysine residue on the target protein in vivo. MethodsLipopolysaccharide (LPS)/ carbonyl cyanide 3-chlorophenylhydrazone (CCCP)-induced fever models were established to evaluate the potential antipyretic effects of CAT. An alkenyl-modified CAT probe was designed to identify and capture potential targets. Binding capacity was tested using in-gel imaging and a cellular thermal shift assay. The underlying antipyretic mechanisms were explored using biochemical and molecular biological methods. Catalpolaglycone (CA) was coupled with protein profile identification and molecular docking analysis to evaluate and identify its binding mode to UCP2. ResultsAfter deglycation of CAT in vivo, the hemiacetal group in CA covalently binds to Lys239 of UCP2 in the mitochondria of the liver via an ɛ-amine nucleophilic addition. This irreversible binding affects proton leakage and improves mitochondrial membrane potential and ADP/ATP transformation efficiency, leading to an antipyretic effect. ConclusionOur findings highlight the potential role of CA in modulating UCP2 activity or function within the mitochondria and open new avenues for investigating the therapeutic effects of CA on mitochondrial homeostasis.

PROKR1–CREB–NR4A2 axis for oxidative muscle fiber specification and improvement of metabolic function

Metabolic disorders are characterized by an imbalance in muscle fiber composition, and a potential therapeutic approach involves increasing the proportion of oxidative muscle fibers. Prokineticin receptor 1 (PROKR1) is a G protein-coupled receptor that plays a role in various metabolic functions, but its specific involvement in oxidative fiber specification is not fully understood. Here, we investigated the functions of PROKR1 in muscle development to address metabolic disorders and muscular diseases. A meta-analysis revealed that the activation of PROKR1 upregulated exercise-responsive genes, particularly nuclear receptor subfamily 4 group A member 2 (NR4A2). Further investigations using ChIP-PCR, luciferase assays, and pharmacological interventions demonstrated that PROKR1 signaling enhanced NR4A2 expression by Gs-mediated phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) in both mouse and human myotubes. Genetic and pharmacological interventions showed that the PROKR1-NR4A2 axis promotes the specification of oxidative muscle fibers in both myocytes by promoting mitochondrial biogenesis and metabolic function. Prokr1-deficient mice displayed unfavorable metabolic phenotypes, such as lower lean mass, enlarged muscle fibers, impaired glucose, and insulin tolerance. These mice also exhibited reduced energy expenditure and exercise performance. The deletion of Prokr1 resulted in decreased oxidative muscle fiber composition and reduced activity in the Prokr1-CREB-Nr4a2 pathway, which were restored by AAV-mediated Prokr1 rescue. In summary, our findings highlight the activation of the PROKR1-CREB-NR4A2 axis as a mechanism for increasing the oxidative muscle fiber composition, which positively impacts overall metabolic function. This study lays an important scientific foundation for the development of effective muscular-metabolic therapeutics with unique mechanisms of action.

Physicochemical Features and Applicability of Newly Fabricated Phytopharmaceutical-Loaded Hydrogel Alginate Microcarriers with Viscoelastic Polyelectrolyte Coatings.

The design of novel polymeric carrier systems with functional coatings is of great interest for delivering various bioactive molecules. Microcapsules coated with polyelectrolyte (PE) films provide additional functionality and fine-tuning advantages essential for controlled drug release. We developed hydrogel microcarriers coated with functional PE films with encapsulated substances of natural origin, resveratrol (RES), curcumin (CUR), and epigallocatechin gallate (EGCG), which have cytotoxic and chemopreventive properties. Alginate (ALG) based microparticles were loaded with phytopharmaceuticals using the emulsification method, and then their surface was modified with PE coatings, such as chitosan (CHIT) or poly(allylamine hydrochloride) (PAH). The morphology and mean diameter of microcarriers were characterised by scanning electron microscopy, encapsulation efficiency was determined by UV-Vis spectroscopy, whereas the physicochemical properties of functional PE layers were studied using quartz crystal microbalance with dissipation monitoring and streaming potential measurements. The release profiles of active compounds from the hydrogel microparticles were described using the Peppas-Sahlin model. The cytotoxic effect of designed delivery systems was studied by evaluating their impact on the proliferation, mitochondrial metabolic function, and lipid peroxidation level of 5637 human bladder cancer cells. The present work demonstrates that the physicochemical and biological features of fabricated microcarriers can be controlled by the type of encapsulated anti-cancer agent and PE coating.

BNIP3 in hypoxia-induced mitophagy: Novel insights and promising target for non-alcoholic fatty liver disease

BNIP3 localizes to the outer mitochondrial membrane, has been demonstrated to be extensively involved in abnormalities to mitochondrial metabolic function and dynamicsand in non-alcoholic fatty liver disease (NAFLD). However, its role in NAFLD under hypoxia remains unclear. This study aimed to investigate the expression and the role of BNIP3 in NAFLD under hypoxia, and explore its involvement in regulating NAFLD mitophagy, fatty acid β-oxidation both in vivo and in vitro. BNIP3-mediated mitophagy level was analyzed using real-time quantitative polymerase chain reaction, Western blotting, immunofluorescence and electron microscopy. The role of BNIP3 in fatty acid β-oxidation was evaluated using lipid droplet staining, triglyceride content determination, and cellular energy metabolism. The results showed that compared with the HFD-2200 m, the body weight, inflammatory liver injury, and lipid deposition were significantly reduced in the HFD-4500 m group (P < 0.05), but autophagy and mitophagy were increased, and the expression of the mitophagy receptor BNIP3 was increased (P < 0.05). Compared to the control group, BNIP3 knockdown in the hypoxia group resulted in decreased levels of CPT1, ATGL, and p-HSL in lipid-accumulating hepatocytes, lipid droplet accumulation and triglyceride content increased (P < 0.05). Moreover, the ability of lipid-accumulating hepatocytes to oxidize fatty acids was reduced by BNIP3 knockdown in the hypoxia group (P < 0.05). Therefore, it can be concluded that, in NAFLD mice under hypoxia, BNIP3-mediated mitophagy promotes fatty acid β-oxidation. This study elucidated the role of BNIP3 in promoting fatty acid β-oxidation in NAFLD under hypoxia, and suggests BNIP3 may serve as a novel potential therapeutic target for NAFLD.

Mitochondrial inhibitors: a new horizon in breast cancer therapy.

Breast cancer, due to resistance to standard therapies such as endocrine therapy, anti-HER2 therapy and chemotherapy, continues to pose a major health challenge. A growing body of research emphasizes the heterogeneity and plasticity of metabolism in breast cancer. Because differences in subtypes exhibit a bias toward metabolic pathways, targeting mitochondrial inhibitors shows great potential as stand-alone or adjuvant cancer therapies. Multiple therapeutic candidates are currently in various stages of preclinical studies and clinical openings. However, specific inhibitors have been shown to face multiple challenges (e.g., single metabolic therapies, mitochondrial structure and enzymes, etc.), and combining with standard therapies or targeting multiple metabolic pathways may be necessary. In this paper, we review the critical role of mitochondrial metabolic functions, including oxidative phosphorylation (OXPHOS), the tricarboxylic acid cycle, and fatty acid and amino acid metabolism, in metabolic reprogramming of breast cancer cells. In addition, we outline the impact of mitochondrial dysfunction on metabolic pathways in different subtypes of breast cancer and mitochondrial inhibitors targeting different metabolic pathways, aiming to provide additional ideas for the development of mitochondrial inhibitors and to improve the efficacy of existing therapies for breast cancer.

Human Umbilical Cord Mesenchymal Stromal Cell-Derived Exosomes Alleviate Hypoxia-Induced Pulmonary Arterial Hypertension in Mice Via Macrophages.

Pulmonary hypertension (PH) is an intractable, severe, and progressive cardiopulmonary disease. Recent findings suggest that human umbilical cord mesenchymal stromal cells (HUCMSCs) and HUCMSC-derived exosomes (HUCMSC-Exos) possess potential therapeutic value for PH. However, whether they have beneficial effects on hypoxic pulmonary hypertension (HPH) is unclear. Exos are released into the extracellular environment by the fusion of intracellular multivesicular bodies with the cell membrane, and they play an important role in cellular communication. Exos ameliorate immune inflammation levels, alter macrophage phenotypes, regulate mitochondrial metabolic function, and inhibit pulmonary vascular remodeling, thereby improving PH. Macrophages are important sources of cytokines and other transmitters and can promote the release of cytokines, vasoactive molecules, and reactive oxygen species, all of which are associated with pulmonary vascular remodeling. Therefore, the aim of this study was to investigate whether HUCMSC-Exos could improve the lung inflammatory microenvironment and inhibit pulmonary vascular remodeling by targeting macrophages and identifying the underlying mechanisms. The results showed that HUCMSC-Exos promoted M2 macrophage polarization, decreased pro-inflammatory factors, increased IL-10 levels, and inhibited IL-33/ST2 axis expression, thereby inhibiting hypoxia-induced proliferation of pulmonary artery smooth muscle cells and ameliorating HPH.

The Na/K-ATPase α1/Src Signaling Axis Regulates Mitochondrial Metabolic Function and Redox Signaling in Human iPSC-Derived Cardiomyocytes.

Na/K-ATPase (NKA)-mediated regulation of Src kinase, which involves defined amino acid sequences of the NKA α1 polypeptide, has emerged as a novel regulatory mechanism of mitochondrial function in metazoans. Mitochondrial metabolism ensures adequate myocardial performance and adaptation to physiological demand. It is also a critical cellular determinant of cardiac repair and remodeling. To assess the impact of the proposed NKA/Src regulatory axis on cardiac mitochondrial metabolic function, we used a gene targeting approach in human cardiac myocytes. Human induced pluripotent stem cells (hiPSC) expressing an Src-signaling null mutant (A420P) form of the NKA α1 polypeptide were generated using CRISPR/Cas9-mediated genome editing. Total cellular Na/K-ATPase activity remained unchanged in A420P compared to the wild type (WT) hiPSC, but baseline phosphorylation levels of Src and ERK1/2 were drastically reduced. Both WT and A420P mutant hiPSC readily differentiated into cardiac myocytes (iCM), as evidenced by marker gene expression, spontaneous cell contraction, and subcellular striations. Total NKA α1-3 protein expression was comparable in WT and A420P iCM. However, live cell metabolism assessed functionally by Seahorse extracellular flux analysis revealed significant reductions in both basal and maximal rates of mitochondrial respiration, spare respiratory capacity, ATP production, and coupling efficiency. A significant reduction in ROS production was detected by fluorescence imaging in live cells, and confirmed by decreased cellular protein carbonylation levels in A420P iCM. Taken together, these data provide genetic evidence for a role of NKA α1/Src in the tonic stimulation of basal mitochondrial metabolism and ROS production in human cardiac myocytes. This signaling axis in cardiac myocytes may provide a new approach to counteract mitochondrial dysfunction in cardiometabolic diseases.

Mitochondrial Mechanisms of Endothelial Cell Damage in Alzheimer’s Disease and Cerebral Amyloid Angiopathy

AbstractBackgroundCerebrovascular endothelial cells (EC) are crucial regulators of cerebral homeostasis and function through the maintenance of the blood‐brain barrier (BBB). ECs damage and/or dysfunction may result in defects in brain clearance and perfusion, microhemorrhages, inflammation, and neurodegeneration. Damage to the ECs may result from cardiovascular risk factors (CVRF) such as high levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy). In Alzheimer’s Disease (AD) and Cerebral Amyloid Angiopathy (CAA), the presence of a CVRF may further exacerbate cerebrovascular function and neurodegeneration. EC do not rely highly on mitochondrial ATP production as they are glycolytic cells. Instead, EC mitochondria serve mostly as signaling organelles, which sense cellular damage and mediate downstream pathways such as inflammation. In this study, we aim to understand the mechanistic nature of endothelial mitochondrial dysfunction and its elicited pathways in Aβ‐ and mixed (Aβ+HHcy)‐mediated vascular pathologies. Understanding the mechanisms by which the mitochondria mediate EC inflammation, may pave the way to new roads for therapeutic advancements in the field.MethodHuman brain microvascular ECs were challenged with Aβ, Hcy, or the combination. Mitochondrial metabolic function, inflammatory cascades and signals, and EC barrier function were assessed.ResultThe presence of Aβ, but not Hcy, induced deficits in mitochondrial respiration and ATP production. In addition, there is evidence of mitochondrial fragmentation and the expression of fission proteins. Preliminary results further suggest a release of mitochondrial danger associated molecular patterns (mtDAMPs) that can promote an inflammatory cascade. The mitochondrial damage and subsequent inflammatory response are associated with an increase in EC barrier permeability and cytokine release, which is exacerbated in the presence of Hcy.ConclusionThe EC dysfunction associated with Aβ is accompanied by severe deficits in mitochondrial metabolism and the release of mtDAMPS. The presence of Hcy exacerbates some of the detrimental effects of Aβ, particularly on mtDAMPS and BBB function, but Hcy does not appear to affect mitochondrial metabolic changes. Overall, our results reveal a novel role for mtDAMPs in vascular dysfunction in the presence of Aβ and/or Hcy.

A new role for phosphoinositides in regulating mitochondrial dynamics

Phosphoinositides are a minor group of membrane-associated phospholipids that are transiently generated on the cytoplasmic leaflet of many organelle membranes and the plasma membrane. There are seven functionally distinct phosphoinositides, each derived via the reversible phosphorylation of phosphatidylinositol in various combinations on the inositol ring. Their generation and termination is tightly regulated by phosphatidylinositol-kinases and –phosphatases. These enzymes can function together in an integrated and coordinated manner, whereby the phosphoinositide product of one enzyme may subsequently serve as a substrate for another to generate a different phosphoinositide species. This regulatory mechanism not only enables the transient generation of phosphoinositides on membranes, but also more complex sequential or bidirectional conversion pathways, and phosphoinositides can also be transferred between organelles via membrane contacts. It is this capacity to fine-tune phosphoinositide signals that makes them ideal regulators of membrane organization and dynamics, through their recruitment of signalling, membrane altering and lipid transfer proteins. Research spanning several decades has provided extensive evidence that phosphoinositides are major gatekeepers of membrane organization, with roles in endocytosis, exocytosis, autophagy, lysosome dynamics, vesicular transport and secretion, cilia, inter-organelle membrane contact, endosome maturation and nuclear function. By contrast, there has been remarkably little known about the role of phosphoinositides at mitochondria – an enigmatic and major knowledge gap, with challenges in reliably detecting phosphoinositides at this site.Here we review recent significant breakthroughs in understanding the role of phosphoinositides in regulating mitochondrial dynamics and metabolic function.

Lncrna Rmrp Regulate Mitochondrial Function in Relapsed and Refractory Acute Myeloid Leukemia

Background: The prognosis of relapsed and refractory acute myeloid leukemia (R/R AML) is extremely poor, primarily due to the development of chemotherapy resistance during the treatment, which leads to its relapse and refractory disease. Extensive studies have established that various dysfunctions in the mitochondria of AML cells play a significant role in promoting chemotherapy resistance and the recurrence of the disease. In our previous investigation, we successfully demonstrated the substantial enrichment of the long non-coding RNA (lncRNA) RMRP in the mitochondria. However, the specific function of RMRP in the mitochondria of R/R AML remains unexplored. The objective of this project is to uncover the function of RMRP in R/R AML mitochondria, aiming to provide innovative insights and a theoretical foundation for surmounting chemotherapy resistance in R/R AML. Methods: In order to investigate the factors that play a crucial role in R/R AML, we employed mass spectrometry (MS) to conduct a screening of factors associated with AML chemoresistance. We utilized quantitative real-time polymerase chain reaction (RT-qPCR) to assess the expression of lncRNA RMRP in both newly diagnosed AML (ND AML) and R/R AML patients. Furthermore, we knocked down the RMRP expression level by transfection with siRNA-NC and siRNA-RMRP. Additionally, we analyzed the correlation between lncRNA RMRP and mitochondria. To explore the involvement of lncRNA RMRP in regulating mitochondrial function in R/R AML, we performed mitochondrial oxidative phosphorylation metabolic function assays subsequent to the inhibition of lncRNA RMRP. Moreover, Western blot analysis was employed to evaluate the relative protein expression levels in AML cell lines. Results: We enrolled 23 AML patients (ND AML, n=12; R/R AML, n=11), and the result of MS shows that R/R AML group expressed higher level of mitochondria related protein. The qPCR results showed that R/R AML had higher expression level of RMRP ( P&amp;lt;0.0001) and mitochondrial genome genes ( P&amp;lt;0.0001) than ND AML patients. The result of co-cultured of BMSCs and AML suggests that mitochondrial transmission of BMSCs to AML existed in AML patients with high RMRP expression level( P&amp;lt;0.0001). The Seahorse metabolomics results suggest that RMRP knock down attenuated Spare Respiratory Capacity ( P=0.01) and Maximal Respiration ( P=0.01), increased the Cytarabine and Doxorubicin sensitivity, and induced alterations in the mitochondrial CpG1 and CpG9 methylation. Knockdown of RMRP is also accompanied by a decrease in mtDNA copy number ( P=0.10, 0.42, and 0.0074, respectively for mtDNA gene MT-ND2, MT-ND3, and MT-CO1), and mitochondrial genome transcription. Under transmission electron microscope (TEM), mitochondria were engulfed by lysosomes in RMRP knockdown AML cells. And results of Western blot showed that the autophagy markers such as LC3B, BNIP3L/Nix, NDP52 was upregulated, and SQSTM1, Parkin was downregulated in RMRP knockdown AML cells, suggesting that RMRP may induce mitophagy through the BNIP3L/Nix pathway, PINK1/Parkin pathway, and NDP52 pathway. Conclusion: In R/R AML, RMRP increased chemotherapy resistance and RMRP high expression was associated with multiple mitochondrial function abnormalities, including altered mitochondrial metabolism, reduced mitophagy, and altered mtDNA epigenetics.