Altered Mitochondrial Respiration Research Articles

Deletion of Sigmar1 leads to increased arterial stiffness and altered mitochondrial respiration resulting in vascular dysfunction

Sigmar1 is a ubiquitously expressed, multifunctional protein known for its cardioprotective roles in cardiovascular diseases. While accumulating evidence indicate a critical role of Sigmar1 in cardiac biology, its physiological function in the vasculature remains unknown. In this study, we characterized the expression of Sigmar1 in the vascular wall and assessed its physiological function in the vascular system using global Sigmar1 knockout (Sigmar1−/−) mice. We determined the expression of Sigmar1 in the vascular tissue using immunostaining and biochemical experiments in both human and mouse blood vessels. Deletion of Sigmar1 globally in mice (Sigmar1−/−) led to blood vessel wall reorganizations characterized by nuclei disarray of vascular smooth muscle cells, altered organizations of elastic lamina, and higher collagen fibers deposition in and around the arteries compared to wildtype littermate controls (Wt). Vascular function was assessed in mice using non-invasive time-transit method of aortic stiffness measurement and flow-mediated dilation (FMD) of the left femoral artery. Sigmar1−/− mice showed a notable increase in arterial stiffness in the abdominal aorta and failed to increase the vessel diameter in response to reactive-hyperemia compared to Wt. This was consistent with reduced plasma and tissue nitric-oxide bioavailability (NOx) and decreased phosphorylation of endothelial nitric oxide synthase (eNOS) in the aorta of Sigmar1−/− mice. Ultrastructural analysis by transmission electron microscopy (TEM) of aorta sections showed accumulation of elongated shaped mitochondria in both vascular smooth muscle and endothelial cells of Sigmar1−/− mice. In accordance, decreased mitochondrial respirometry parameters were found in ex-vivo aortic rings from Sigmar1 deficient mice compared to Wt controls. These data indicate a potential role of Sigmar1 in maintaining vascular homeostasis.

Altered mitochondrial respiration in peripheral blood mononuclear cells of post-acute sequelae of SARS-CoV-2 infection

Peripheral blood mononuclear cells (PBMC) mitochondrial respiration was measured ex vivo from participants without a history of COVID (n = 19), with a history of COVID and full recovery (n = 20), and with PASC (n = 20). Mean mitochondrial basal respiration, ATP-linked respiration, maximal respiration, spare respiration capacity, ATP-linked respiration, and non-mitochondrial respiration were highest in COVID + PASC+ (p ≤ 0.04). Every unit increase in non-mitochondrial respiration, ATP-linked respiration, basal respiration, spare respiration capacity, and maximal respiration increased the predicted odds of PASC between 1 % and 6 %. Mitochondrial dysfunction in PBMCs may be contributing to the etiology of PASC.

Oocyte and cumulus cell cooperativity and metabolic plasticity under the direction of oocyte paracrine factors.

Mammalian oocytes develop and mature in a mutually dependent relationship with surrounding cumulus cells. The oocyte actively regulates cumulus cell differentiation and function by secreting soluble paracrine oocyte-secreted factors (OSFs). We characterized the molecular mechanisms by which two model OSFs, cumulin and BMP15, regulate oocyte maturation and cumulus-oocyte cooperativity. Exposure to these OSFs during mouse oocyte maturation in vitro altered the proteomic and multispectral autofluorescence profiles of both the oocyte and cumulus cells. In oocytes, cumulin significantly upregulated proteins involved in nuclear function. In cumulus cells, both OSFs elicited marked upregulation of a variety of metabolic processes (mostly anabolic), including lipid, nucleotide, and carbohydrate metabolism, whereas mitochondrial metabolic processes were downregulated. The mitochondrial changes were validated by functional assays confirming altered mitochondrial morphology, respiration, and content while maintaining ATP homeostasis. Collectively, these data demonstrate that cumulin and BMP15 remodel cumulus cell metabolism, instructing them to upregulate their anabolic metabolic processes, while routine cellular functions are minimized in the oocyte during maturation, in preparation for ensuing embryonic development.NEW & NOTEWORTHY Oocyte-secreted factors (OSFs) promote oocyte and cumulus cell cooperativity by altering the molecular composition of both cell types. OSFs downregulate protein catabolic processes and upregulate processes associated with DNA binding, translation, and ribosome assembly in oocytes. In cumulus cells, OSFs alter mitochondrial number, morphology, and function, and enhance metabolic plasticity by upregulating anabolic pathways. Hence, the oocyte via OSFs, instructs cumulus cells to increase metabolic processes on its behalf, thereby subduing oocyte metabolism.

The synergism of cytosolic acidosis and reduced NAD+/NADH ratio is responsible for lactic acidosis-induced vascular smooth muscle cell impairment in sepsis

BackgroundDuring sepsis, serve vascular dysfunctions lead to life-threatening multiple organ failure, due to vascular smooth muscle cells (VSMC) impairments, resulting in vasoplegia, hypotension and hypoperfusion. In addition, septic patients have an altered cell metabolism that leads to lactic acidosis. Septic patients suffering from lactic acidosis have a high risk of mortality. In addition, septic survivors are at risk of secondary vascular disease. The underlying mechanisms of whether and how lactic acidosis leads to the changes in VSMCs is not well understood. The aim of this study was to comprehensively investigate the effect of lactic acidosis on VSMCs and additionally compare the effects with those induced by pure acidosis and sodium lactate.MethodsPrimary human aortic smooth muscle cells (HAoSMCs) were treated for 48 h with lactic acidosis (LA_pH 6.8), hydrochloric acid (HCl_pH 6.8), sodium lactate (Na+-lactate_pH 7.4) and the respective controls (ctrl._pH 7.4; hyperosmolarity control: mannitol_pH 7.4) and comparatively analyzed for changes in (i) transcriptome, (ii) energy metabolism, and (iii) phenotype.ResultsBoth types of acidosis led to comparable and sustained intracellular acidification without affecting cell viability. RNA sequencing and detailed transcriptome analysis revealed more significant changes for lactic acidosis than for hydrochloric acidosis, with lactate being almost ineffective, suggesting qualitative and quantitative synergism of acidosis and lactate. Bioinformatic predictions in energy metabolism and phenotype were confirmed experimentally. Lactic acidosis resulted in strong inhibition of glycolysis, glutaminolysis, and altered mitochondrial respiration which reduced cellular ATP content, likely due to increased TXNIP expression and altered NAD+/NADH ratio. Hydrochloric acidosis induced significantly smaller effects without changing the NAD+/NADH ratio, with the ATP content remaining constant. These metabolic changes led to osteo-/chondrogenic/senescent transdifferentiation of VSMCs, with the effect being more pronounced in lactic acidosis than in pure acidosis.ConclusionsOverall, lactic acidosis exerted a much stronger effect on energy metabolism than pure acidosis, whereas lactate had almost no effect, reflecting the qualitative and quantitative synergism of acidosis and lactate. As a consequence, lactic acidosis may lead to acute functional impairments of VSMC, sustained perturbations of the transcriptome and cellular dedifferentiation. Moreover, these effects may contribute to the acute and prolonged vascular pathomechanisms in septic patients.

Mitochondrial dynamics and myocardial metabolism alterations during anthracycline-cardiotoxicity

Abstract Background Despite the increased effectiveness of chemotherapeutic agents, cancer represents a lethal illness worldwide and anticancer strategies still present severe adverse effects. Cardiac dysfunction is a dose-dependent common affliction of anthracyclines treatment. Anthracycline-induced cardiotoxicity (AIC) and consequent heart failure has recently been suggested to be mediated by mitochondrial dysfunction; however, the molecular mechanisms driving this connection are incompletely understood. Purpose To study the early molecular changes (before onset of overt heart failure phenotype) related to mitochondrial biology and cardiac metabolism in a mouse model of AIC. Methods CD1 mice underwent 5 weekly i.p. injections (5 mg/kg) of doxorubicin. Cardiac anatomic and functional changes were evaluated by echocardiography at 1, 9 and 15 weeks-after last doxorubicin injection. At each time-point, some animals were sacrificed and hearts were collected for characterisation of mitochondrial dynamics and metabolic substrate utilization by western blot, PCR, immunocytochemistry, high-resolution respirometry, transmission electron microscopy and PET/CT. Results Doxorubicin administration resulted in an early reduction in left ventricle (LV) mass which was the result of marked cardiomyocyte atrophy as early as 1 week after doxorubicin completion. Cardiac atrophy preceded functional abnormalities, which were apparent in the form of reduction in LV ejection fraction at 9 weeks. Mitochondrial dynamics (fusion/fission) and membranes were altered as early as 1 week after doxorubicin completion (i.e. long before changes in LV function). Moreover, mitochondrial respiratory rate was decreased in these animals, together with a decrease in mitochondrial DNA content and biogenesis at early timepoints. Early (1 week) mitochondrial molecular changes were accompanied by altered cardiac metabolism (i.e. switch in substrate utilization to glucose). Conclusions Here we have characterized the dynamics of molecular, anatomical and functional changes during AIC. LV systolic dysfunction is a late phenomenon happening long after molecular and metabolic changes in the heart. Cardiomyocyte atrophy, altered mitochondrial dynamics and respiration, and metabolic switching occur long before overt heart failure phenotype. These early changes appear as promising therapeutic targets to prevent overt AIC and heart failure.

Nicotine affects mitochondrial structure and function in human airway smooth muscle cells.

Exposure to cigarette smoke and e-cigarettes, with nicotine as the active constituent, contributes to increased health risks associated with asthma. Nicotine exerts its functional activity via nicotinic acetylcholine receptors (nAChRs), and the alpha7 subtype (α7nAChR) has recently been shown to adversely affect airway dynamics. The mechanisms of α7nAChR action in airways, particularly in the context of airway smooth muscle (ASM), a key cell type in asthma, are still under investigation. Mitochondria have garnered increasing interest for their role in regulating airway tone and adaptations to cellular stress. Here mitochondrial dynamics such as fusion versus fission, and mitochondrial Ca2+ ([Ca2+]m), play an important role in mitochondrial homeostasis. There is currently no information on effects and mechanisms by which nicotine regulates mitochondrial structure and function in ASM in the context of asthma. We hypothesized that nicotine disrupts mitochondrial morphology, fission-fusion balance, and [Ca2+]m regulation, with altered mitochondrial respiration and bioenergetics in the context of asthmatic ASM. Using human ASM (hASM) cells from nonasthmatics, asthmatics, and smokers, we examined the effects of nicotine on mitochondrial dynamics and [Ca2+]m. Fluorescence [Ca2+]m imaging of hASM cells with rhod-2 showed robust responses to 10 μM nicotine, particularly in asthmatics and smokers. In both asthmatics and smokers, nicotine increased the expression of fission proteins while decreasing fusion proteins. Seahorse analysis showed blunted oxidative phosphorylation parameters in response to nicotine in these groups. α7nAChR siRNA blunted nicotine effects, rescuing [Ca2+]m, changes in mitochondrial structural proteins, and mitochondrial dysfunction. These data highlight mitochondria as a target of nicotine effects on ASM, where mitochondrial disruption and impaired buffering could permit downstream effects of nicotine in the context of asthma.NEW & NOTEWORTHY Asthma is a major healthcare burden, which is further exacerbated by smoking. Recognizing the smoking risk of asthma, understanding the effects of nicotine on asthmatic airways becomes critical. Surprisingly, the mechanisms of nicotine action, even in normal and especially asthmatic airways, are understudied. Accordingly, the goal of this research is to investigate how nicotine influences asthmatic airways in terms of mitochondrial structure and function, via the a7nAChR.

Analysis of oxygen consumption rates in zebrafish reveals differences based on sex, age and physical activity recovery.

Introduction: Mitochondrial dysfunction is linked to a variety of human diseases. Understanding the dynamic alterations in mitochondrial respiration at various stages of development is important to our understanding of disease progression. Zebrafish provide a system for investigating mitochondrial function and alterations during different life stages. The purpose of this study was to investigate our ability to measure mitochondrial oxygen consumption rates in zebrafish embryos, larvae, and adults as an indicator of mitochondrial function. Methods: Basal respiration of entire zebrafish embryos (5dpf), larvae (0.6-0.9cm), young adults (3-month-old), and old adults (12-month-old) was measured using an Oroboros Oxygraph, with a stirrer speed of 26rpm. For embryos and larvae, "leak" respiration (plus oligomycin), maximum respiration (plus uncoupler), non-mitochondrial respiration (plus inhibitors), and complex IV activity were also measured. To induce physical activity in adult fish, the stirrer speed was increased to 200rpm. Results and Discussion: We demonstrate the ability to accurately measure respiration rates in zebrafish at various ages using the Oroboros Oxygraph. When comparing zebrafish embryos to larvae, embryos have a higher maximum respiration. Three-month-old zebrafish males have higher basal respiration than females, while 12-month-old zebrafish females exhibit greater rates of respiration than males and younger females. When the stirrer speed was increased, respiration rates decrease, but with differences depending on sex. This study demonstrates a simple and accessible method to assess zebrafish physiology by mitochondrial oxygen consumption measurements in an unmodified Oroboros Oxygraph. The method should facilitate studies to understand the intricate interplay between mitochondrial function, development, and aging.

CHCHD2 mediates glioblastoma cell proliferation, mitochondrial metabolism, hypoxia‑induced invasion and therapeutic resistance.

Glioblastoma (GBM) is the most common and malignant primary brain tumor affecting adults and remains incurable. The mitochondrial coiled‑coil‑helix‑coiled‑coil‑helix domain‑containing protein 2 (CHCHD2) has been demonstrated to mediate mitochondrial respiration, nuclear gene expression and cell migration; however, evidence of this in GBM is lacking. In the present study, it was hypothesized that CHCHD2 may play a functional role in U87 GBM cells expressing the constitutively active epidermal growth factor receptor variant III (EGFRvIII). The amplification of the CHCHD2 gene was found to be associated with a decreased patient overall and progression‑free survival. The CHCHD2 mRNA levels were increased in high‑vs. low‑grade glioma, IDH‑wt GBMs, and in tumor vs. non‑tumor tissue. Additionally, CHCHD2 protein expression was greatest in invasive, EGFRvIII‑expressing patient‑derived samples. The CRISPR‑Cas9‑mediated knockout of CHCHD2 in EGFRvIII‑expressing U87 cells resulted in an altered mitochondrial respiration and glutathione status, in decreased cell growth and invasion under both normoxic and hypoxic conditions, and in an enhanced sensitivity to cytotoxic agents. CHCHD2 was distributed in both the mitochondria and nuclei of U87 and U87vIII cells, and the U87vIII cells exhibited a greater nuclear expression of CHCHD2 compared to isogenic U87 cells. Incubation under hypoxic conditions, serum starvation and the reductive unfolding of CHCHD2 induced the nuclear accumulation of CHCHD2 in both cell lines. Collectively, the findings of the present study indicate that CHCHD2 mediates a variety of GBM characteristics, and highlights mitonuclear retrograde signaling as a pathway of interest in GBM cell biology.

Cell type and sex specific mitochondrial phenotypes in iPSC derived models of Alzheimer's disease.

Mitochondrial dysfunction is observed in Alzheimer's disease (AD). Altered mitochondrial respiration, cytochrome oxidase (COX) Vmax, and mitophagy are observed in human subjects and animal models of AD. Models derived from induced pluripotent stem cells (iPSCs) may not recapitulate these phenotypes after reprogramming from differentiated adult cells. We examined mitochondrial function across iPSC derived models including cerebral organoids, forebrain neurons, and astrocytes. iPSCs were reprogrammed from fibroblasts either from the University of Kansas Alzheimer's Disease Research Center (KU ADRC) cohort or purchased from WiCell. A total of four non-demented and four sporadic AD iPSC lines were examined. Models were subjected to mitochondrial respiration analysis using Seahorse XF technology, spectrophotometric cytochrome oxidase (COX) Vmax assays, fluorescent assays to determine mitochondrial mass, mitochondrial membrane potential, calcium, mitochondrial dynamics, and mitophagy levels. AD pathological hallmarks were also measured. iPSC derived neurons and cerebral organoids showed reduced COX Vmax in AD subjects with more profound defects in the female cohort. These results were not observed in astrocytes. iPSC derived neurons and astrocytes from AD subjects had reduced mitochondrial respiration parameters with increased glycolytic flux. iPSC derived neurons and astrocytes from AD subjects showed sex dependent effects on mitochondrial membrane potential, mitochondrial superoxide production, and mitochondrial calcium. iPSC derived neurons from AD subjects had reduced mitochondrial localization in lysosomes with sex dependent effects on mitochondrial mass, while iPSC derived astrocytes from female AD subjects had increased mitochondrial localization to lysosomes. Both iPSC derived neurons and astrocytes from AD subjects showed altered mitochondrial dynamics. iPSC derived neurons had increased secreted Aβ, and sex dependent effects on total APP protein expression. iPSC derived astrocytes showed sex dependent changes in GFAP expression in AD derived cells. Overall, iPSC derived models from AD subjects show mitochondrial phenotypes and AD pathological hallmarks in a cell type and sex dependent manner. These results highlight the importance of sex as a biological variable in cell culture studies.

Abstract P3200: SH3BGR Affects Energy Metabolism In Neonatal Rat Cardiomyocytes

Background: The SH3 domain-binding glutamic acid-rich (SH3BGR) gene maps to the genetic fragment associated with Down's syndrome (DS), one of the predominant causes of congenital heart diseases (CHD). We have previously shown that SH3BGR augments cardiomyocyte apoptosis, however, its possible role in cardiomyocyte metabolism is not known yet. Methods: Functional effects of SH3BGR knockdown or overexpression were studied in neonatal rat ventricular cardiomyocytes (NRVCMs) using seahorse metabolic assays, quantitative real-time PCR, immunoblotting, and immunofluorescence microscopy. Results: Since we found significantly altered cardiac levels of SH3BGR in human patients suffering from cardiac hypertrophy, which is reportedly associated with mitochondrial dysfunction, we determined if loss- or gain-of-function of SH3BGR impacts mitochondrial metabolism. We observed significant reduction in oxidative phosphorylation complexes and associated genes. Interestingly, seahorse metabolic activity assays revealed impairment in mitochondrial bioenergetics by both knockdown and overexpression of SH3BGR suggesting the importance of its stead-state levels in cardiomyocyte mitochondrial metabolism. NRVCMs showed inability to meet high energy demand on altered SH3BGR expression indicated by higher proton leak and impeded ATP production. These observations suggest altered mitochondrial respiration, which was further supported by alterations in the expression levels of respective metabolic genes. Combining these results with our previous findings suggests that SH3BGR plays an important role in cardiomyocyte oxidative phosphorylation, cell viability and apoptosis. Conclusion: Our data provide novel insights into potential role of SH3BGR in cardiomyocyte mitochondrial bioenergetics. These findings will help us understand how genetic deletion or SH3BGR mutations are associated with CHD e.g., using CRISPR/CAS mediated genetic manipulations using human patient derived induced pluripotent stem cells.

Computational Modeling of Substrate-Dependent Mitochondrial Respiration and Bioenergetics in the Heart and Kidney Cortex and Outer Medulla.

Integrated computational modeling provides a mechanistic and quantitative framework to characterize alterations in mitochondrial respiration and bioenergetics in response to different metabolic substrates in-silico. These alterations play critical roles in the pathogenesis of diseases affecting metabolically active organs such as heart and kidney. Therefore, the present study aimed to develop and validate thermodynamically constrained integrated computational models of mitochondrial respiration and bioenergetics in the heart and kidney cortex and outer medulla (OM). The models incorporated the kinetics of major biochemical reactions and transport processes as well as regulatory mechanisms in the mitochondria of these tissues. Intrinsic model parameters such as Michaelis-Menten constants were fixed at previously estimated values, while extrinsic model parameters such as maximal reaction and transport velocities were estimated separately for each tissue. This was achieved by fitting the model solutions to our recently published respirometry data measured in isolated rat heart and kidney cortex and OM mitochondria utilizing various NADH- and FADH2-linked metabolic substrates. The models were validated by predicting additional respirometry and bioenergetics data, which were not used for estimating the extrinsic model parameters. The models were able to predict tissue-specific and substrate-dependent mitochondrial emergent metabolic system properties such as redox states, enzyme and transporter fluxes, metabolite concentrations, membrane potential, and respiratory control index under diverse physiological and pathological conditions. The models were also able to quantitatively characterize differential regulations of NADH- and FADH2-linked metabolic pathways, which contribute differently toward regulations of oxidative phosphorylation and ATP synthesis in the heart and kidney cortex and OM mitochondria.

Transcriptomic profiling of reward and sensory brain areas in perinatal fentanyl exposed juvenile mice.

Use of the synthetic opioid fentanyl increased ~300% in the last decade, including among women of reproductive ages. Adverse neonatal outcomes and long-term behavioral disruptions are associated with perinatal opioid exposure. Our previous work demonstrated that perinatal fentanyl exposed mice displayed enhanced negative affect and somatosensory circuit and behavioral disruptions during adolescence. However, little is known about molecular adaptations across brain regions that underlie these outcomes. We performed RNA sequencing across three reward and two sensory brain areas to study transcriptional programs in perinatal fentanyl exposed juvenile mice. Pregnant dams received 10 μg/ml fentanyl in the drinking water from embryonic day 0 (E0) through gestational periods until weaning at postnatal day 21 (P21). RNA was extracted from nucleus accumbens (NAc), prelimbic cortex (PrL), ventral tegmental area (VTA), somatosensory cortex (S1) and ventrobasal thalamus (VBT) from perinatal fentanyl exposed mice of both sexes at P35. RNA sequencing was performed, followed by analysis of differentially expressed genes (DEGs) and gene co-expression networks. Transcriptome analysis revealed DEGs and gene modules significantly associated with exposure to perinatal fentanyl in a sex-wise manner. The VTA had the most DEGs, while robust gene enrichment occurred in NAc. Genes enriched in mitochondrial respiration were pronounced in NAc and VTA of perinatal fentanyl exposed males, extracellular matrix (ECM) and neuronal migration enrichment were pronounced in NAc and VTA of perinatal fentanyl exposed males, while genes associated with vesicular cycling and synaptic signaling were markedly altered in NAc of perinatal fentanyl exposed female mice. In sensory areas from perinatal fentanyl exposed females, we found alterations in mitochondrial respiration, synaptic and ciliary organization processes. Our findings demonstrate distinct transcriptomes across reward and sensory brain regions, with some showing discordance between sexes. These transcriptome adaptations may underlie structural, functional, and behavioral changes observed in perinatal fentanyl exposed mice.

Mitochondrial Phenotypes in iPSC AD Models

AbstractBackgroundMitochondrial dysfunction is observed in Alzheimer’s disease (AD). Altered mitochondrial respiration, cytochrome oxidase (COX) Vmax, and mitophagy are observed in human subjects and animal models of AD. Models derived from induced pluripotent stem cells (iPSCs) may not recapitulate these phenotypes after reprogramming from differentiated adult cells. We examined mitochondrial function across iPSC derived models including cerebral organoids, forebrain neurons, and astrocytes. Postmortem brain tissue was used as a comparison.MethodiPSCs were reprogrammed from fibroblasts either from the University of Kansas Alzheimer’s Disease Research Center (KU ADRC) cohort or purchased from WiCell. Postmortem brain samples were from the KU ADRC cohort when available. A total of four non‐demented and four sporadic AD iPSC lines were examined. Postmortem brain tissue was derived from 9 ND and 12 AD subjects. iPSCs were differentiated into neurons, astrocytes, or cerebral organoids using StemCell Technologies protocols and reagents. iPSC derived models and postmortem brain tissue were subjected to mitochondrial respiration analysis using Seahorse XF technology and spectrophotometric COX Vmax assays. iPSC derived neurons and astrocytes underwent fluorescent assays to determine mitochondrial mass, mitochondrial membrane potential, and mitophagy levels.ResultiPSC derived neurons and cerebral organoids showed reduced COX Vmax in AD subjects. These results were not observed in astrocytes. Postmortem human brain samples showed reduced COX Vmax in AD subjects. iPSC derived neurons had reduced mitochondrial respiration parameters, mitochondrial mass, mitochondrial membrane potential, and mitochondrial superoxide production. iPSC derived astrocytes had reduced mitochondrial respiration parameters but increased mitochondrial membrane potential and no change in mitochondrial superoxide production.ConclusioniPSC derived models from AD subjects show mitochondrial dysfunction phenotypes like what is observed in postmortem brain. As iPSCs do not maintain their epigenetic signatures after reprogramming the observed phenotypes are likely due to other somatic factors.

Compartmentalized activities of HMGCS1 control cervical cancer radiosensitivity

The underlying mechanisms by which cellular metabolism affects cervical cancer cell radiosensitivity remain poorly understood. Here, we found that loss of 3-hydroxy-3-methylglutaryl coenzyme A synthase 1 (HMGCS1), a key enzyme catalyzing the conversion of acetoacetyl-CoA to HMG-CoA in the cholesterol biosynthesis pathway, sensitizes the cervical cancer cells to radiation. We observed a compartmentalized cellular distribution of HMGCS1 in nuclei, cytosol, and mitochondria of cervical cancer cells and found that cytosolic HMGCS1 and mitochondrial HMGCS1 contribute together to the regulation of radiosensitivity. Mechanistically, we show that cytosolic HMGCS1 regulates radiosensitivity via manipulating the cholesterol metabolism, while mitochondrial HMGCS1 controls mitochondrial gene expression, thereby sustaining the mitochondrial function of cervical cancer cells. Together, our study identifies HMGCS1 as a novel regulator of radiosensitivty in cervical cancer cells, providing a molecular link between altered cholesterol metabolism, mitochondrial respiration, and radiosensitivity. Thus, targeting HMGCS1 may improve the therapeutic outcome of cervical cancer radiotherapy.

Transcriptomic profiles of muscular dystrophy with myositis (mdm) in extensor digitorum longus, psoas, and soleus muscles from mice

BackgroundTitinopathies are inherited muscular diseases triggered by genetic mutations in the titin gene. Muscular dystrophy with myositis (mdm) is one such disease caused by a LINE repeat insertion, leading to exon skipping and an 83-amino acid residue deletion in the N2A-PEVK region of mouse titin. This region has been implicated in a number of titin—titin ligand interactions, hence are important for myocyte signaling and health. Mice with this mdm mutation develop a severe and progressive muscle degeneration. The range of phenotypic differences observed in mdm mice shows that the deletion of this region induces a cascade of transcriptional changes extending to numerous signaling pathways affected by the titin filament. Previous research has focused on correlating phenotypic differences with muscle function in mdm mice. These studies have provided understanding of the downstream physiological effects resulting from the mdm mutation but only provide insights on processes that can be physiologically observed and measured. We used differential gene expression (DGE) to compare the transcriptomes of extensor digitorum longus (EDL), psoas and soleus muscles from wild-type and mdm mice to develop a deeper understand of these tissue-specific responses.ResultsThe overall expression pattern observed shows a well-differentiated transcriptional signature in mdm muscles compared to wild type. Muscle-specific clusters observed within the mdm transcriptome highlight the level of variability of each muscle to the deletion. Differential gene expression and weighted gene co-expression network analysis showed a strong directional response in oxidative respiration-associated mitochondrial genes, which aligns with the poor shivering and non-shivering thermogenesis previously observed. Sln, which is a marker associated with shivering and non-shivering thermogenesis, showed the strongest expression change in fast-fibered muscles. No drastic changes in MYH expression levels were reported, which indicated an absence of major fiber-type switching events. Overall expression shifts in MYH isoforms, MARPs, and extracellular matrix associated genes demonstrated the transcriptional complexity associated with mdm mutation. The expression alterations in mitochondrial respiration and metabolism related genes in the mdm muscle dominated over other transcriptomic changes, and likely account for the late stage cellular responses in the mdm muscles.ConclusionsWe were able to demonstrate that the complex nature of mdm mutation extends beyond a simple rearrangement in titin gene. EDL, psoas and soleus exemplify unique response modes observed in skeletal muscles with mdm mutation. Our data also raises the possibility that failure to maintain proper energy homeostasis in mdm muscles may contribute to the pathogenesis of the degenerative phenotype in mdm mice. Understanding the full disease-causing molecular cascade is difficult using bulk RNA sequencing techniques due to intricate nature of the disease. The development of the mdm phenotype is temporally and spatially regulated, hence future studies should focus on single fiber level investigations.

1367-P: A Submembrane Cytoskeleton-Dependent Mechanism Modulates Mitochondrial Homeostasis and Skeletal Muscle Bioenergetics

Human variants in the ubiquitously expressed cytoskeletal scaffolding protein Ankyrin B (AnkB) , which is enriched in skeletal muscle (SKM) , have been identified as risk factors for diabetes and obesity. Mice harboring pathogenic variants exhibit reductions in levels of the predominant 220-kDa AnkB isoform in several tissues, including SKM and impaired systemic glucose regulation. To understand the specific metabolic role of AnkB in SKM, we developed a conditional knock-out mouse in which 220-kDa AnkB is knocked out only in SKM (SKM-AnkB-KO) . SKM-AnkB-KO mice display glucose mishandling that persists with age, as well as decreases in activity and exercise capacity, measured by indirect calorimetry and voluntary running wheel analysis. Histological evaluations of aged SKM-AnkB-KO mice showed general disorganization of muscle fibers and the presence of centralized nuclei. This is accompanied by increases in lean muscle mass and in the cross-sectional area of mitochondria-rich soleus muscle, suggesting the development of muscle hypertrophy over time. Electron microscopy analysis revealed the presence of hyperfused mitochondria in SKM-AnkB-KO mice. This morphological change was concomitant with up-regulation of transcripts in pathways associated with energetic stress, including AMPK signaling, redox signaling and mTOR signaling, assessed by RNA-seq, and with alterations in mitochondrial respiration in isolated muscle fibers. Interestingly, SKM-AnkB-KO mice mi SKM-AnkB-KO mice also exhibit increases in protein levels of electron transport chain proteins. Through proteomics and biochemical analysis, we uncovered that AnkB directly associates with several mitochondrial proteins and co-fractionates with mitochondrial in SKM. Together, these data suggest a direct role of AnkB in SKM mitochondrial metabolism that is distinct from its previously characterized role in insulin signaling in other tissues and cytoskeletal organization in SKM. Disclosure K.Voos: n/a. J.Tzeng: None. H.Choi: None. T.Pharr: None. D.Lorenzo: None. Funding American Diabetes Association; National Institutes of Health (1T32GM119999-01A1)

A diabetic milieu increases ACE2 expression and cellular susceptibility to SARS-CoV-2 infections in human kidney organoids and patient cells

SummaryIt is not well understood why diabetic individuals are more prone to develop severe COVID-19. To this, we here established a human kidney organoid model promoting early hallmarks of diabetic kidney disease development. Upon SARS-CoV-2 infection, diabetic-like kidney organoids exhibited higher viral loads compared with their control counterparts. Genetic deletion of the angiotensin-converting enzyme 2 (ACE2) in kidney organoids under control or diabetic-like conditions prevented viral detection. Moreover, cells isolated from kidney biopsies from diabetic patients exhibited altered mitochondrial respiration and enhanced glycolysis, resulting in higher SARS-CoV-2 infections compared with non-diabetic cells. Conversely, the exposure of patient cells to dichloroacetate (DCA), an inhibitor of aerobic glycolysis, resulted in reduced SARS-CoV-2 infections. Our results provide insights into the identification of diabetic-induced metabolic programming in the kidney as a critical event increasing SARS-CoV-2 infection susceptibility, opening the door to the identification of new interventions in COVID-19 pathogenesis targeting energy metabolism.

Dysregulated Mitochondrial Respiration Occurs With Aging

Numerous metabolic changes occur in skeletal muscle with aging. At the crossroads of these changes lies the cellular energy highway, the mitochondrial reticulum. Aging is associated with altered patterns of mitochondrial dynamics and morphology, which are thought to be primarily driven by mitochondrial fragmentation. This fragmentation phenomenon may also lead to altered mitochondrial respiration with aging. We used a mouse model and measured parameters of respiration in muscle mitochondrial preparations to evaluate the hypothesis of aging‐related effects on metabolism and energy substrate partitioning. Mixed skeletal muscle of C57/Bl6 mice (n=32) was excised from single hind‐limbs of young males (YM) (n=8), old males (OM) (n=8), young females (YF) (n=8) and old females (OF) (n=8). Tissues were homogenized in an isotonic solution supplemented with 0.15% protease inhibitor. Mitochondria were isolated by differential centrifugation and their concentrations were determined by BCA analysis. Isolated mitochondria (0.1mg of mitochondria/mL of respiration medium) were then respired using a Clarke‐Type Electrode at 25°C with 100 nM of ADP and either 10 mM Pyruvate+ 2.5 mM Malate (P+M) or 40 uM Palmitoyl‐L‐Carnitine+ 1 mM Malate (PC+M) as substrates. Respiratory Control Ratios (RCR) were calculated as the quotient of State 3 (ADP‐stimulated respiration) and State 4 (recovery rate post ATP synthesis) respiratory rates (State 3/State 4) and compared between substrate and age. ADP to oxygen ratios (P:O) were calculated by dividing the amount of ADP added to the respiratory chamber by the oxygen consumption rate (nmol of O2/mg of mitochondria/minute) of state 3 respiration. RCR in both P+M and PC+M significantly decreased with aging [P+M (p<0.05): YM (4.1±0.9) vs. OM (2.7±1.1); YF (5.1±1.4) vs. OF (3.2±2)] [PC+M (p<0.05): YM (4.2±0.9) vs. OM (2.4±0.7); YF (3.4.±0.5) vs. OF (2.2±0.9)]. P:O in both P+M and PC+M did not significantly change with aging [P+M: (p=0.69): YM (2.9±0.3) vs. OM (2.9±0.2); YF: (p=0.07): (3±0.1) vs. OF (2.9±0.1)] [PC+M:(p=0.65); YM (2.9±0.4) vs. OM (2.9±0.3); (p=0.22): YF (2.9±0.2) vs. OF (2.7±0.3)]. While there were no significant changes in the P:O the age related decreases in the RCR were characterized by a lower capacity to respire derivatives of both CHO and FA. Decreased State 3 respiration and RCR typically corresponds to loss of respiratory capacity and loosening of coupling efficiency. Because exercise increases mitochondrial function and biogenesis it may be prudent to study older animals after exercise training. We plan to further investigate the structural changes in the mitochondrial reticulum and proteome that may be altering mitochondrial function in aging.

Dinitramine induces implantation failure by cell cycle arrest and mitochondrial dysfunction in porcine trophectoderm and luminal epithelial cells

The herbicide market is growing rapidly, as weed control is a significant challenge in agriculture. Many studies have reported the toxicity of herbicides to non-target organisms. Dinitramine is a dinitroaniline herbicide that is particularly toxic to aquatic organisms. However, little is known about the effects of dinitramine on the female reproductive system. Therefore, in the present study, we utilized porcine trophectoderm (pTr) cells and porcine endometrial luminal epithelial (pLE) cells to verify the reproductive toxicity of dinitramine. Dinitramine reduced the viability of both cell types, by triggering cell cycle arrest, especially at the sub-G1 phase, and increasing apoptosis, inhibiting DNA replication. Dinitramine disrupted intracellular calcium homeostasis and induced oxidative stress by producing reactive oxygen species, leading to the loss of mitochondrial membrane potential and alteration of mitochondrial respiration. Mitogen-activated protein kinase pathways were altered, and migration decreased in pTr and pLE cells after dinitramine treatment; the expression of pregnancy-related genes in these cells was decreased. Thus, dinitramine reduced the viability and migratory capacity of both cell types, and this could interrupt the early stages of pregnancy.

Distinct effects of interleukin-6 and interferon-γ on differentiating human cortical neurons

Translational evidence suggests that cytokines involved in maternal immune activation (MIA), such as interleukin-6 (IL-6) and interferon-γ (IFN-γ), can cross the placenta, injure fetal brain, and predispose to neuropsychiatric disorders. To elaborate developmental neuronal sequelae of MIA, we differentiated human pluripotent stem cells to cortical neurons over a two-month period, exposing them to IL-6 or IFN-γ. IL-6 impacted expression of genes regulating extracellular matrix, actin cytoskeleton and TGF-β signaling while IFN-γ impacted genes regulating antigen processing, major histocompatibility complex and endoplasmic reticulum biology. IL-6, but not IFN-γ, altered mitochondrial respiration while IFN-γ, but not IL-6, induced reduction in dendritic spine density. Pre-treatment with folic acid, which has known neuroprotective and anti-inflammatory properties, ameliorated IL-6 effects on mitochondrial respiration and IFN-γ effects on dendritic spine density. These findings suggest distinct mechanisms for how fetal IL-6 and IFN-γ exposure influence risk for neuropsychiatric disorders, and how folic acid can mitigate such risk.