Mitochondrial DNA Levels Research Articles

Deficiency of myeloid NPC1 exacerbates liver injury and fibrosis by impairing macrophage efferocytosis.

Niemann-Pick C1 (NPC1), a lysosomal cholesterol transport protein, is required for efficient efferocytosis. Patients with Npc1 mutation are frequently accompanied with hepatic symptoms, including hepatomegaly, elevated liver transaminases, or even acute liver failure, but the pathogenic mechanism remains unknown. Our work aims to characterize the functional role of myeloid NPC1 in liver injury and elucidate its underlying mechanism. Analyses of injured livers from patients with liver diseases and mouse models were conducted to examine NPC1 expression. Myeloid cell-specific Npc1 knockout mice were constructed to determine the functional role of macrophage NPC1 in liver injury. Isolated macrophages were subjected to in vitro mechanistical assays. We found that NPC1 is mainly expressed in hepatic macrophages. Its mRNA and protein expression are significantly elevated in injured livers from both patients and mouse models. Tissue-specific deletion of myeloid Npc1 increased liver inflammation, levels of serum liver function enzymes, and liver fibrosis in mouse models of liver injury induced by carbon tetrachloride (CCl4) injection and methionine-and-choline-deficient (MCD) diets. Further analyses indicate that Npc1 deficiency in mouse models of liver injury resulted in increased levels of serum HMGB1 and mitochondrial DNA, promoted hepatic macrophage proinflammatory activation, M1 polarization, led to overproduction of hepatic inflammatory cytokines/chemokines, e.g. CCL2 and STING/NFκB pathway activation. In vitro mechanistical studies reveal that Npc1-deficient macrophages exhibited inefficient efferocytosis, partly due to impaired cargo degradation. These findings indicate that elevated expression of myeloid NPC1 in liver diseases protects liver from injury by promoting macrophage efferocytosis of damaged cells. Dysfunction of NPC1 aggravates liver injury, suggesting that NPC1 may be a potential therapeutic target for treating liver diseases.

Mitochondrial DNA in plasma and long-term physical recovery of critically ill patients: an observational study

BackgroundPost-intensive care syndrome (PICS) poses a notable public health concern, with survivors of critical illness experiencing long-term physical, psychological, and cognitive challenges. Mitochondrial dysfunction has gained attention for its potential involvement in PICS. However, the long-term impact of mitochondrial status on patient recovery remains uncertain. A single-centre retrospective analysis was conducted in Leeuwarden, the Netherlands, between May and November 2019, within a mixed ICU survivor cohort. Patients were assessed for mitochondrial markers (mtDNA damage represented by the presence of mtDNA fragmentation and mitochondrial DNA levels evaluated by the ratio of mtDNA and nuclear DNA), clinical factors, and long-term outcomes measured by the physical functioning (PF) domain of health-related quality of life.ResultsA total of 43 patients were included in this study divided into recovery and non-recovery groups based on age-adjusted PF scores at 12 months post-ICU. Nineteen patients scored below these thresholds. No significant differences in mitochondrial markers between groups were identified. Furthermore, no significant correlations were found between mtDNA levels and mtDNA damage at baseline and 12 months with PF scores. However, mtDNA levels decreased over time in the recovery (p-value < < 0.01) and non-recovery groups (p-value < 0.01).ConclusionNo significant correlation was found between mitochondrial markers and physical functioning scores. This study underscores the multifactorial nature of PICS and the need for a comprehensive understanding of its metabolic and cellular components. While mitochondrial markers may play a role in PICS, they operate within a framework influenced by various factors. This exploratory study serves as a foundation for future investigations aimed at developing targeted interventions to enhance the quality of life for ICU survivors grappling with PICS.

Evaluation of β-Actin and Mitochondrial DNA Levels in Determining the Age of Suidae Remains.

African Swine Fever (ASF) is an infectious disease affecting pigs and wild boars, causing significant economic losses. Epidemiological surveillance plays an important role in minimizing its impact. The aim of this study was to evaluate the usefulness of β-actin DNA and mitochondrial DNA (mtDNA) levels in determining the age of animal remains from the Suidae family, which could be helpful in epidemiological investigations. The study included selected tissues and internal organs of Sus scrofa domesticus, exposed to natural environmental conditions or kept in stable 4 °C conditions, to assess the levels of β-actin and mtDNA over a period of 18 months. The levels of both tested parameters exhibited the expected decreasing trend over time. However, in most tissues, some discrepancies from this general pattern were observed. The results obtained for bone marrow showed a consistent downward trend and a strong correlation between mtDNA and β-actin, with long-term detectability (up to the 13th month of the study). Therefore, bone marrow could be considered a matrix of choice for age assessment. However, due to various influencing factors, further studies are required.

HbA1c and leukocyte mtDNA levels as major factors associated with post-COVID-19 syndrome in type 2 diabetes patients

Post-COVID-19 syndrome (PCS) is an emerging health problem in people recovering from COVID-19 infection within the past 3–6 months. The current study aimed to define the predictive factors of PCS development by assessing the mitochondrial DNA (mtDNA) levels in blood leukocytes, inflammatory markers and HbA1c in type 2 diabetes patients (T2D) with regard to clinical phenotype, gender, and biological age. In this case-control study, 65 T2D patients were selected. Patients were divided into 2 groups depending on PCS presence: the PCS group (n = 44) and patients who did not develop PCS (n = 21) for up to 6 months after COVID-19 infection. HbA1c and mtDNA levels were the primary factors linked to PCS in different models. We observed significantly lower mtDNA content in T2D patients with PCS compared to those without PCS (1.26 ± 0.25 vs. 1.44 ± 0.24; p = 0.011). In gender-specific and age-related analyses, the mt-DNA amount did not differ significantly between the subgroups. According to the stepwise multivariate logistic regression analysis, low mtDNA content and HbA1c were independent variables associated with PCS development, regardless of oxygen, glucocorticoid therapy and COVID-19 severity. The top-performing model for PCS prediction was the gradient boosting machine (GBM). HbA1c and mtDNA had a notably greater influence than the other variables, indicating their potential as prognostic biomarkers.

High mitochondrial DNA content is a key determinant of stemness, proliferation, cell migration, and cancer metastasis in vivo

Here, we examined the potential role of mitochondrial DNA (mtDNA) levels in conveying aggressive phenotypes in cancer cells, using two widely-used breast cell lines as model systems (MCF7[ER+] and MDA-MB-231[ER-]). These human breast cancer cell lines were fractionated into mtDNA-high and mtDNA-low cell sub-populations by flow cytometry, using SYBR Gold as a vital probe to stain mitochondrial nucleoids in living cells. Enrichment of mtDNA-high and mtDNA-low cell sub-populations was independently validated, using a specific DNA-binding mAb probe (AC-30-10), and mitochondrial-based functional assays. As predicted, mtDNA-high MCF7 cells showed significant increases in mitochondrial mass, membrane potential, and superoxide production, as well as increased mitochondrial respiration and ATP production. Moreover, mtDNA-high MCF7 cells demonstrated increases in stemness features, such as anchorage-independent growth and CD44 levels, as well as drug-resistance to Gemcitabine and Tamoxifen. Proliferation rates were also significantly increased, with a dramatic shift towards the S- and G2/M-phases of the cell cycle; this was indeed confirmed by RNA-Seq analysis. Complementary results were obtained with MDA-MB-231 cells. More specifically, mtDNA-high MDA-MB-231 cells showed increases in stemness features and ATP production, as well as rapid cell cycle progression. Moreover, mtDNA-high MDA-MB-231 cells also exhibited increases in both cell migration and invasion, suggesting a role for mtDNA in distant metastasis. To test this hypothesis more directly, a preclinical in vivo model was utilized. For this purpose, MDA-MB-231 tumour cell grafts were treated with an established mtDNA synthesis inhibitor, namely Alovudine (3’-deoxy-3’-fluorothymidine). As expected, drug-induced depletion of mtDNA led to a shift from mitochondrial to glycolytic metabolism. Interestingly, Alovudine very effectively reduced the formation of spontaneous metastases by nearly 70%, but minimally inhibited tumour growth by approximately 20%. Taken together, these data suggest that high mtDNA content is a key driver of stemness, proliferation, and migration, as well as cancer cell metastasis.

The DNA sensor AIM2 mediates psoriasiform inflammation by inducing type 3 immunity.

Psoriasis is a chronic and recurrent inflammatory skin disease characterized by abnormal proliferation and differentiation of keratinocytes and activation of immune cells. However, the molecular driver that triggers this immune response in psoriatic skin remains unclear. The inflammation-related gene absent in melanoma 2 (AIM2) was identified as a susceptibility gene/locus associated with psoriasis. In this study, we investigated the role of AIM2 in the pathophysiology of psoriasis. We found elevated levels of mitochondrial DNA in patients with psoriasis, along with high expression of AIM2 in both the human psoriatic epidermis and a mouse model of psoriasis induced by topical imiquimod (IMQ) application. Genetic ablation of AIM2 reduced the development of IMQ-induced psoriasis by decreasing the production of type 3 cytokines (such as IL-17A and IL-23) and infiltration of immune cells into the inflammatory site. Furthermore, we demonstrate that IL-17A induced AIM2 expression in keratinocytes. Finally, the genetic absence of inflammasome components downstream AIM2, ASC, and caspase-1 alleviated IMQ-induced skin inflammation. Collectively, our data show that AIM2 is involved in developing psoriasis through its canonical activation.

Decreased plasma cell-free mitochondrial DNA may be a new biomarker of tachycardia-induced cardiomyopathy in patients with atrial fibrillation

ObjectivesTo determine cell-free mitochondrial DNA (mt-cfDNA) levels in tachycardia-induced cardiomyopathy (TIC) and non-TIC among atrial fibrillation (AF) cases. BackgroundsTIC is a reversible cardiomyopathy resulting from tachyarrhythmias, such as AF. The exact cause of TIC is not fully understood, but mitochondrial dysfunction has been reported in a variety of cardiomyopathies and may be involved in TIC as well. AF is recognized to be associated with systemic inflammation, and studies have shown that in patients with AF have elevated levels of mt-cfDNA increased, and this increase is linked to systemic inflammation. MethodsWe enrolled 67 patients with TIC (TIC group) and 671 patients without TIC (non-TIC group), who underwent catheter ablation for AF at our hospital between November 2009 and September 2016 and did not meet the exclusion criteria. We performed quantitative PCR analysis of plasma mt-cfDNA and nuclear-cfDNA and compared clinical factors and these measurements between the two groups. ResultsLevels of mt-cfDNA were significantly lower in the TIC group than in the non-TIC group (1110.01 vs. 1918.71 copies/μg plasma, P = 0.027), while levels of nuclear-cfDNA were comparable between these two groups. In particular, mt-cfDNA (P = 0.0003, odds ratio [OR] 2.54), non-paroxysmal AF (P < 0.0001, OR 3.07), and diabetes mellitus (P = 0.006, OR 2.36) were identified as independent factors associated with TIC. ConclusionThere are lower mt-cfDNA in TIC, and decreased plasma levels of circulating mt-cfDNA may be a new biomarker and involve in related mechanisms for AF associated TIC. Condensed abstractTachycardia-induced cardiomyopathy (TIC) is a reversible cardiomyopathy caused by tachyarrhythmias, such as atrial fibrillation (AF) tachycardia. The pathogenesis of TIC remains incompletely understood, and there is currently no method to predict its development in patients. In this study, we show that cell-free mitochondrial DNA (mt-cfDNA) levels were significantly lower in the TIC group than in the non-TIC group. Persistent AF, coexisting diabetes mellitus, and decreased mt-cfDNA levels were independently associated with TIC. Decreased mt-cfDNA levels may serve as a novel biomarker for predicting TIC in patients with AF.

Forkhead box O-1 regulates the biological behavior of BMP-2-induced human bone mesenchymal stem cells through mitochondrial dynamics and autophagy.

This study explored the mechanism by which forkhead box O-1 (FoxO1) modulates the biological behaviors of bone mesenchymal stem cell (BMSC). Human BMSCs were cultured for 7 days in DMEM containing bone morphogenetic protein-2 (BMP-2) and treated with a short hairpin-FoxO1 plasmid. The study assessed cell proliferation, migration, apoptosis, adenosine triphosphate (ATP) levels, mitochondrial DNA levels, membrane potential, autophagy, and the levels of FoxO1, apoptosis-associated proteins, osteogenic differentiation-associated proteins, mitochondrial fusion and fission proteins and mitochondrial autophagy-related proteins. The cells were also treated with the mitochondrial fusion activator MASM7 and the mitochondrial autophagy activator carbonyl cyanide 3-chlorophenylhydrazone. The study evaluated whether mitochondrial dynamics and autophagy activation could rescue the FoxO1 knockdown-induced changes in BMSC biological behaviors, mitochondrial dynamics, and mitochondrial autophagy. BMP-2-induced BMSCs exhibited up-regulated FoxO1 expression, enhanced proliferation and migration, and induced osteogenic differentiation, while FoxO1 knockdown inhibited BMP-2-induced BMSC proliferation, migration and osteogenic differentiation, increased apoptosis, and affected mitochondrial dynamics and autophagy. Promoting mitochondrial fusion partially reversed the regulatory effects of FoxO1 downregulation on mitochondrial autophagy and the inhibitory effects of FoxO1 silencing on BMP-2-induced BMSC biological behaviors. Activated mitochondrial autophagy facilitated the homeostasis of mitochondrial dynamics and partially counteracted the inhibitory effects of FoxO1 knockdown on BMP-2-induced BMSC biological behaviors. In conclusion, FoxO1 regulates mitochondrial dynamics and autophagy to modulate the osteogenic differentiation of BMP-2-induced human BMSCs.

Mitochondrial DNA Leakage Promotes Persistent Pancreatic Acinar Cell Injury in Acute Pancreatitis viathe cGAS-STING-NF-κB Pathway.

Previous research has shown that the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway in macrophages can promote severe acute pancreatitis through the release of inflammatory factors. The role of this pathway in pancreatic acinar cells, however, has not been studied, and understanding its mechanism could be crucial. We analysed plasma from 50 acute pancreatitis (AP) patients and 10 healthy donors using digital PCR, which links mitochondrial DNA (mtDNA) levels to the severity of AP. Single-cell sequencing of the pancreas during AP revealed differentially expressed genes and pathways in acinar cells. Experimental studies using mouse and cell models, which included mtDNA staining and quantitative PCR, revealed mtDNA leakage and the activation of STING-related pathways, indicating potential inflammatory mechanisms in AP. In conclusion, our study revealed that the mtDNA-STING-nuclear factor κB(NF-κB) pathway in pancreatic acinar cells could be a novel pathogenic factor in AP.

Identification of human circulating factors following remote ischemic conditioning (RIC): Potential impact on stroke

Remote ischemic conditioning (RIC) is a procedure consisting of short cycles of ischemia applied in a limb that activates endogenous protection in distant organs, such as the brain. Despite the promising outcomes of RIC, the biochemical factors governing inter-organ communication remain largely unexplored, particularly in humans. A pilot study on 20 healthy humans was performed to identify potential circulating biochemical factors involved in RIC signalling. Blood was collected before and immediately, 4 and 22 h after the end of RIC. To characterize the responses triggered by RIC, a combination of biochemical and proteomic analysis, along with functional in vitro tests in human cells, were performed.RIC did not alter the levels of nitric oxide, bilirubin and cell-free mitochondrial DNA. In contrast, carboxyhaemoglobin levels increased following RIC at all time points and young subset, suggesting endogenous production of carbon monoxide that is a cytoprotective gasotransmitter. Additionally, the levels of glutathione and cysteinylglycine bound to proteins also increased after RIC, while glutathione catabolism decreased. Plasma proteomic analysis identified overall 828 proteins. Several steps of statistical analysis (Student's t-test, repeated measures ANOVA, with Holm corrected pairwise p-values <0.05 threshold and fold change higher or lower than 100 %) leaded to the identification of 9 proteins with altered circulating levels in response to RIC at 4h and 22h. All 9 proteins are from extracellular space or exosomes, being involved in inflammation, angiogenesis or metabolism control. In addition, RIC-conditioned plasma from young subjects protected microglial cell culture against inflammatory stimuli, indicating an anti-inflammatory effect of RIC. Nevertheless, other functional tests in neurons or endothelial cells had no effect. Overall, we present some evidence for RIC-induced anti-inflammatory and antioxidant responses in healthy human subjects, in particular in young subjects. This study is a first step towards the disclosure of signalling factors involved in RIC-mediated inter-organ communication.

Role of Microglial Mitophagy in Alleviating Postoperative Cognitive Dysfunction: a Mechanistic Study.

Postoperative cognitive dysfunction (POCD), a common complication following anesthesia and surgery, is influenced by hippocampal neuroinflammation and microglial activation. Mitophagy, a process regulating inflammatory responses by limiting the accumulation of damaged mitochondria, plays a significant role. This study aimed to determine whether regulating microglial mitophagy and the cGAS-STING pathway could alleviate cognitive decline after surgery. Exploratory laparotomy was performed to establish a POCD model using mice. Western blotting, immunofluorescence staining, transmission electron microscopy, and mt-Keima assays were used to examine microglial mitophagy and the cGAS-STING pathway. Quantitative polymerase chain reaction (qPCR) was used to detect inflammatory mediators and cytosolic mitochondrial DNA (mtDNA) levels in BV2 cells. Exploratory laparotomy triggered mitophagy and enhanced the cGAS-STING pathway in mice hippocampi. Pharmacological treatment reduced microglial activation, neuroinflammation, and cognitive impairment after surgery. Mitophagy suppressed the cGAS-STING pathway in mice hippocampi. In vitro, microglia-induced inflammation was mediated by mitophagy and the cGAS-STING pathway. Small interfering RNA (siRNA) of PINK1 hindered mitophagy activation and facilitated the cytosolic release of mtDNA, resulting in the initiation of the cGAS-STING pathway and innate immune response. Microglial mitophagy inhibited inflammatory responses via the mtDNA-cGAS-STING pathway inducing microglial mitophagy and inhibiting the mtDNA-cGAS-STING pathway may be an effective therapeutic approach for patients with POCD.

Investigating developmental characteristics of biopsied blastocysts stratified by mitochondrial copy numbers using time-lapse monitoring

BackgroundFor in vitro fertilization (IVF), mitochondrial DNA (mtDNA) levels in the trophectodermal (TE) cells of biopsied blastocysts have been suggested to be associated with the cells’ developmental potential. However, scholars have reached differing opinions regarding the use of mtDNA levels as a reliable biomarker for predicting IVF outcomes. Therefore, this study aims to assess the association of mitochondrial copy number measured by mitoscore associated with embryonic developmental characteristics and ploidy.MethodsThis retrospective study analyzed the developmental characteristics of embryos and mtDNA levels in biopsied trophectodermal cells. The analysis was carried out using time-lapse monitoring and next-generation sequencing from September 2021 to September 2022. Five hundred and fifteen blastocysts were biopsied from 88 patients undergoing IVF who met the inclusion criteria. Embryonic morphokinetics and morphology were evaluated at 118 h after insemination using all recorded images. Blastocysts with appropriate morphology on day 5 or 6 underwent TE biopsy and preimplantation genetic testing for aneuploidy (PGT-A). Statistical analysis involved generalized estimating equations, Pearson’s chi-squared test, Fisher’s exact test, and Kruskal–Wallis test, with a significance level set at P < 0.05.ResultsTo examine differences in embryonic characteristics between blastocysts with low versus high mitoscores, the blastocysts were divided into quartiles based on their mitoscore. Regarding morphokinetic characteristics, no significant differences in most developmental kinetics and observed cleavage dysmorphisms were discovered. However, blastocysts in mitoscore group 1 had a longer time for reaching 3-cell stage after tPNf (t3; median: 14.4 h) than did those in mitoscore group 2 (median: 13.8 h) and a longer second cell cycle (CC2; median: 11.7 h) than did blastocysts in mitoscore groups 2 (median: 11.3 h) and 4 (median: 11.4 h; P < 0.05). Moreover, blastocysts in mitoscore group 4 had a lower euploid rate (22.6%) and a higher aneuploid rate (59.1%) than did those in the other mitoscore groups (39.6–49.3% and 30.3–43.2%; P < 0.05). The rate of whole-chromosomal alterations in mitoscore group 4 (63.4%) was higher than that in mitoscore groups 1 (47.3%) and 2 (40.1%; P < 0.05). A multivariate logistic regression model was used to analyze associations between the mitoscore and euploidy of elective blastocysts. After accounting for factors that could potentially affect the outcome, the mitoscore still exhibited a negative association with the likelihood of euploidy (adjusted OR = 0.581, 95% CI: 0.396–0.854; P = 0.006).ConclusionsBlastocysts with varying levels of mitochondrial DNA, identified through biopsies, displayed similar characteristics in their early preimplantation development as observed through time-lapse imaging. However, the mitochondrial DNA level determined by the mitoscore can be used as a standalone predictor of euploidy.

HOXC11-mediated regulation of mitochondrial function modulates chemoresistance in colorectal cancer

BackgroundChemoresistance remains a significant challenge in colorectal cancer (CRC) treatment, necessitating a deeper understanding of its underlying mechanisms. HOXC11 has emerged as a potential regulator in various cancers, but its role in CRC chemoresistance remains unclear.MethodsSulforhodamine B assay was employed to assess the cell viability of CRC cells following treatment with chemotherapeutic drugs. Immunofluorescence staining was performed to examine the subcellular localization of HOXC11 in normal and chemoresistant CRC cells. The Seahorse mito stress test was conducted to evaluate the mitochondrial respiratory function of CRC cells. Real-time PCR was utilized to measure the expression level and copy number of mitochondrial DNA (mtDNA).ResultsOur findings revealed that HOXC11 was overexpressed in CRC cells compared to normal colorectal cells and correlated with poorer prognosis in CRC patients. Knockout of HOXC11 reversed acquired chemoresistance in CRC cells. Furthermore, we observed a functional subset of HOXC11 localized to the mitochondria in chemoresistant CRC cells, which regulated mitochondrial function by modulating mtDNA transcription, thereby affecting chemoresistance.ConclusionsIn summary, our study reveals that HOXC11 regulates mitochondrial function through the modulation of mtDNA transcription, impacting chemoresistance in colorectal cancer cells. These findings underscore the significance of understanding the molecular mechanisms underlying chemoresistance and highlight the potential therapeutic implications of targeting mitochondrial function in CRC treatment.

Comparative mitochondrial genomics analysis of selected species of Schizothoracinae sub family to explore the differences at mitochondrial DNA level

A comprehensive analysis of the whole mitochondrial genomes of the Schizothoracinae subfamily of the family Cyprinidae has been revealed for the first time. The species analyzed include Schizothorax niger, Schizothorax esocinus, Schizothorax labiatus and Schizothorax plagoistomus. The total mitochondrial DNA (mtDNA) length was determined to be 16585 bp, 16583 bp, 16582 bp and 16576 bp, respectively with 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and 2 non-coding area genes. The combined mean base compositions of the four species were as follows: A: 29.91 % T: 25.47 % G: 17.65 % C 27.01 %. The range of the GC content is 45–44 %, respectively. All protein coding genes (PCGs) commenced with the typical ATG codon, except for the cytochrome c oxidase subunit 1 (COX1) gene with GTG. The analysis of vital amino acid biosynthesis genes (COX1, ATPase 6, ATPase 8) in four different species revealed no significant differences. All 13 PCGs had Ka/Ks ratios that were all lesser than one, demonstrating purifying selection on those molecules. These tRNA genes were predicted to fold into the typical cloverleaf secondary structures with normal base pairing and ranged in size from 66 to 75 nucleotides. Additionally, the phylogenetic tree analysis revealed that S. esocinus species that was most alike to S. labiatus. This study provides critical data for phylogenetic analysis of the Schizothoracinae subfamily, which will help to resolve taxonomic difficulties and identify evolutionary links. Detailed mtDNA data are an invaluable resource for studying genetic diversity, population structure, and gene flow. Understanding genetic makeup can help inform conservation plans, identify unique populations, and track genetic variation to ensure effective preservation.

Low levels of supercoiled mitochondrial DNA are involved in heart failure induced by transverse aortic constriction in mice via an inflammatory response mediated by ZBP1

BackgroundInflammation in the myocardium plays a critical role in cardiac remodeling and the pathophysiology of heart failure (HF). Previous studies have shown that mitochondrial DNA (mtDNA) can exist in different topological forms. However, the specific influence of the ratio of supercoiled/relaxed mtDNA on the inflammatory response in cardiomyocytes remains poorly understood. The aim of this study was to elucidate the differential effects of different mtDNA types on cardiomyocyte inflammation through regulation of ZBP1. Materials and methodsA mouse model of HF was established by transverse aortic constriction (TAC) or doxorubicin (Doxo) induction. Histopathological changes were assessed by HE staining. ELISA was used to measure cytokine levels (IL-1β and IL-6). Southern blot analysis was performed to examine the different topology of mtDNA. Pearson correlation analysis was used to determine the correlation between the ratio of supercoiled/relaxed mtDNA and inflammatory cytokines. Reverse transcription quantitative PCR (RT-qPCR) was used to measure the mRNA expression levels of cytokines (IL-1β, IL-6) and Dloop, as an mtDNA marker. ResultsThe ratio of supercoiled to relaxed mtDNA was significantly increased in the myocardium of Doxo-induced mice, whereas no significant changes were observed in TAC-induced mice. The levels of IL-1β and IL-6 were positively correlated with the cytoplasmic mtDNA supercoiled/relaxed circle ratio. Different mtDNA topology has different effects on inflammatory pathways. Low supercoiled mtDNA primarily activates the NF-κB (Ser536) pathway via ZBP1, whereas high supercoiled mtDNA significantly affects the STAT1 and STAT2 pathways. The RIPK3-NF-κB pathway, as a downstream target of ZBP1, mediates the inflammatory response induced by low supercoiled mtDNA. Knockdown of TLR9 enhances the expression of ZBP1, p-NF-κB, and RIPK3 in cardiomyocytes treated with low supercoiled mtDNA, indicating the involvement of TLR9 in the anti-inflammatory role of ZBP1 in low supercoiled mtDNA-induced inflammation. ConclusionDifferent ratios of supercoiled to relaxed mtDNA influence the inflammatory response of cardiomyocytes and contribute to HF through the involvement of ZBP1. ZBP1, together with its downstream inflammatory mechanisms, mediates the inflammatory response induced by a low ratio of supercoiled mtDNA.

Mitochondrial Biomarkers and Metabolic Syndrome in Bipolar Disorder

The object of this study is test whether mitochondrial blood-based biomarkers are associated with markers of metabolic syndrome in bipolar disorder, hypothesizing higher lactate but unchanged cell-free circulating mitochondrial DNA levels in bipolar disorder patients with metabolic syndrome. In a cohort study, primary testing from the FondaMental Advanced Centers of Expertise for bipolar disorder (FACE-BD) was conducted, including 837 stable bipolar disorder patients. The I-GIVE validation cohort consists of 237 participants: stable and acute bipolar patients, non-psychiatric controls, and acute schizophrenia patients. Multivariable regression analyses show significant lactate association with triglycerides, fasting glucose and systolic and diastolic blood pressure. Significantly higher levels of lactate were associated with presence of metabolic syndrome after adjusting for potential confounding factors. Mitochondrial-targeted metabolomics identified distinct metabolite profiles in patients with lactate presence and metabolic syndrome, differing from those without lactate changes but with metabolic syndrome. Circulating cell-free mitochondrial DNA was not associated with metabolic syndrome. This thorough analysis mitochondrial biomarkers indicate the associations with lactate and metabolic syndrome, while showing the mitochondrial metabolites can further stratify metabolic profiles in patients with BD. This study is relevant to improve the identification and stratification of bipolar patients with metabolic syndrome and provide potential personalized-therapeutic opportunities.

Fit for purpose: Selecting the best mitochondrial DNA for the job

The factors determining levels of pathogenic mitochondrial DNA in cells and tissues are critical to disease pathology but remain poorly understood and contentious. In Nature, Kotrys etal. published a single-cell-based analysis casting fresh light on this thorny problem and introduced a powerful new investigative tool.

Shikonin blocks CAF-induced TNBC metastasis by suppressing mitochondrial biogenesis through GSK-3β/NEDD4-1 mediated phosphorylation-dependent degradation of PGC-1α

BackgroundTriple-negative breast cancer (TNBC) is characterized by its high metastatic potential, which results in poor patient survival. Cancer-associated fibroblasts (CAFs) are crucial in facilitating TNBC metastasis via induction of mitochondrial biogenesis. However, how to inhibit CAF-conferred mitochondrial biogenesis is still needed to explore.MethodsWe investigated metastasis using wound healing and cell invasion assays, 3D-culture, anoikis detection, and NOD/SCID mice. Mitochondrial biogenesis was detected by MitoTracker green FM staining, quantification of mitochondrial DNA levels, and blue-native polyacrylamide gel electrophoresis. The expression, transcription, and phosphorylation of peroxisome-proliferator activated receptor coactivator 1α (PGC-1α) were detected by western blotting, chromatin immunoprecipitation, dual-luciferase reporter assay, quantitative polymerase chain reaction, immunoprecipitation, and liquid chromatography-tandem mass spectrometry. The prognostic role of PGC-1α in TNBC was evaluated using the Kaplan–Meier plotter database and clinical breast cancer tissue samples.ResultsWe demonstrated that PGC-1α indicated lymph node metastasis, tumor thrombus formation, and poor survival in TNBC patients, and it was induced by CAFs, which functioned as an inducer of mitochondrial biogenesis and metastasis in TNBC. Shikonin impeded the CAF-induced PGC-1α expression, nuclear localization, and interaction with estrogen-related receptor alpha (ERRα), thereby inhibiting PGC-1α/ERRα-targeted mitochondrial genes. Mechanistically, the downregulation of PGC-1α was mediated by synthase kinase 3β-induced phosphorylation of PGC-1α at Thr295, which associated with neural precursor cell expressed developmentally downregulated 4e1 recognition and subsequent degradation by ubiquitin proteolysis. Mutation of PGC-1α at Thr295 negated the suppressive effects of shikonin on CAF-stimulated TNBC mitochondrial biogenesis and metastasis in vitro and in vivo.ConclusionsOur findings indicate that PGC-1α is a viable target for blocking TNBC metastasis by disrupting mitochondrial biogenesis, and that shikonin merits potential for treatment of TNBC metastasis as an inhibitor of mitochondrial biogenesis through targeting PGC-1α.

Outcomes of combined mitochondria and mesenchymal stem cells-derived exosome therapy in rat acute respiratory distress syndrome and sepsis

BACKGROUND The treatment of acute respiratory distress syndrome (ARDS) complicated by sepsis syndrome (SS) remains challenging. AIM To investigate whether combined adipose-derived mesenchymal-stem-cells (ADMSCs)-derived exosome (EXAD) and exogenous mitochondria (mitoEx) protect the lung from ARDS complicated by SS. METHODS In vitro study, including L2 cells treated with lipopolysaccharide (LPS) and in vivo study including male-adult-SD rats categorized into groups 1 (sham-operated-control), 2 (ARDS-SS), 3 (ARDS-SS + EXAD), 4 (ARDS-SS + mitoEx), and 5 (ARDS-SS + EXAD + mitoEx), were included in the present study. RESULTS In vitro study showed an abundance of mitoEx found in recipient-L2 cells, resulting in significantly higher mitochondrial-cytochrome-C, adenosine triphosphate and relative mitochondrial DNA levels (P &lt; 0.001). The protein levels of inflammation [interleukin (IL)-1β/tumor necrosis factor (TNF)-α/nuclear factor-κB/toll-like receptor (TLR)-4/matrix-metalloproteinase (MMP)-9/oxidative-stress (NOX-1/NOX-2)/apoptosis (cleaved-caspase3/cleaved-poly (ADP-ribose) polymerase)] were significantly attenuated in lipopolysaccharide (LPS)-treated L2 cells with EXAD treatment than without EXAD treatment, whereas the protein expressions of cellular junctions [occluding/β-catenin/zonula occludens (ZO)-1/E-cadherin] exhibited an opposite pattern of inflammation (all P &lt; 0.001). Animals were euthanized by 72 h post-48 h-ARDS induction, and lung tissues were harvested. By 72 h, flow cytometric analysis of bronchoalveolar lavage fluid demonstrated that the levels of inflammatory cells (Ly6G+/CD14+/CD68+/CD11b/c+/myeloperoxidase+) and albumin were lowest in group 1, highest in group 2, and significantly higher in groups 3 and 4 than in group 5 (all P &lt; 0.0001), whereas arterial oxygen-saturation (SaO2%) displayed an opposite pattern of albumin among the groups. Histopathological findings of lung injury/fibrosis area and inflammatory/DNA-damaged markers (CD68+/γ-H2AX) displayed an identical pattern of SaO2% among the groups (all P &lt; 0.0001). The protein expressions of inflammatory (TLR-4/MMP-9/IL-1β/TNF-α)/oxidative stress (NOX-1/NOX-2/p22phox/oxidized protein)/mitochondrial-damaged (cytosolic-cytochrome-C/dynamin-related protein 1)/autophagic (beclin-1/Atg-5/ratio of LC3B-II/LC3B-I) biomarkers exhibited a similar manner, whereas antioxidants [nuclear respiratory factor (Nrf)-1/Nrf-2]/cellular junctions (ZO-1/E-cadherin)/mitochondrial electron transport chain (complex I-V) exhibited an opposite manner of albumin among the groups (all P &lt; 0.0001). CONCLUSION Combined EXAD-mitoEx therapy was better than merely one for protecting the lung against ARDS-SS induced injury.

Outcomes of combined mitochondria and mesenchymal stem cells-derived exosome therapy in rat acute respiratory distress syndrome and sepsis.

The treatment of acute respiratory distress syndrome (ARDS) complicated by sepsis syndrome (SS) remains challenging. To investigate whether combined adipose-derived mesenchymal-stem-cells (ADMSCs)-derived exosome (EXAD) and exogenous mitochondria (mitoEx) protect the lung from ARDS complicated by SS. In vitro study, including L2 cells treated with lipopolysaccharide (LPS) and in vivo study including male-adult-SD rats categorized into groups 1 (sham-operated-control), 2 (ARDS-SS), 3 (ARDS-SS + EXAD), 4 (ARDS-SS + mitoEx), and 5 (ARDS-SS + EXAD + mitoEx), were included in the present study. In vitro study showed an abundance of mitoEx found in recipient-L2 cells, resulting in significantly higher mitochondrial-cytochrome-C, adenosine triphosphate and relative mitochondrial DNA levels (P < 0.001). The protein levels of inflammation [interleukin (IL)-1β/tumor necrosis factor (TNF)-α/nuclear factor-κB/toll-like receptor (TLR)-4/matrix-metalloproteinase (MMP)-9/oxidative-stress (NOX-1/NOX-2)/apoptosis (cleaved-caspase3/cleaved-poly (ADP-ribose) polymerase)] were significantly attenuated in lipopolysaccharide (LPS)-treated L2 cells with EXAD treatment than without EXAD treatment, whereas the protein expressions of cellular junctions [occluding/β-catenin/zonula occludens (ZO)-1/E-cadherin] exhibited an opposite pattern of inflammation (all P < 0.001). Animals were euthanized by 72 h post-48 h-ARDS induction, and lung tissues were harvested. By 72 h, flow cytometric analysis of bronchoalveolar lavage fluid demonstrated that the levels of inflammatory cells (Ly6G+/CD14+/CD68+/CD11b/c+/myeloperoxidase+) and albumin were lowest in group 1, highest in group 2, and significantly higher in groups 3 and 4 than in group 5 (all P < 0.0001), whereas arterial oxygen-saturation (SaO2%) displayed an opposite pattern of albumin among the groups. Histopathological findings of lung injury/fibrosis area and inflammatory/DNA-damaged markers (CD68+/γ-H2AX) displayed an identical pattern of SaO2% among the groups (all P < 0.0001). The protein expressions of inflammatory (TLR-4/MMP-9/IL-1β/TNF-α)/oxidative stress (NOX-1/NOX-2/p22phox/oxidized protein)/mitochondrial-damaged (cytosolic-cytochrome-C/dynamin-related protein 1)/autophagic (beclin-1/Atg-5/ratio of LC3B-II/LC3B-I) biomarkers exhibited a similar manner, whereas antioxidants [nuclear respiratory factor (Nrf)-1/Nrf-2]/cellular junctions (ZO-1/E-cadherin)/mitochondrial electron transport chain (complex I-V) exhibited an opposite manner of albumin among the groups (all P < 0.0001). Combined EXAD-mitoEx therapy was better than merely one for protecting the lung against ARDS-SS induced injury.