Mitochondrial Gene Expression Research Articles

Preoperative Increases in T2-Weighted Fat-Suppressed Magnetic Resonance Imaging Signal Intensities Associated With Advanced Tissue Degeneration and Mitochondrial Dysfunction in Rotator Cuff Tears

PurposeThe purpose of this study was to investigate the relationship between magnetic resonance imaging (MRI) signal intensities and mitochondrial function in patients undergoing arthroscopic rotator cuff repair, assessed through histological and genetic profiling of tendon tissue. MethodsThis study, conducted between April 2022 and January 2023, included 20 patients undergoing rotator cuff repair for atraumatic/degenerative tears. Rotator cuff tendon edge samples were obtained during arthroscopic rotator cuff repair. Patients were classified based on signal intensity from preoperative T2-weighted fat suppressed MRI. Specifically, they were categorized as having either high or low signal intensity at the rotator cuff tendon edge, with the deltoid muscle serving as a reference. Comparative analyses specifically compared the histological features and genetic profiles of the tendon tissue at the rotator cuff tendon edge. Histological evaluation of harvested tendon specimens during arthroscopic rotator cuff repair employed the modified Bonar score. Real-time polymerase chain reaction was used to assess expression of various mitochondrial and apoptosis-related genes. The mitochondrial morphology of the rotator cuff torn site was examined using electron microscopy. ResultsThe higher signal intensity group showed significantly higher modified Bonar scores (p=0.0068), decreased mitochondrial gene expression, increased TUNEL-positive cells (p=0.032), lower SOD activity (p=0.011), reduced ATP5A (p=0.031), and increased cleaved caspase-9 activity (p=0.026) when compared to the lower signal intensity group. Electron microscopy revealed fewer mitochondrial cristae in the higher signal intensity group. ConclusionOur results suggest correlations between high MRI signal intensities and the presence of degeneration, mitochondrial dysfunction, and increased apoptosis in rotator cuff tissues. This underscores the utility of MRI signal intensity as an indicator of tissue condition. Clinical RelevanceBuilding on the established premise that elevated preoperative MRI signal intensities may indicate higher rates of postoperative rotator cuff re-tears, the current study substantiates these findings from a mitochondrial function perspective.

Mitochondrial microRNA profiles are altered in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) during hibernation.

Thirteen-lined ground squirrels (TLGSs) are obligate hibernators that cycle between torpor (low metabolic rate and body temperature) and interbout euthermia (IBE; typical euthermic body temperature and metabolism) from late autumn to spring. Many physiological changes occur throughout hibernation, including a reduction in liver mitochondrial metabolism during torpor, which is reversed during arousal to interbout euthermia. Nuclear-encoded microRNA (miRNA, small posttranscriptional regulator molecules) differ in abundance throughout TLGS hibernation and have been shown to regulate mitochondrial gene expression in mammalian cell culture (where they are referred to as mitomiRs). This study characterized differences in mitomiR profiles from TLGS liver mitochondria isolated during summer, torpor, and IBE, and predicted their mitochondrial targets. Using small RNA sequencing, differentially abundant mitomiRs were identified between hibernation states, and using quantitative PCR analysis, we quantified the expression of predicted mitochondrial mRNA targets. Most differences in mitomiR abundances were seasonal (i.e., between summer and winter) with only one mitomiR differentially abundant between IBE and torpor. Multiple factor analysis (MFA) revealed three clusters divided by hibernation states, where clustering was predominantly driven by mitomiR abundances. Nine of these differentially abundant mitomiRs had predicted mitochondrial RNA targets, including subunits of electron transfer system complexes I and IV, 12S rRNA, and two tRNAs. Overall, mitomiRs were predicted to suppress the expression of their mitochondrial targets and may have some involvement in regulating protein translation in mitochondria. This study found differences in mitomiR abundances between seasons and hibernation states of TLGS and suggests potential mechanisms for regulating the mitochondrial electron transfer system.NEW & NOTEWORTHY During the hibernation season, thirteen-lined ground squirrels periodically increase metabolism remarkably between torpor and interbout euthermia (IBE). This process involves rapid reactivation of mitochondrial respiration. We predicted that mitochondrial microRNA (mitomiRs) might be altered during this response. We found that the abundance of 38 liver mitomiRs differs based on hibernation state (summer, IBE, and torpor). Small RNA sequencing identified mitomiR profiles, including some mitomiRs that are predicted to bind to mitochondrial RNAs.

Cytosine methylation flags mitochondrial RNA for degradation

Mitochondrial double-stranded RNA (dsRNA) can form spontaneously in mitochondria, blocking mitochondrial gene expression and triggering an immune response. A recent study by Kim, Tan, et al. identified a safeguard mechanism in which NOP2/Sun RNA methyltransferase 4 (NSUN4)-mediated RNA methylation (m5C) recruits the RNA degradation machinery to prevent dsRNA formation.

Deciphering tissue-specific expression profiles of mitochondrial genome-encoded tRNAs and rRNAs through transcriptomic profiling in buffalo.

Mitochondria, essential for cellular energy production through oxidative phosphorylation (OXPHOS), integrate mt-DNA and nuclear-encoded genes. This cooperation extends to the mitochondrial translation machinery, involving crucial mtDNA-encoded RNAs: 22 tRNAs (mt-tRNAs) as adapters and two rRNAs (mt-rRNAs) for ribosomal assembly, enabling mitochondrial-encoded mRNA translation. Disruptions in mitochondrial gene expression can strongly impact energy generation and overall animal health. Our study investigates the tissue-specific expression patterns of mt-tRNAs and mt-rRNAs in buffalo. To investigate the expression patterns of mt-tRNAs and mt-rRNAs in different tissues and gain a better understanding of tissue-specific variations, RNA-seq was performed on various tissues, such as the kidney, heart, brain, and ovary, from post-pubertal female buffaloes. Subsequently, we identified transcripts that were differentially expressed in various tissue comparisons. The findings reveal distinct expression patterns among specific mt-tRNA and mt-rRNA genes across various tissues, with some exhibiting significant upregulation and others demonstrating marked downregulation in specific tissue contexts. These identified variations reflect tissue-specific physiological roles, underscoring their significance in meeting the unique energy demands of each tissue. Notably, the brain exhibits the highest mtDNA copy numbers and an abundance of mitochondrial mRNAs of our earlier findings, potentially linked to the significant upregulation of mt-tRNAs in brain. This suggests a plausible association between mtDNA replication and the regulation of mtDNA gene expression. Overall, our study unveils the tissue-specific expression of mitochondrial-encoded non-coding RNAs in buffalo. As we proceed, our further investigations into tissue-specific mitochondrial proteomics and microRNA studies aim to elucidate the intricate mechanisms within mitochondria, contributing to tissue-specific mitochondrial attributes. This research holds promise to elucidate the critical role of mitochondria in animal health and disease.

Inhibition of K63 ubiquitination by G-Protein pathway suppressor 2 (GPS2) regulates mitochondria-associated translation

G-Protein Pathway Suppressor 2 (GPS2) is an inhibitor of non-proteolytic K63 ubiquitination mediated by the E2 ubiquitin-conjugating enzyme Ubc13. Previous studies have associated GPS2-mediated restriction of ubiquitination with the regulation of insulin signaling, inflammatory responses and mitochondria-nuclear communication across different tissues and cell types. However, a detailed understanding of the targets of GPS2/Ubc13 activity is lacking. Here, we have dissected the GPS2-regulated K63 ubiquitome in mouse embryonic fibroblasts and human breast cancer cells, unexpectedly finding an enrichment for proteins involved in RNA binding and translation on the outer mitochondrial membrane. Validation of selected targets of GPS2-mediated regulation, including the RNA-binding protein PABPC1 and translation factors RPS1, RACK1 and eIF3M, revealed a mitochondrial-specific strategy for regulating the translation of nuclear-encoded mitochondrial proteins via non-proteolytic ubiquitination. Removal of GPS2-mediated inhibition, either via genetic deletion or stress-induced nuclear translocation, promotes the import-coupled translation of selected mRNAs leading to the increased expression of an adaptive antioxidant program. In light of GPS2 role in nuclear-mitochondria communication, these findings reveal an exquisite regulatory network for modulating mitochondrial gene expression through spatially coordinated transcription and translation.

Mitochondrial NME6: A Paradigm Change within the NME/NDP Kinase Protein Family?

Eukaryotic NMEs/NDP kinases are a family of 10 multifunctional proteins that occur in different cellular compartments and interact with various cellular components (proteins, membranes, and DNA). In contrast to the well-studied Group I NMEs (NME1-4), little is known about the more divergent Group II NMEs (NME5-9). Three recent publications now shed new light on NME6. First, NME6 is a third mitochondrial NME, largely localized in the matrix space, associated with the mitochondrial inner membrane. Second, while its monomeric form is inactive, NME6 gains NDP kinase activity through interaction with mitochondrial RCC1L. This challenges the current notion that mammalian NMEs require the formation of hexamers to become active. The formation of complexes between NME6 and RCC1L, likely heterodimers, seemingly obviates the necessity for hexamer formation, stabilizing a NDP kinase-competent conformation. Third, NME6 is involved in mitochondrial gene maintenance and expression by providing (d)NTPs for replication and transcription (in particular the pyrimidine nucleotides) and by a less characterized mechanism that supports mitoribosome function. This review offers an overview of NME evolution and structure and highlights the new insight into NME6. The new findings position NME6 as the most comprehensively studied protein in NME Group II and may even suggest it as a new paradigm for related family members.

The predictive value of peripheral blood cell mitochondrial gene expression in identifying the prognosis in pediatric sepsis at preschool age.

Sepsis represents a severe manifestation of infection often accompanied by metabolic disorders and mitochondrial dysfunction. Notably, mitochondrial DNA copy number (mtDNA-CN) and the expression of specific mitochondrial genes have emerged as sensitive indicators of mitochondrial function. To investigate the utility of mitochondrial gene expression in peripheral blood cells for distinguishing severe infections and predicting associated outcomes, we conducted a prospective cohort study. We established a prospective cohort comprising 74 patients with non-sepsis pneumonia and 67 cases of sepsis induced by respiratory infections, aging from 2 to 6 years old. We documented corresponding clinical data and laboratory information and collected blood samples upon initial hospital admission. Peripheral blood cells were promptly isolated, and both total DNA and RNA were extracted. We utilized absolute quantification PCR to assess mtDNA-CN, as well as the expression levels of mt-CO1, mt-ND1, and mt-ATP6. Subsequently, we extended these comparisons to include survivors and non-survivors among patients with sepsis using univariate and multivariate analyses. Receiver operating characteristic (ROC) curves were constructed to assess the diagnostic potential. The mtDNA-CN in peripheral blood cells was significantly lower in the sepsis group. Univariate analysis revealed a significant reduction in the expression of mt-CO1, mt-ND1, and mt-ATP6 in patients with sepsis. However, multivariate analysis did not support the use of mitochondrial function in peripheral blood cells for sepsis diagnosis. In the comparison between pediatric sepsis survivors and non-survivors, univariate analysis indicated a substantial reduction in the expression of mt-CO1, mt-ND1, and mt-ATP6 among non-survivors. Notably, total bilirubin (TB), mt-CO1, mt-ND1, and mt-ATP6 levels were identified as independent risk factors for sepsis-induced mortality. ROC curves were then established for these independent risk factors, revealing areas under the curve (AUCs) of 0.753 for TB (95% CI 0.596-0.910), 0.870 for mt-CO1 (95% CI 0.775-0.965), 0.987 for mt-ND1 (95% CI 0.964-1.000), and 0.877 for mt-ATP6 (95% CI 0.793-0.962). MtDNA-CN and mitochondrial gene expression are closely linked to the severity and clinical outcomes of infectious diseases. Severe infections lead to impaired mitochondrial function in peripheral blood cells. Notably, when compared to other laboratory parameters, the expression levels of mt-CO1, mt-ND1, and mt-ATP6 demonstrate promising potential for assessing the prognosis of pediatric sepsis.

The human mitochondrial mRNA structurome reveals mechanisms of gene expression.

The human mitochondrial genome encodes crucial oxidative phosphorylation system proteins, pivotal for aerobic energy transduction. They are translated from nine monocistronic and two bicistronic transcripts whose native structures remain unexplored, posing a gap in understanding mitochondrial gene expression. In this work, we devised the mitochondrial dimethyl sulfate mutational profiling with sequencing (mitoDMS-MaPseq) method and applied detection of RNA folding ensembles using expectation-maximization (DREEM) clustering to unravel the native mitochondrial messenger RNA (mt-mRNA) structurome in wild-type (WT) and leucine-rich pentatricopeptide repeat-containing protein (LRPPRC)-deficient cells. Our findings elucidate LRPPRC's role as a holdase contributing to maintaining mt-mRNA folding and efficient translation. mt-mRNA structural insights in WT mitochondria, coupled with metabolic labeling, unveil potential mRNA-programmed translational pausing and a distinct programmed ribosomal frameshifting mechanism. Our data define a critical layer of mitochondrial gene expression regulation. These mt-mRNA folding maps provide a reference for studying mt-mRNA structures in diverse physiological and pathological contexts.

A Th17 cell-intrinsic glutathione/mitochondrial-IL-22 axis protects against intestinal inflammation

The intestinal tract generates significant reactive oxygen species (ROS), but the role of Tcell antioxidant mechanisms in maintaining intestinal homeostasis is poorly understood. We used Tcell-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), which impaired glutathione (GSH) production, crucially reducing IL-22 production by Th17 cells in the lamina propria, which is critical for gut protection. Under steady-state conditions, Gclc deficiency did not alter cytokine secretion; however, C.rodentium infection induced increased ROS and disrupted mitochondrial function and TFAM-driven mitochondrial gene expression, resulting in decreased cellular ATP. These changes impaired the PI3K/AKT/mTOR pathway, reducing phosphorylation of 4E-BP1 and consequently limiting IL-22 translation. The resultant low IL-22 levels led to poor bacterial clearance, severe intestinal damage, and high mortality. Our findings highlight a previously unrecognized, essential role of Th17 cell-intrinsic GSH in promoting mitochondrial function and cellular signaling for IL-22 protein synthesis, which is critical for intestinal integrity and defense against gastrointestinal infections.

Enhanced neuroprotective effect of verapamil-loaded hyaluronic acid modified carbon quantum dots in an in-vitro model of amyloid-induced Alzheimer's disease

This study aims to investigate the molecular mechanisms and the neuroprotective effect of hyaluronic acid modified verapamil-loaded carbon quantum dots (VRH-loaded HA-CQDs) against an in-vitro Alzheimer's disease model induced by amyloid beta (Aβ) in SH-SY5Y and Neuro 2a neuroblastoma cells. Briefly, different HA-CQDs were prepared using hydrothermal method and optimized by Box-Behnken design to maximize quantum yield and minimize particle size. Serum stable negatively charged VRH-loaded HA-CQDs was successfully prepared by admixing the optimized HA-CQDs and VRH with association efficiency and loading capacity of 81.25 ± 3.65 % and 5.11 ± 0.81 %, respectively. Cells were pretreated with VRH solution or loaded-HA-CQDs followed by exposure to Aβ. Compared to the control group, amyloidosis led to reduction in cellular proliferation, mitochondrial membrane potential, expression of cytochrome P450, cytochrome c oxidase, CREB-regulated transcriptional coactivator 3, and mitotic index, along with marked increase in reactive oxygen species (ROS) and inflammatory cytokines. Pretreatment with VRH, either free or loaded HA-CQDs, enhanced cell survival, mitochondrial membrane potential, mitotic index, and gene expression. It also reduced inflammation and ROS. However, VRH-loaded HA-CQDs exhibited superior effectiveness in the measured parameters. These findings suggest that VRH-loaded HA-CQDs have enhanced therapeutic potential compared to free VRH in mitigating amyloidosis negative features.

GTPBP8 plays a role in mitoribosome formation in human mitochondria

Mitochondrial gene expression relies on mitoribosomes to translate mitochondrial mRNAs. The biogenesis of mitoribosomes is an intricate process involving multiple assembly factors. Among these factors, GTP-binding proteins (GTPBPs) play important roles. In bacterial systems, numerous GTPBPs are required for ribosome subunit maturation, with EngB being a GTPBP involved in the ribosomal large subunit assembly. In this study, we focus on exploring the function of GTPBP8, the human homolog of EngB. We find that ablation of GTPBP8 leads to the inhibition of mitochondrial translation, resulting in significant impairment of oxidative phosphorylation. Structural analysis of mitoribosomes from GTPBP8 knock-out cells shows the accumulation of mitoribosomal large subunit assembly intermediates that are incapable of forming functional monosomes. Furthermore, fPAR-CLIP analysis reveals that GTPBP8 is an RNA-binding protein that interacts specifically with the mitochondrial ribosome large subunit 16 S rRNA. Our study highlights the role of GTPBP8 as a component of the mitochondrial gene expression machinery involved in mitochondrial large subunit maturation.

Metabolic remodeling and calcium handling abnormality in induced pluripotent stem cell-derived cardiomyocytes in dilated phase of hypertrophic cardiomyopathy with MYBPC3 frameshift mutation

Hypertrophic cardiomyopathy (HCM) is an inherited disorder characterized by left ventricular hypertrophy and diastolic dysfunction, and increases the risk of arrhythmias and heart failure. Some patients with HCM develop a dilated phase of hypertrophic cardiomyopathy (D-HCM) and have poor prognosis; however, its pathogenesis is unclear and few pathological models exist. This study established disease-specific human induced pluripotent stem cells (iPSCs) from a patient with D-HCM harboring a mutation in MYBPC3 (c.1377delC), a common causative gene of HCM, and investigated the associated pathophysiological mechanisms using disease-specific iPSC-derived cardiomyocytes (iPSC-CMs). We confirmed the expression of pluripotent markers and the ability to differentiate into three germ layers in D-HCM patient-derived iPSCs (D-HCM iPSCs). D-HCM iPSC-CMs exhibited disrupted myocardial sarcomere structures and an increased number of damaged mitochondria. Ca2+ imaging showed increased abnormal Ca2+ signaling and prolonged decay time in D-HCM iPSC-CMs. Cell metabolic analysis revealed increased basal respiration, maximal respiration, and spare-respiratory capacity in D-HCM iPSC-CMs. RNA sequencing also showed an increased expression of mitochondrial electron transport system-related genes. D-HCM iPSC-CMs showed abnormal Ca2+ handling and hypermetabolic state, similar to that previously reported for HCM patient-derived iPSC-CMs. Although further studies are required, this is expected to be a useful pathological model for D-HCM.

P-549 Differentially expressed genes in the immune cells obtained from preovulatory follicles undergoing progestin-primed ovarian stimulation and GnRH-antagonist: Insights from single-cell RNA sequencing analysis

Abstract Study question Could controlled ovarian stimulation protocol (progestin-primed ovarian stimulation [PPOS] vs GnRH-antagonist [GnRH-ant]) affect the gene expression of human immune cells (ICs) in preovulatory follicles? Summary answer Proinflammatory cytokine-related gene expression was significantly higher and proinflammatory pathways were also more prominently involved in ICs in PPOS than the GnRH-ant. What is known already PPOS has garnered attention, with several studies suggesting reproductive outcomes comparable to GnRH-ant. However, in our previous study, pregnancy outcomes with PPOS were significantly lower than GnRH-ant (38th ESHRE Annual Meeting [O-130]) and single-cell RNA sequencing (scRNA-seq) of granulosa cells revealed increased mitochondrial DNA gene expression in PPOS than GnRH-ant (39th ESHRE Annual Meeting [P-281]). Previous studies suggested that ICs play a pivotal role in follicular and embryonic developments, via proinflammatory cytokines such as TNF-α, IL-1β, and IL-6. Notably, changes in gene expressions of ICs under different controlled ovarian stimulation (COS) protocols remain unresolved to date. Study design, size, duration From November 2021 to January 2022, all samples were obtained from the patients who provided written informed consent before the inclusion and underwent COS cycles at a single-institution. scRNA-seq was performed on 6,828 cells corresponding to ICs of the MII-oocyte follicles (56 from PPOS and 39 from GnRH-ant), involving 16 patients with normal ovarian reserves (aged < 40 years, AMH ≥1.1 ng/mL) undergoing PPOS with chlormadinone acetate (n = 8) or GnRH-ant with cetrorelix (n = 8). Participants/materials, setting, methods The ICs were collected via follicular aspiration from 16 patients. The follicular fluid (FF) from each follicle was collected and all FF were mixed together in MII-oocyte for sequencing. Sample sequencing was performed using Chromium Next GEM Single Cell V(D)J Reagent Kits (10X Genomics, Pleasanton, CA, USA). Libraries were sequenced using a DNBSEQ-G400, with reads subsequently analyzed using Cellxgene. The clustering was performed using fastcluster in scDblFinder. Functional enrichment analysis was carried out using Enrichr. Main results and the role of chance Two population of ICs were used in this investigation: PPOS and GnRH-ant of MII-oocyte. After the quality control, 4,447 cells for PPOS and 2,381 cells for GnRH-ant, respectively, were analyzed. Following single-cell clustering based on the Seurat package, cell proportion analysis revealed eight clusters, which consist of monocyte, myeloid dendritic cell, T cell, natural killer cell and naive B cell, in the PPOS and GnRH-ant groups. No significant difference was observed in gene expression and cell distribution in each cluster. Differentially expressed genes (DEGs) were identified in the two IC population. Interestingly, a significant increase in expression of proinflammatory cytokine including IL-1β and IL-6 was observed in PPOS compared to GnRH-ant, particularly in CD14-positive monocytes. Functional significance of DEGs was validated by identifying significantly regulated pathways based on Kyoto Encyclopedia of Genes and Genomes categories, revealing involvement in TNF signaling, IL-17 signaling, and NF-κB signaling pathways. Limitations, reasons for caution Our conclusions are limited due to the inclusion of Japanese patients at a single-institution. The results need to be validated across different centers and other ethnicities. Due to the small sample size, these results should prompt further study to confirm our findings. Wider implications of the findings This is the first report assessing the DEGs of ICs under different COS protocols at the single-cell level. This study suggests that progestin for PPOS induces elevation of the proinflammatory cytokine gene expression of ICs in preovulatory follicles, which may be associated with reproductive outcomes. Trial registration number Not applicable

P-172 Mitochondria are dysregulated in poor quality oocytes and can be targeted in vitro to alter oocyte quality

Abstract Study question Does mitochondrial gene expression differ between good and poor quality oocytes and can mitochondria be targeted in vitro to alter oocyte quality? Summary answer Mitochondrial-related genes are dysregulated in poor quality oocytes and mitochondrial inhibitors can be used during IVM to alter oocyte quality. What is known already Good oocyte quality is essential for successful pregnancy yet we still have a poor understanding of the optimal composition of a good egg. Mitochondrial number and activity are important for oocyte quality and during maturation the ultrastructure of the mitochondria changes to a hooded or ring form. We have observed differences in the timing of this morphological change between good and poor quality oocytes in a sheep model. Oligomycin treatment of oocytes decreases the proportion of hooded mitochondria and increases mitochondrial membrane potential (MMP) indicating that hooded mitochondria may form during maturation to reduce mitochondrial activity prior to fertilisation. Study design, size, duration Gene expression differences were compared between poorer quality peripubertal lamb oocytes and adult oocytes harvested during estrus from pools of ∼80 abattoir-derived ovaries on three separate dates for each age. Denuded oocytes (GV stage) were frozen in three pools of ∼20 per age and total RNA isolated. Sheep oocytes (∼30 per group) were treated with oligomycin, FCCP or vehicle control in final 2 hours of IVM on five separate dates, fertilised and cultured. Participants/materials, setting, methods RNA sequencing cleaned reads were mapped using HISAT2 on sheep genome version ARS-UI_Ramb_v2. Count matrix were generated using featureCount and differential gene expression was performed using DESeq2. After normalisation, Wald test was used to define genes differentially expressed. Genes were considered significantly altered with a FDR < 5% after Benjamini-Hochberg correction. Mean cleavage rate was compared to vehicle control in five replicate experiments using one-way ANOVA with Benjamini, Krieger and Yekutieli FDR < 5%. Main results and the role of chance Oligomycin (ATP synthase inhibitor) decreased cleavage rate compared to control (P < 0.001) while FCCP (electron transport chain uncoupler) had no effect. There were 163 genes up-regulated and 194 genes down-regulated in lamb GV oocytes compared to adult GV stage (FDR <5%). Metascape pathway analysis of the up-regulated genes in lamb oocytes showed the most enriched pathway was Thermogenesis which included these genes: ACOT9, AJUBA, ATP5ME, ATP5MF, CALML4, CREB3L3, CRPPA, DNAJC15, DPM3, FMC1, LDHA, NDUFA12, NDUFAF2, NDUFS5, NT5C, PHGDH, POLR2G, PPARG, PRPS1L1, SDHAF3, SLIRP, TIMM8B and UBB. Six of these up-regulated genes were in the top 20 most highly expressed genes. Thermogenesis is the process by which the mitochondrial electron transport chain becomes uncoupled to dissipate heat and may also control production of free radicals by mitochondria and their response to oxidants. These genes and other mitochondrial related genes that were up-regulated in lamb GV oocytes (CKS2, DCN, PCP4L1, SLC51B) are involved in formation of the electron transport chain complexes, oxidative phosphorylation, ATP synthesis, mitochondrial organization and regulation of membrane potential. Together these results indicate that mitochondrial function is dysregulated in poorer quality oocytes and that treatment with mitochondrial inhibitors during IVM can alter oocyte quality. Limitations, reasons for caution This study was carried out in sheep oocytes and results would need to be confirmed in human oocytes if possible. Further research is needed to determine if decreasing MMP can improve oocyte quality and embryo development. Wider implications of the findings This animal model study indicates that mitochondria may be targeted during IVM to alter oocyte quality and this knowledge could be applied to develop ways of increasing human IVM and ART success rates. Trial registration number not applicable

Overexpression of mitochondrial fission or mitochondrial fusion genes enhances resilience and extends longevity.

The dynamicity of the mitochondrial network is crucial for meeting the ever-changing metabolic and energy needs of the cell. Mitochondrial fission promotes the degradation and distribution of mitochondria, while mitochondrial fusion maintains mitochondrial function through the complementation of mitochondrial components. Previously, we have reported that mitochondrial networks are tubular, interconnected, and well-organized in young, healthy C. elegans, but become fragmented and disorganized with advancing age and in models of age-associated neurodegenerative disease. In this work, we examine the effects of increasing mitochondrial fission or mitochondrial fusion capacity by ubiquitously overexpressing the mitochondrial fission gene drp-1 or the mitochondrial fusion genes fzo-1 and eat-3, individually or in combination. We then measured mitochondrial function, mitochondrial network morphology, physiologic rates, stress resistance, and lifespan. Surprisingly, we found that overexpression of either mitochondrial fission or fusion machinery both resulted in an increase in mitochondrial fragmentation. Similarly, both mitochondrial fission and mitochondrial fusion overexpression strains have extended lifespans and increased stress resistance, which in the case of the mitochondrial fusion overexpression strains appears to be at least partially due to the upregulation of multiple pathways of cellular resilience in these strains. Overall, our work demonstrates that increasing the expression of mitochondrial fission or fusion genes extends lifespan and improves biological resilience without promoting the maintenance of a youthful mitochondrial network morphology. This work highlights the importance of the mitochondria for both resilience and longevity.

Liver Mitochondrial Morphology and Gene Expression as Markers of Liver Reserve: Prognostic Implications for Native Liver Survival in Biliary Atresia

PurposeHepatocyte mitochondrial morphology and gene expression were compared between biliary atresia (BA), infantile cholestasis (IC), and normal liver (NL) as prognostic indicators. MethodsSpecimens of liver at portoenterostomy (PE) for BA, from intrahepatic bile duct paucity patients for IC, and from choledochal cyst or hepatoblastoma patients for NL were collected prospectively (P) beginning in 2021 (P-BA = 11, P-IC = 9, P-NL = 7) and retrospectively (R) from paraffin-embedded tissue going back to 1981 (R-BA = 25, R-IC = 9, R-NL = 4). The P-cohort had transmission electron microscopy (TEM) to image mitochondria, immunoblotting for heat shock protein 60 (HSP60), and quantitative PCR (qPCR) for HSP60 and mitochondrial functional genes. Both cohorts had immunofluorescence for HSP60 quantified as a ratio to albumin-positive hepatocytes (ALB) with HSP60/ALB<1.0 as a cutoff limit using ImageJ. ResultsHSP60 was significantly lower in BA/IC than NL on qPCR (BA: p < 0.01, IC: p < 0.05) and lower in BA than IC/NL on immunoblotting (p < 0.05). HSP60/ALB was significantly lower in BA than NL/IC (p < 0.001). Despite BA subjects being matched for types of BA and ages at PE, HSP60/ALB did not correlate with jaundice clearance (JC; T-Bil<1.2 mg/dL) but was significantly higher in native liver survivors (NLS) after PE compared with liver transplant (LTx) cases (p < 0.05) and significantly lower in LTx cases achieving JC than NLS achieving JC (p < 0.05). TEM showed BA had significantly more mitochondrial inclusion bodies (p < 0.05) and significantly larger cristae (p < 0.01) than IC/NL. qPCR in BA showed significant repression of mitochondrial functional genes for mRNA stabilization and energy facilitation. ConclusionHSP60/ALB correlates with NLS after PE for BA. Level of evidenceII

The genomic mosaic of mitochondrial dysfunction: Decoding nuclear and mitochondrial epigenetic contributions to maternally inherited diabetes and deafness pathogenesis

AimsMaternally inherited diabetes and deafness (MIDD) is a complex disorder characterized by multiorgan clinical manifestations, including diabetes, hearing loss, and ophthalmic complications. This pilot study aimed to elucidate the intricate interplay between nuclear and mitochondrial genetics, epigenetic modifications, and their potential implications in the pathogenesis of MIDD. Main methodsA comprehensive genomic approach was employed to analyze a Sicilian family affected by clinically characterized MIDD, negative to the only known causative m.3243 A > G variant, integrating whole-exome sequencing and whole-genome bisulfite sequencing of both nuclear and mitochondrial analyses. Key findingsRare and deleterious variants were identified across multiple nuclear genes involved in retinal homeostasis, mitochondrial function, and epigenetic regulation, while complementary mitochondrial DNA analysis revealed a rich tapestry of genetic diversity across genes encoding components of the electron transport chain and ATP synthesis machinery. Epigenetic analyses uncovered significant differentially methylated regions across the genome and within the mitochondrial genome, suggesting a nuanced landscape of epigenetic modulation. SignificanceThe integration of genetic and epigenetic data highlighted the potential crosstalk between nuclear and mitochondrial regulation, with specific mtDNA variants influencing methylation patterns and potentially impacting the expression and regulation of mitochondrial genes. This pilot study provides valuable insights into the complex molecular mechanisms underlying MIDD, emphasizing the interplay between nucleus and mitochondrion, tracing the way for future research into targeted therapeutic interventions and personalized approaches for disease management.

Embryo thermal manipulation enhances mitochondrial function in the skeletal muscle of heat-stressed broilers by regulating transient receptor potential V2 expression

Heat stress induces mitochondrial dysfunction, thereby impeding skeletal muscle development and significantly impacting the economic efficiency of poultry production. This study aimed to investigate the effects of embryo thermal manipulation (TM, 41.5°C, 65% RH, 3 h/d during 16-18th embryonic age) on the mitochondrial function of the pectoralis major (PM) in broiler chickens exposed to thermoneutral (24 ± 1°C, 60% RH) or cyclic heat stress (35 ± 1°C, 60% RH, 12 h/d) from day 22 to 28, and to explore potential mechanisms involving transient receptor potential V2 (TRPV2). Additionally, in vitro experiments were conducted to assess the regulatory effects of TRPV2 pharmacological activation and inhibition on mitochondrial function in primary myotubes. The results revealed that TM had no discernible effect on the body weight and feed intake of broiler chickens under heat stress conditions (P > 0.05). However, it did delay the increase in rectal temperature and accelerate the decrease in serum T3 levels (P < 0.05). Furthermore, TM promoted the development of PM muscle fibers, significantly increasing myofiber diameter and cross-sectional area (P < 0.05). Under heat stress conditions, TM significantly upregulated the expression of mitochondrial electron transport chain (ETC) genes and TRPV2 in broiler PM muscle (P < 0.05), with a clear positive correlation observed between the two (P < 0.05). In vitro, pharmacological activation of TRPV2 not only increased its own expression but also enhanced mitochondrial ETC genes expression and oxidative phosphorylation function by upregulating intracellular calcium ion levels (P < 0.05). Conversely, TRPV2 inhibition had the opposite effect. Overall, this study underscores the potential of prenatal thermal manipulation in regulating postnatal broiler skeletal muscle development and mitochondrial function through the modulation of TRPV2 expression.

Timed topical dexamethasone eye drops improve mitochondrial function to prevent severe retinopathy of prematurity.

Pathological neovascularization in retinopathy of prematurity (ROP) can cause visual impairment in preterm infants. Current ROP treatments which are not preventative and only address late neovascular ROP, are costly and can lead to severe complications. We showed that topical 0.1% dexamethasone eye drops administered prior to peak neovessel formation prevented neovascularization in five extremely preterm infants at high risk for ROP and suppressed neovascularization by 30% in mouse oxygen-induced retinopathy (OIR) modeling ROP. In contrast, in OIR, topical dexamethasone treatment before any neovessel formation had limited efficacy in preventing later neovascularization, while treatment after peak neovessel formation had a non-statistically significant trend to exacerbating disease. Optimally timed topical dexamethasone suppression of neovascularization in OIR was associated with increased retinal mitochondrial gene expression and decreased inflammatory marker expression, predominantly found in immune cells. Blocking mitochondrial ATP synthetase reversed the inhibitory effect of dexamethasone on neovascularization in OIR. This study provides new insights into topical steroid effects in retinal neovascularization and into mitochondrial function in phase II ROP, and suggests a simple clinical approach to prevent severe ROP.

Intravenous calcitriol administration improves the liver redox status and attenuates ferroptosis in mice with high-fat diet-induced obesity complicated with sepsis

Obesity aggravates ferroptosis, and vitamin D (VD) may inhibit ferroptosis. We hypothesized that weight reduction and/or calcitriol administration have benefits against the sepsis-induced liver redox imbalance and ferroptosis in obese mice. Mice were fed a high-fat diet for 11 weeks, then half of the mice continued to consume the diet, while the other half were transferred to a low-energy diet for 5 weeks. After feeding the respective diets for 16 weeks, sepsis was induced by cecal ligation and puncture (CLP). Septic mice were divided into four experimental groups: OS group, obese mice injected with saline; OD group, obese mice with calcitriol; WS group, weight-reduction mice with saline; and WD group, weight-reduction mice with calcitriol. Mice in the respective groups were euthanized at 12 or 24 h after CLP. Results showed that the OS group had the highest inflammatory mediators and lipid peroxide levels in the liver. Calcitriol treatment reduced iron content, enhanced the reduced glutathione/oxidized glutathione ratio, upregulated nuclear factor erythroid 2-related factor 2, ferroptosis-suppressing protein 1, and solute carrier family 7 member 11 expression levels. Also, mitochondrion-associated nicotinamide adenine dinucleotide phosphate oxidase 1, peroxisome proliferator-activated receptor-γ coactivator 1, hypoxia-inducible factor-1α, and heme oxidase-1 expression levels increased in the late phase of sepsis. These results were not noted in the WS group. These findings suggest that calcitriol treatment elicits a more-balanced glutathione redox status, alleviates liver ferroptosis, and enhances mitochondrial biogenesis-associated gene expressions. Weight reduction alone had minimal influences on liver ferroptosis and mitochondrial biogenesis in obese mice with sepsis.