Respiratory Chain Research Articles

Abstract Mo045: The metabolic landscape of Fontan-associated liver disease

Introduction: Congenital heart diseases (CHDs) are the most common birth defects in the U.S. There are growing numbers of infants and children living with single ventricle congenital heart disease (SVCHD), a unique congenital cardiac anomaly in which there is only one functional ventricle. The Fontan surgery is the current standard of care for SVCHD. Although operative survival has improved over the years, it is now evident that these surviving patients are facing new life-threatening challenges: severe complications resulting from the Fontan operation, including one of the most evident consequences-hepatic fibrosis, which is now recognized as Fontan-Associated Liver Disease (FALD). Unfortunately, the mechanisms underlying FALD remain little understood. Improved understanding in this knowledge gap will offer crucial insights into FALD pathophysiology that can lead to targeted therapies for SVCHD patients. Goal: To determine if liver metabolism changes significantly in FALD and compare these liver metabolomics changes to metabolic gene expression (our snRNA-ATAC-seq data) and reported plasma metabolomics results, and validate the key findings. Methods: We recently generated the first single-cell transcriptomic landscape of human FALD using snRNA-ATAC-seq, which revealed profound metabolic reprogramming in FALD. Here we utilized metabolomics to further understand this. Liver samples/biopsies were collected from healthy donors and FALD patients. The extracted metabolites were analyzed using C18 and HILIC HPLC/MS columns. Raw data files were acquired by high resolution MS on Orbitrap and processed by Compound Discoverer. Further statistical and bioinformatics analyses were performed to identify altered metabolites and potential metabolic pathways involved in FALD. Results: Significant changes of metabolite levels of multiple metabolic pathways were observed in Fontan livers, often consistent with the gene expression results. Overall, these data unveiled profound metabolic abnormalities in livers of patients with early-stage FALD. Conclusions: In conclusion, our investigation revealed alterations in metabolites involved in glycolysis/gluconeogenesis, mitochondrial electron transport chain, and amino acids in early-stage FALD. The observed correlations between the metabolomics and gene expression results reveal new insights into the mechanisms underlying FALD. FALD is not only a hepatic fibrosis disease, but also features severe dysregulation of liver metabolism.

Mitochondrial fragmentation in early differentiation of human MB135 myoblasts: Role of mitochondrial ROS production in the absence of depolarization

AimsStudy of the role of mitochondria-generated reactive oxygen species (mtROS) and mitochondrial polarization in mitochondrial fragmentation at the initial stages of myogenesis. Main methodsMitochondrial morphology, Drp1 protein phosphorylation, mitochondrial electron transport chain components content, mtROS and mitochondrial lipid peroxidation levels, and mitochondrial polarization were evaluated on days 1 and 2 of human MB135 myoblasts differentiation. A mitochondria-targeted antioxidant SkQ1 was used to elucidate the effect of mtROS on mitochondria. Key findingsIn immortalized human MB135 myoblasts, mitochondrial fragmentation began on day 1 of differentiation before the myoblast fusion. This fragmentation was preceded by dephosphorylation of p-Drp1 (Ser-637). On day 2, an increase in the content of some mitochondrial proteins was observed, indicating mitochondrial biogenesis stimulation. Furthermore, we found that myogenic differentiation, even on day 1, was accompanied both by an increased production of mtROS, and lipid peroxidation of the inner mitochondrial membrane. SkQ1 blocked these effects and partially reduced the level of mitochondrial fragmentation, but did not affect the dephosphorylation of p-Drp1 (Ser-637). Importantly, mitochondrial fragmentation at early stages of MB135 differentiation was not accompanied by depolarization, as an important stimulus for mitochondrial fragmentation. SignificanceMitochondrial fragmentation during early myogenic differentiation depends on mtROS production rather than mitochondrial depolarization. SkQ1 only partially inhibited mitochondrial fragmentation, without significant effects on mitophagy or early myogenic differentiation.

Abstract Tu055: Prominent effects of p38 MAPK inhibition on the phosphoproteome of a guinea pig model of heart failure and sudden cardiac death

Introduction: The p38 mitogen-activated protein kinase (p38 MAPK), activated by stress, plays a role in cardiac hypertrophy and heart failure (HF). The precise mechanism of action of p38 MAPK remains unclear, which could explain the mixed outcomes in P38 inhibitor trials. Here, we examine the impact of p38 MAPK inhibition on cardiac function and the proteome/phosphoproteome of the failing heart. Objective: To ascertain p38 MAPK’s impact on the progression of HF by inhibiting it with SB203580 (SB) in a guinea pig model of HF and sudden cardiac death. Methods: We assessed the proteomic changes in a previously described guinea pig HF model of ascending aortic constriction and once-daily isoproterenol administration (ACi). Three groups were included: sham-operated controls (Control), ACi, and ACi plus SB treatment (ACiSB). Cardiac function was evaluated by M-Mode echocardiography and proteome/phosphoproteome analysis was performed by multiplexed TMT-labeling and LC-MS/MS. Results: Inhibition of p38 MAPK with SB attenuated but did not fully block HF progression. At 5 weeks, fractional shortening was significantly improved in the ACiSB group compared to ACi (p=0.0037). Furthermore, it significantly decreased cardiac hypertrophy and lung edema (p-values <0.0001 and 0.0003 respectively). Compared to our previous study on the impact of a mitochondrially-targeted antioxidant (MitoTEMPO), SB protected a smaller subset of protein expression changes in HF; However, its impact on the phosphoproteome was substantial, extending beyond known p38 substrates. SB suppressed HF-induced inflammatory pathway proteins, modestly protected against downregulation of some mitochondrial respiratory chain proteins, and prevented maladaptive phosphorylation of several proteins involved in excitation-contraction coupling, including the ryanodine receptor, phospholamban, and cytoskeletal protein phosphorylation. Conclusion: p38 MAPK inhibition offered significant protection against HF, primarily affecting the phosphoproteome with modest effects on protein expression. The data suggests that narrowly targeted p38 MAPK-driven phosphoproteome modifications contribute to cardiac decompensation, despite broad protein expression changes in HF. Differences between MitoTEMPO and SB effects on the HF proteome highlight the plethora of remodeling pathways triggered by mitochondrial ROS including p38 MAPK activation. Our findings emphasize the importance of preventing p38 MAPK to preserve cardiac function.

Abstract Mo108: Sex-specific Impact of Glutathione Precursor-supplemented Diet on the Aging Mouse Heart

Common features of the aging heart include dysregulated metabolism, inflammation, and fibrosis, which can all contribute to diastolic dysfunction. Imbalanced oxidative stress can exacerbate each of these conditions, worsening the age-related cardiac defects. Therefore, we hypothesized that increasing natural antioxidant defenses (glutathione) may favor a healthier cardiac aging phenotype. Twenty-one month-old mice were fed for 12 weeks with a diet supplemented in two glutathione precursors, Glycine and N-Acetyl Cysteine (GlyNAC) or with a control diet. Heart function was measured noninvasively and longitudinally at baseline and then every 6 weeks. We found that GlyNAC reduced the age-associated increase in left atrial volume in old male, but not in old female mice ( 12% ± 3% in GlyNAC vs 20% ± 2% in control males (*); 11% ± 3% in GlyNAC vs 16% ± 3% in control females; * P=0.03). It also improved exercise performance in males. Subsequent unbiased cardiac proteome analysis identified that GlyNAC modestly changed global protein expression in males and pointed to the significant changes in two pathways: mitochondria-metabolism and extracellular matrix. Further analysis performed on mitochondrial extracts determined that GlyNAC diet led to an increase in Ndufb8, a subunit of the mitochondrial respiratory chain complex (1.76±0.3 in GlyNAC males vs 0.7±0.1 in control males, P=0.02). In the GlyNAC old male mitochondria, we found an improved catalytic activity for CPT1b (7.51±0.79 vs 3.74±0.32 in control males; P=0.002) and an increased CrAT activity (1.99±0.23 vs 1.06±0.08 in control males; P=0.005). Both enzymes are involved in fatty acid metabolism. Hearts from GlyNAC-fed old males exhibited enrichment in Fmod, a protein that can inhibit collagen fibril formation (1.08±0.03 vs 0.89±0.05, P=0.01), possibly reducing extracellular matrix stiffness. In females, the expression or enzymatic activities of these proteins were not affected by the GlyNAC-supplemented diet. In summary, our study supports the concept that aged male and female hearts exhibit basic phenotypical differences that may affect the response to interventions. These observations may have direct relevance in cardiac gerontology.

New functions of pirin proteins and a 2-ketoglutarate: Ferredoxin oxidoreductase ortholog in Bacteroides fragilis metabolism and their impact on antimicrobial susceptibility to metronidazole and amixicile.

The understanding of how central metabolism and fermentation pathways regulate antimicrobial susceptibility in the anaerobic pathogen Bacteroides fragilis is still incomplete. Our study reveals that B. fragilis encodes two iron-dependent, redox-sensitive regulatory pirin protein genes, pir1 and pir2. The mRNA expression of these genes increases when exposed to oxygen and during growth in iron-limiting conditions. These proteins, Pir1 and Pir2, influence the production of short-chain fatty acids and modify the susceptibility to metronidazole and amixicile, a new inhibitor of pyruvate: ferredoxin oxidoreductase in anaerobes. We have demonstrated that Pir1 and Pir2 interact directly with this oxidoreductase, as confirmed by two-hybrid system assays. Furthermore, structural analysis using AlphaFold2 predicts that Pir1 and Pir2 interact stably with several central metabolism enzymes, including the 2-ketoglutarate:ferredoxin oxidoreductases Kor1AB and Kor2CDAEBG. We used a series of metabolic mutants and electron transport chain inhibitors to demonstrate the extensive impact of bacterial metabolism on metronidazole and amixicile susceptibility. We also show that amixicile is an effective antimicrobial against B. fragilis in an experimental model of intra-abdominal infection. Our investigation led to the discovery that the kor2AEBG genes are essential for growth and have dual functions, including the formation of 2-ketoglutarate via the reverse TCA cycle. However, the metabolic activity that bypasses the function of Kor2AEBG following the addition of phospholipids or fatty acids remains undefined. Overall, our study provides new insights into the central metabolism of B. fragilis and its regulation by pirin proteins, which could be exploited for the development of new narrow-spectrum antimicrobials in the future.

Controlling Pericellular Oxygen Tension in Cell Culture Reveals Distinct Breast Cancer Responses to Low Oxygen Tensions.

In oxygen (O2)-controlled cell culture, an indispensable tool in biological research, it is presumed that the incubator setpoint equals the O2 tension experienced by cells (i.e., pericellular O2). However, it is discovered that physioxic (5% O2) and hypoxic (1% O2) setpoints regularly induce anoxic (0% O2) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O2 consumption is the driving factor. RNA-seq analysis revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to cellular O2 consumption. A reaction-diffusion model is developed to predict pericellular O2 tension a priori, demonstrating that the effect of cellular O2 consumption has the greatest impact in smaller volume culture vessels. By controlling pericellular O2 tension in cell culture, it is found that hypoxia vs. anoxia induce distinct breast cancer transcriptomic and translational responses, including modulation of the hypoxia-inducible factor (HIF) pathway and metabolic reprogramming. Collectively, these findings indicate that breast cancer cells respond non-monotonically to low O2, suggesting that anoxic cell culture is not suitable for modeling hypoxia. Furthermore, it is shown that controlling atmospheric O2 tension in cell culture incubators is insufficient to regulate O2 in cell culture, thus introducing the concept of pericellular O2-controlled cell culture.

Nervonic acid triggered ovarian inflammation by inducing mitochondrial oxidative stress to activate NLRP3/ IL-1β pathway

IntroductionMetabolic syndrome is a serious public health concern across the globe. However, the typical metabolites and mechanisms underlying the decreased fertility related to metabolic syndrome is still elusive. ObjectivesThe aim of the present study was to explore the typical metabolites and mechanisms underlying the decreased fertility related with metabolic syndrome. MethodsUtilizing metabolomics, a comparative analysis was conducted on fatty acid compositions in various tissues of sows with high and low reproductive performance. Additionally, serum fatty acid compositions in a metabolic syndrome model (obese mice) induced by a high-fat diet (HFD) were investigated to elucidate the lipid metabolites associated with metabolic syndrome. Furthermore, the impact of nervonic acid (NA) on ovarian function was examined using rodent animal models (rats and mice). Through biological techniques such as transcriptomics, CUT&Tag, and analysis of post-translational protein modifications, the molecular mechanisms underlying NA mediated ovarian inflammation were further elucidated based on models utilizing ovarian granulosa cells from pigs, humans, and mice. Finally, validation was performed on ovaries from patients diagnosed with polycystic ovary syndrome. ResultsIn vitro, targeted serum lipidomic analysis revealed that sows with low embryo survival rates exhibited abnormal lipid metabolism characterized by abnormal accumulation of NA in the liver, ovary, and adipose tissue. Additionally, elevated NA levels trigger ovarian inflammation to cause ovarian dysfunction in both sows and rats. Mechanistically, NA induce mitochondrial oxidative stress through inhibiting respiratory chain proteins CYTB and NDFUB8 to activate NLRP3 inflammasome, which triggers procaspase-1 into active caspase-1, and convert the cytokine precursors pro-IL-1β into biologically active IL-1β in ovarian granulosa cells. Notably, we evidenced that NA promotes IL-1β activities by increasing H3K9ac modification level of IL-1β promoter regions and regulating the expression of the transcription factor AP-1. Finally, we found that the decreased expression of CerS2 in ovaries and the increased level of chemokine CXCL14 may be the cause of abnormal NA accumulation. Surprisingly, individuals with polycystic ovary syndrome, obesity, non-alcoholic fatty liver or gestational diabetes mellitus exhibit a high level of serum NA. ConclusionCollectively, our current study suggests that NA is a typical metabolite of metabolic syndrome, which strongly influences the ovarian function and embryo survival and also provides that interfering with mitochondrial ROS production is a potential strong strategy for target solving abnormal NA accumulation.

Impaired energy metabolism and altered brain histoarchitecture characterized by inhibition of glycolysis and mitochondrial electron transport-linked enzymes in rats exposed to diisononyl phthalate

The brain is an energy demanding organ, constituting about 20 % of the body's resting metabolic rate. An efficient energy metabolism is critical to neuronal functions. Glucose serves as the primary essential energy source for the adult brain and plays a critical role in supporting neural growth and development. Endocrine disrupting chemicals (EDCs) such as phthalates has been shown to have a negative impact on neurological functions. The impact of diisononyl phthalate (DiNP) on neural energy transduction using cellular energy metabolizing enzymes as indicators was examined. Over the course of 14 days, eighteen (18) albino rats divided into three groups (1,2 and 3) of six albino rats were given Tween-80/saline, 20 and 200 mg/kg body weight respectively. In the brain, we assessed histological changes as well as activities of selected enzymes of energy metabolism such as the glycolytic pathway, citric acid cycle and mitochondrial electron transport-linked complexes. Activities of the glycolytic and TCA cycle enzymes assayed were significantly decreased except citrate synthase activity with no statistically significant change following the administration of DiNP. Also, respiratory chain complexes (Complex I-IV) activities were significantly reduced when compared to control. DiNP exposure altered the histological integrity of various brain sections. These include degenerated Purkinje neurons, distortion of the granular layer and Purkinje cell layer. Data from this study indicated impaired brain energy metabolism via down-regulation of enzymes of cellular respiration of the glycolytic and oxidative phosphorylation pathways and altered brain histoarchitecture orchestrated by DiNP exposure.

Ectonucleotidases in Ischemia Reperfusion Injury: Unravelling the Interplay With Mitochondrial Dysfunction in Liver Transplantation

Ischemia-reperfusion injury (IRI) profoundly impacts organ transplantation, especially in orthotopic liver transplantation (OLT). Disruption of the mitochondrial respiratory chain during ischemia leads to ATP loss and ROS production. Reperfusion exacerbates mitochondrial damage, triggering the release of damage-associated molecular patterns (DAMPs) and inflammatory responses. Mitochondrial dysfunction, a pivotal aspect of IRI, is explored in the context of the regulatory role of ectonucleotidases in purinergic signaling and immune responses. CD39, by hydrolyzing ATP and ADP; and CD73, by converting AMP to adenosine, emerge as key players in mitigating liver IRI, particularly through ischemic preconditioning and adenosine receptor signaling. Despite established roles in vascular health and immunity, the impact of ectonucleotidases on mitochondrial function during hepatic IRI is unclear. This review aims to elucidate the interplay between CD39/73 and mitochondria, emphasizing their potential as therapeutic targets for liver transplantation.This article explores the role of CD39/73 in tissue hypoxia, emphasizing adenosine production during inflammation. CD39 and CD73 upregulation under hypoxic conditions regulate immune responses, demonstrating protective effects in various organ-specific ischemic models. However, prolonged adenosine activation may have dual effects, beneficial in acute settings but detrimental in chronic hypoxia.Herein, we raise questions about ectonucleotidases influencing mitochondrial function during hepatic IRI, drawing parallels with cancer cell responses to chemotherapy.The review underscores the need for comprehensive research into the intricate interplay between ectonucleotidases, mitochondrial dynamics, and their therapeutic implications in hepatic IRI, providing valuable insights for advancing transplantation outcomes.

Regulation of mitochondrial metabolism by hibernating bear serum: Insights into seasonal metabolic adaptations

Hibernating animals undergo a unique and reversible decrease in their whole-body metabolism, which is often accompanied by a suppression of mitochondrial respiration. However, the precise mechanisms underlying these seasonal shifts in mitochondrial metabolism remain unclear. In this study, the effect of the serum from active and hibernating Japanese black bears on mitochondrial respiration was assessed. Stromal–vascular cells were obtained from bear white adipose tissue and cultured with or without an adipocyte differentiation cocktail. When the oxygen consumption was measured in the presence of bear serum, the hibernating bear serum reduced maximal respiration by 15.5 % (p < 0.05) and spare respiratory capacity by 46.0 % (p < 0.01) in the differentiated adipocytes in comparison to the active bear serum. Similar reductions of 23.4 % (p = 0.06) and 40.6 % (p < 0.05) respectively were observed in undifferentiated cells, indicating the effect is cell type-independent. Blue native PAGE analysis revealed that hibernating bear serum suppressed cellular metabolism independently of the assembly of mitochondrial respiratory chain complexes. RNA-seq analysis identified 1094 differentially expressed genes (fold change>1.5, FDR<0.05) related to insulin signaling and glucose metabolism pathways. These findings suggest that the rapid alterations in mitochondrial metabolism during hibernation are likely induced by a combination of reduced insulin signaling and suppressed mitochondrial function, rather than changes in respiratory complex assembly.

Metabolic Deficits in the Retina of a Familial Dysautonomia Mouse Model.

Neurodegenerative retinal diseases such as glaucoma, diabetic retinopathy, Leber's hereditary optic neuropathy (LHON), and dominant optic atrophy (DOA) are marked by progressive death of retinal ganglion cells (RGC). This decline is promoted by structural and functional mitochondrial deficits, including electron transport chain (ETC) impairments, increased oxidative stress, and reduced energy (ATP) production. These cellular mechanisms associated with progressive optic nerve atrophy have been similarly observed in familial dysautonomia (FD) patients, who experience gradual loss of visual acuity due to the degeneration of RGCs, which is thought to be caused by a breakdown of mitochondrial structures, and a disruption in ETC function. Retinal metabolism plays a crucial role in meeting the elevated energetic demands of this tissue, and recent characterizations of FD patients' serum and stool metabolomes have indicated alterations in central metabolic processes and potential systemic deficits of taurine, a small molecule essential for retina and overall eye health. The present study sought to elucidate metabolic alterations that contribute to the progressive degeneration of RGCs observed in FD. Additionally, a critical subpopulation of retinal interneurons, the dopaminergic amacrine cells, mediate the integration and modulation of visual information in a time-dependent manner to RGCs. As these cells have been associated with RGC loss in the neurodegenerative disease Parkinson's, which shares hallmarks with FD, a targeted analysis of the dopaminergic amacrine cells and their product, dopamine, was also undertaken. One dimensional (1D) proton (1H) nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and retinal histology methods were employed to characterize retinae from the retina-specific Elp1 conditional knockout (CKO) FD mouse model (Pax6-Cre; Elp1LoxP/LoxP). Metabolite alterations correlated temporally with progressive RGC degeneration and were associated with reduced mitochondrial function, alterations in ATP production through the Cahill and mini-Krebs cycles, and phospholipid metabolism. Dopaminergic amacrine cell populations were reduced at timepoints P30-P90, and dopamine levels were 25-35% lower in CKO retinae compared to control retinae at P60. Overall, this study has expanded upon our current understanding of retina pathology in FD. This knowledge may apply to other retinal diseases that share hallmark features with FD and may help guide new avenues for novel non-invasive therapeutics to mitigate the progressive optic neuropathy in FD.

Streptomyces-Secreted Fluvirucin B6 as a Potential Bio-Fungicide for Managing Banana Fusarium Wilt and Mycotoxins and Modulating the Soil Microbial Community Structure.

Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc TR4) is the most destructive soil-borne fungal disease. Until now, there has been a lack of effective measures to control the disease. It is urgent to explore biocontrol agents to control Foc TR4 and the secretion of mycotoxin. In this study, fluvirucin B6 was screened from Streptomyces solisilvae using an activity-guided method. Fluvirucin B6 exhibited strong antifungal activity against Foc TR4 (0.084 mM of EC50 value) and significantly inhibited mycelial growth and spore germination. Further studies demonstrated that fluvirucin B6 could cause the functional loss of mitochondria, the disorder of metabolism of Foc TR4 cells, and the decrease of enzyme activities in the tricarboxylic acid cycle and electron transport chain, ultimately inhibiting mycotoxin metabolism. In a pot experiment, the application of fluvirucin B6 significantly decreased the incidence of banana Fusarium wilt and the amount of Foc TR4 and controlled fungal toxins in the soil. Additionally, fluvirucin B6 could positively regulate the changes in the structure of the banana rhizosphere microbial community, significantly enriching beneficial microbes associated with disease resistance. In summary, this study identifies fluvirucin B6, which plays versatile roles in managing fungal diseases and mycotoxins.

Dissecting structure and function of the monovalent cation/H+ antiporters Mdm38 and Ylh47 in Saccharomyces cerevisiae.

The inner membrane of mitochondria contains numerous ion transporters, including those facilitating H+ transport by the electron transport chain and ATP synthase to maintain membrane potential. Letm1 in the inner membrane of mitochondria in animals functions as a Ca2+/H+ antiporter. However, this study reveals that homologous antiporters in mitochondria of yeast, Mdm38 and Ylh47, do not transport Ca2+ but instead are selective for K+ and Na+. Additionally, the identification of conserved amino acids crucial for antiporter activity further expanded our understanding of the structure and function of the Letm1-Mdm38-Ylh47 antiporter family.

MGA deletion leads to Richter's transformation by modulating mitochondrial OXPHOS.

Richter's transformation (RT) is a progression of chronic lymphocytic leukemia (CLL) to aggressive lymphoma. MGA (Max gene associated), a functional MYC suppressor, is mutated at 3% in CLL and 36% in RT. However, genetic models and molecular mechanisms of MGA deletion that drive CLL to RT remain elusive. We established an RT mouse model by knockout of Mga in the Sf3b1/Mdr CLL model using CRISPR-Cas9 to determine the role of Mga in RT. Murine RT cells exhibited mitochondrial aberrations with elevated oxidative phosphorylation (OXPHOS). Through RNA sequencing and functional characterization, we identified Nme1 (nucleoside diphosphate kinase) as an Mga target, which drives RT by modulating OXPHOS. Given that NME1 is also a known MYC target without targetable compounds, we found that concurrent inhibition of MYC and electron transport chain complex II substantially prolongs the survival of RT mice in vivo. Our results suggest that the Mga-Nme1 axis drives murine CLL-to-RT transition via modulating OXPHOS, highlighting a potential therapeutic avenue for RT.

Esaxerenone Attenuates Aldosterone-Induced Mitochondrial Damage-Mediated Pyroptosis in Mouse Aorta and Rat Vascular Smooth Muscle Cells.

Vascular smooth muscle cell (VSMC) injury caused by the inflammatory response plays a key role in cardiovascular disease (CVD), and the vasoprotective effects of mineralocorticoid receptor blockers (MRBs) support the role of mineralocorticoid receptor (MR) activation. C57BL/6 mice and VSMCs isolated from rats were treated with aldosterone and esaxerenone. Caspase-1, GSDMD-N, IL-1β, and NR3C2 expression and DNA damage in aortic VSMCs were detected using immunohistochemistry, Western blotting, and TUNEL staining. Mitochondrial changes were detected by transmission electron microscopy (TEM). Reactive oxygen species (ROS), MitoTracker, JC-I, mitochondrial respiratory chain complexes I-V, and NR3C2 were detected using immunofluorescence and flow cytometry. Pyroptosis was detected with scanning electron microscopy (SEM). After aldosterone treatment, the number of TUNEL-positive cells increased significantly, and the expression of caspase-1, GSDMD-N, and IL-1β increased. TEM revealed mitochondrial damage, and SEM revealed specific pyroptotic changes, such as cell membrane pore changes and cytoplasmic extravasation. Increased ROS levels and nuclear translocation of NR3C2 were also observed. These pyroptosis-related changes were reversed by esaxerenone. Aldosterone activates the MR and mediates mitochondrial damage, thereby inducing pyroptosis in VSMCs via the NLRP3/caspase-1 pathway. Esaxerenone inhibits MR activation and reduces mitochondrial damage and oxidative stress, thereby inhibiting pyroptosis.

Vimentin regulates mitochondrial ROS production and inflammatory responses of neutrophils.

The intermediate filament vimentin is present in immune cells and is implicated in proinflammatory immune responses. Whether and how it supports antimicrobial activities of neutrophils are not well established. Here, we developed an immortalized neutrophil model to examine the requirement of vimentin. We demonstrate that vimentin restricts the production of proinflammatory cytokines and reactive oxygen species (ROS), but enhances phagocytosis and swarming. We observe that vimentin is dispensable for neutrophil extracellular trap (NET) formation, degranulation, and inflammasome activation. Moreover, gene expression analysis demonstrated that the presence of vimentin was associated with changes in expression of multiple genes required for mitochondrial function and ROS overproduction. Treatment of wild-type cells with rotenone, an inhibitor for complex I of the electron transport chain, increases the ROS levels. Likewise, treatment with mitoTEMPO, a SOD mimetic, rescues the ROS production in cells lacking vimentin. Together, these data show vimentin regulates neutrophil antimicrobial functions and alters ROS levels through regulation of mitochondrial activity.

The Uncoupling Effect of 17β-Estradiol Underlies the Resilience of Female-Derived Mitochondria to Damage after Experimental TBI.

Current literature finds females have improved outcomes over their male counterparts after severe traumatic brain injury (TBI), while the opposite seems to be true for mild TBI. This begs the question as to what may be driving these sex differences after TBI. Estrogen is thought to be neuroprotective in certain diseases, and its actions have been shown to influence mitochondrial function. Mitochondrial impairment is a major hallmark of TBI, and interestingly, this dysfunction has been shown to be more severe in males than females after brain injury. This suggests estrogen could be playing a role in promoting "mitoprotection" following TBI. Despite the existence of estrogen receptors in mitochondria, few studies have examined the direct role of estrogen on mitochondrial function, and no studies have explored this after TBI. We hypothesized ex vivo treatment of isolated mitochondria with 17β-estradiol (E2) would improve mitochondrial function after experimental TBI in mice. Total mitochondria from the ipsilateral (injured) and contralateral (control) cortices of male and female mice were isolated 24 h post-controlled severe cortical impact (CCI) and treated with vehicle, 2 nM E2, or 20 nM E2 immediately before measuring reactive oxygen species (ROS) production, bioenergetics, electron transport chain complex (ETC) activities, and β-oxidation of palmitoyl carnitine. Protein expression of oxidative phosphorylation (OXPHOS) complexes was also measured in these mitochondrial samples to determine whether this influenced functional outcomes with respect to sex or injury. While mitochondrial ROS production was affected by CCI in both sexes, there were other sex-specific patterns of mitochondrial injury 24 h following severe CCI. For instance, mitochondria from males were more susceptible to CCI-induced injury with respect to bioenergetics and ETC complex activities, whereas mitochondria from females showed only Complex II impairment and reduced β-oxidation after injury. Neither concentration of E2 influenced ETC complex activities themselves, but 20 nM E2 appeared to uncouple mitochondria isolated from the contralateral cortex in both sexes, as well as the injured ipsilateral cortex of females. These studies highlight the significance of measuring mitochondrial dysfunction in both sexes after TBI and also shed light on another potential neuroprotective mechanism in which E2 may attenuate mitochondrial dysfunction after TBI in vivo.

Elucidation of the Role of L-Arginine and Nω-Nitro-L-Arginine in the Treatment of Rats with Different Levels of Hypoxic Tolerance and Exposure to Lead Nitrate

Background/Aims: Individual resistance to hypoxia is an important feature of the physiological profile of an organism, particularly in relation to lead-induced toxicity. Methods: Our study focused on evaluating parameters of mitochondrial oxygen consumption, microsomal oxidation, intensity of lipoperoxidation processes and antioxidant defences in the liver of rats with low (LR) and high (HR) resistance to hypoxia to elucidate the mechanisms of action of L-arginine and the NO synthase inhibitor L-NNA before or after exposure to lead nitrate. Results: Our study suggests that the redistribution of oxygen-dependent processes towards mitochondrial processes under the influence of the nitric oxide precursor amino acid L-arginine is an important mechanism for maintaining mitochondrial respiratory chain function during per os lead nitrate exposure (3.6 mg lead nitrate/kg bw per day for 30 days). Animals were given L-arginine at a dose of 600 mg/kg bw (i.p., 30 min) before and after exposure to lead nitrate or the NO synthase inhibitor Nω-nitro-L-arginine (L-NNA) at a dose of 35 mg/kg bw (i.p., 30 min) before and after exposure to lead nitrate. Our experiments demonstrated the efficacy of using lead nitrate to simulate lead-related toxic processes via Pb levels in liver tissue; we demonstrated significantly reduced levels of nitrites and nitrates, i.e. stable metabolites of the nitric oxide system, in both LR and HR animals. The effect of the amino acid L-arginine stabilised the negative effects of lead nitrate exposure in both groups of LR and HR rats. We observed the efficiency of mitochondrial energy supply processes and showed a greater vulnerability of NADH-dependent oxidation during lead nitrate exposure in the liver of HR rats. Conclusion: L-arginine initiated the processes of oxidation of NADH-dependent substrates in the LR group, whereas in the HR group this directionality of processes was more effective when the role of the nitric oxide system was reduced (use of L-NNA). Our study of key antioxidant enzyme activities in rat liver tissue during lead nitrate exposure revealed changes in the catalase-peroxidase activity ratio. We found different activities of antioxidant enzymes in the liver tissue of rats treated with lead nitrate and L-arginine or L-NNA, with a significant increase in GPx activity in the LR group when L-arginine was administered both before and after exposure to lead nitrate.

Acute Metabolic Stress Induces Lymphatic Dysfunction Through KATP Channel Activation.

Lymphatic dysfunction is an underlying component of multiple metabolic diseases, including diabetes, obesity, and metabolic syndrome. We investigated the roles of KATP channels in lymphatic contractile dysfunction in response to acute metabolic stress induced by inhibition of the mitochondrial electron transport chain. Ex vivo popliteal lymphatic vessels from mice were exposed to the electron transport chain inhibitors antimycin A and rotenone, or the oxidative phosphorylation inhibitor/protonophore, CCCP. Each inhibitor led to a significant reduction in the frequency of spontaneous lymphatic contractions and calculated pump flow, without a significant change in contraction amplitude. Contraction frequency was restored by the KATP channel inhibitor, glibenclamide. Lymphatic vessels from mice with global Kir6.1 deficiency or expressing a smooth muscle-specific dominant negative Kir6.1 channel were resistant to inhibition. Antimycin A inhibited the spontaneous action potentials generated in lymphatic muscle and this effect was reversed by glibenclamide, confirming the role of KATP channels. Antimycin A, but not rotenone or CCCP, increased dihydrorhodamine fluorescence in lymphatic muscle, indicating ROS production. Pretreatment with tiron or catalase prevented the effect of antimycin A on wild-type lymphatic vessels, consistent with its action being mediated by ROS. Our results support the conclusion that KATP channels in lymphatic muscle can be directly activated by reduced mitochondrial ATP production or ROS generation, consequent to acute metabolic stress, leading to contractile dysfunction through inhibition of the ionic pacemaker controlling spontaneous lymphatic contractions. We propose that a similar activation of KATP channels contributes to lymphatic dysfunction in metabolic disease.

Metabolic Insights into Neuropsychiatric Illnesses and Ketogenic Therapies: A Transcriptomic View.

The disruption of brain energy metabolism, leading to alterations in synaptic signaling, neural circuitry, and neuroplasticity, has been implicated in severe mental illnesses such as schizophrenia, bipolar disorder, and major depressive disorder. The therapeutic potential of ketogenic interventions in these disorders suggests a link between metabolic disturbances and disease pathology; however, the precise mechanisms underlying these metabolic disturbances, and the therapeutic effects of metabolic ketogenic therapy, remain poorly understood. In this study, we conducted an in silico analysis of transcriptomic data to investigate perturbations in metabolic pathways in the brain across severe mental illnesses via gene expression profiling. We also examined dysregulation of the same pathways in rodent or cell culture models of ketosis, comparing these expression profiles to those observed in the disease states. Our analysis revealed significant perturbations across all metabolic pathways, with the greatest perturbations in glycolysis, the tricarboxylic acid (TCA) cycle, and the electron transport chain (ETC) across all three disorders. Additionally, we observed some discordant gene expression patterns between disease states and ketogenic intervention studies, suggesting a potential role for ketone bodies in modulating pathogenic metabolic changes. Our findings highlight the importance of understanding metabolic dysregulation in severe mental illnesses and the potential therapeutic benefits of ketogenic interventions in restoring metabolic homeostasis. This study provides insights into the complex relationship between metabolism and neuropsychiatric disorders and lays the foundation for further experimental investigations aimed at appreciating the implications of the present transcriptomic findings as well as developing targeted therapeutic strategies.