Maximal Respiratory Capacity Research Articles

ND-13 preserves mitochondrial integrity via the RhoA/ROCK1/Drp1-S616 pathway providing acute cardioprotection in myocardial infarction

Abstract Background Ischemia-reperfusion injury (IRI) following acute myocardial infarction is a major contributor to heart failure and mortality worldwide. Drp1-driven mitochondrial fragmentation disrupts mitochondrial function, increases cardiomyocyte death, and aggravates cardiac dysfunction. We investigated whether ND-13, a novel DJ-1-derived peptide, can acutely protect the heart by maintaining mitochondrial homeostasis and modulating Drp1 activity during IRI. Methods The effects of ND-13 on Drp1 activity were assessed by studying mitochondrial dynamics, function and survival signalling pathways. Cardioprotective effects of ND-13 were assessed using in vitro, ex vivo and in vivo models of IRI. Results ND-13 demonstrated acute cardioprotective effects in murine adult cardiomyocytes subjected to simulated IRI by a 42% reduction in cell death. The excessive mitochondrial fission following IRI was attenuated by inhibiting Drp1 phosphorylation at the Ser616 residue by reduced activity of the RhoA/ROCK1 pathway. This in turn enhanced mitochondrial function as seen through increased mitochondrial respiration rates (basal, maximal and spare respiratory capacity), increased ATP production, maintained mitochondrial membrane potential and reduced reactive oxygen species levels. The acute cardioprotective effect of ND-13 was reproduced in both an ex vivo Langendorff based model of IRI with a 43% reduction infarct size and an in vivo murine model of IRI with a 35% reduction of infarct size. These results indicate the robust effect of the peptide and its potential for clinical translation through intravenous delivery. Conclusion In this study, we demonstrate ND-13 as a small peptide that modulates mitochondrial dynamics through the RhoA/ROCK1/DRP1-S616 pathway, which highlights a potential new therapeutic target for myocardial IRI.

Gestational Diabetes-like Fuels Impair Mitochondrial Function and Long-Chain Fatty Acid Uptake in Human Trophoblasts

In the parent, gestational diabetes mellitus (GDM) causes both hyperglycemia and hyperlipidemia. Despite excess lipid availability, infants exposed to GDM are at risk for essential long-chain polyunsaturated fatty acid (LCPUFA) deficiency. Isotope studies have confirmed less LCPUFA transfer from the parent to the fetus, but how diabetic fuels impact placental fatty acid (FA) uptake and lipid droplet partitioning is not well-understood. We evaluated the effects of high glucose conditions, high lipid conditions, and their combination on trophoblast growth, viability, mitochondrial bioenergetics, BODIPY-labeled fatty acid (FA) uptake, and lipid droplet dynamics. The addition of four carbons or one double bond to FA acyl chains dramatically affected the uptake in both BeWo and primary isolated cytotrophoblasts (CTBs). The uptake was further impacted by media exposure. The combination-exposed trophoblasts had more mitochondrial protein (p = 0.01), but impaired maximal and spare respiratory capacities (p < 0.001 and p < 0.0001), as well as lower viability (p = 0.004), due to apoptosis. The combination-exposed trophoblasts had unimpaired uptake of BODIPY C12 but had significantly less whole-cell and lipid droplet uptake of BODIPY C16, with an altered lipid droplet count, area, and subcellular localization, whereas these differences were not seen with individual high glucose or lipid exposure. These findings bring us closer to understanding how GDM perturbs active FA transport to increase the risk of adverse outcomes from placental and neonatal lipid accumulation alongside LCPUFA deficiency.

Inhibition of CILP2 Improves Glucose Metabolism and Mitochondrial Dysfunction in Sarcopenia via the Wnt Signalling Pathway.

Skeletal muscle is the primary organ involved in insulin-mediated glucose metabolism. Elevated levels of CILP2 are a significant indicator of impaired glucose tolerance and are predominantly expressed in skeletal muscle. It remains unclear whether CILP2 contributes to age-related muscle atrophy through regulating the glucose homeostasis and insulin sensitivity. Initially, the expression levels of CILP2 were assessed in elderly mice and patients with sarcopenia. Lentiviral vectors were used to induce either silencing or overexpression of CILP2 in C2C12 myoblast cells. The effects of CILP2 on proliferation, myogenic differentiation, insulin sensitivity and glucose uptake were evaluated using immunofluorescence, western blotting, real-time quantitative polymerase chain reaction, RNA sequencing, glucose uptake experiments, dual-luciferase reporter assays and co-immunoprecipitation (CO-IP). An adeno-associated virus-9 containing a muscle-specific promoter was injected into SAMP8 senile mice to observe the efficacy of CILP2 knockout. We found that there was more CLIP2 expressed in the skeletal muscle of ageing mice (+1.1-fold, p < 0.01) and in patients with sarcopenia (+2.5-fold, p < 0.01) compared to the control group. Following the overexpression of CILP2, Ki67 (-65%, p < 0.01), PCNA (-32%, p < 0.05), MyoD1 (-89%, p < 0.001), MyoG (-31%, p < 0.05) and MyHC (-85%, p < 0.001), which indicate proliferation and differentiation potential, were significantly reduced. In contrast, MuRF-1 (+59%, p < 0.05), atrogin-1 (+43%, p < 0.05) and myostatin (+31%, p < 0.05), the markers of muscular atrophy, were significantly increased. Overexpression of CILP2 decreased insulin sensitivity, glucose uptake (-18%, p < 0.001), GLUT4 translocation to the membrane and the maximum respiratory capacity of mitochondria. Canonical Wnt signalling was identified through RNA sequencing as a potential pathway for CILP2 regulation in C2C12, and Wnt3a was confirmed as an interacting protein of CILP2 in the CO-IP assay. The addition of recombinant Wnt3a protein reversed the inhibitory effects on myogenesis and glucose metabolism caused by CILP2 overexpression. Conversely, CILP2 knockdown promoted myogenesis and glucose metabolism. CILP2 knockdown improved muscle atrophy in mice, characterized by significant increases in time to exhaustion (+42%, p < 0.001), grip strength (+19%, p < 0.01), muscle mass (+15%, p < 0.001) and mean muscle cross-sectional area (+37%, p < 0.01). CILP2 knockdown enhanced glycogen synthesis (+83%, p < 0.001) and the regeneration of oxidative and glycolytic muscle fibres in SAMP8 ageing mice via the Wnt/β-catenin signalling pathway. Our results indicate that CILP2 interacts with Wnt3a to suppress the Wnt/β-catenin signalling pathway and its downstream cascade, leading to impaired insulin sensitivity and glucose metabolism in skeletal muscle. Targeting CILP2 inhibition could offer potential therapeutic benefits for sarcopenia.

A comparative study of bioenergetic metabolism on mammary epithelial cells from humans and Göttingen Minipigs

Mammary epithelial cells (MECs) of humans (h) and Göttingen Minipigs (mp) were analyzed to compare their ability to perform ATP production by oxidative phosphorylation and glycolysis. The ATP production under basal and stressor situations highlights the same metabolic potential of both primary cell lines. However, quantitively the ATP production rate of hMECs was higher than mpMECs. Conversely, oxidative cell respiration in mpMECs exploits a maximum respiratory capacity to support pathophysiological circumstances or stress conditions that could require an excessive effort of cell metabolism. Since mpMECs primarily utilize an oxidative metabolism similar to hMECs, the metabolic characterization conducted allows us to confirm that mpMECs represent a potential alternative cellular model in the translational medicine approach.

Reduced bioenergetics and mitochondrial fragmentation in human primary cytotrophoblasts induced by an EGFR-targeting chemical mixture

Exposures to complex environmental chemical mixtures during pregnancy reach and target the feto-placental unit. This study investigates the influence of environmental chemical mixtures on placental bioenergetics. Recognizing the essential role of the epidermal growth factor receptor (EGFR) in placental development and its role in stimulating glycolysis and mitochondrial respiration in trophoblast cells, we explored the effects of chemicals known to disrupt EGFR signaling on cellular energy production. Human primary cytotrophoblasts (hCTBs) and a first-trimester extravillous trophoblast cell line (HTR-8/SVneo) were exposed to a mixture of EGFR-interfering chemicals, including atrazine, bisphenol S, niclosamide, PCB-126, PCB-153, and trans-nonachlor. An RNA sequencing approach revealed that the mixture altered the transcriptional signature of genes involved in cellular energetics. Next, the impact of the mixture on cellular bioenergetics was evaluated using a combination of mitochondrial and glycolytic stress tests, ATP production, glucose consumption, lactate synthesis, and super-resolution imaging. The chemical mixture did not alter basal oxygen consumption but diminished the maximum respiratory capacity in a dose-dependent manner, indicating a disruption of mitochondrial function. The respiratory capacity and ATP production were increased by EGF, while the Chem-Mix reduced both EGF- and non-EGF-mediated oxygen consumption rate in hCTBs. A similar pattern was observed in the glycolytic medium acidification, with EGF increasing the acidification, and the Chem-Mix blocking EGF-induced glycolytic acidification. Furthermore, direct stochastic optical reconstruction microscopy (dSTORM) imaging demonstrated that the Chem-Mix led to a reduction of the mitochondrial network architecture, with findings supported by a decrease in the abundance of OPA1, a mitochondrial membrane GTPase involved in mitochondrial fusion. In conclusion, we demonstrated that a mixture of EGFR-disrupting chemicals alters mitochondrial remodeling, resulting in disturbed cellular bioenergetics, reducing the capacity of human cytotrophoblast cells to generate energy. Future studies should investigate the mechanism by which mitochondrial dynamics are disrupted and the pathological significance of these findings.

Chronic undernutrition impairs renal mitochondrial respiration accompanied by intense ultrastructural damage in juvenile rats

This study investigated whether chronic undernutrition alters the mitochondrial structure and function in renal proximal tubule cells, thus impairing fluid transport and homeostasis. We previously showed that chronic undernutrition downregulates the renal proximal tubules (Na++K+)ATPase, the main molecular machine responsible for fluid transport and ATP consumption. Male rats received a multifactorial deficient diet, the so-called Regional Basic Diet (RBD), mimicking those used in impoverished regions worldwide, from weaning to a juvenile age (3 months). The diet has a low content (8 %) of poor-quality proteins, low lipids, and no vitamins compared to control (CTR). We investigated citrate synthase activity, mitochondrial respiration (oxygraphy) in phosphorylating and non-phosphorylating conditions with different substrates/inhibitors, potential across the internal membrane (Δψ), and anion superoxide/H2O2 formation. The data were correlated with ultrastructural alterations evaluated using transmission electron microscopy (TEM) and focused ion beam scanning electron microscopy (FIB-SEM). Citrate synthase activity decreased (∼50 %) in RBD rats, accompanied by a similar reduction in respiration in non-phosphorylating conditions, maximum respiratory capacity, and ATP synthesis. The Δψ generation and its dissipation after carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone remained unmodified in the survival mitochondria. H2O2 production increased (∼100 %) after Complex II energization. TEM demonstrated intense matrix vacuolization and disruption of cristae junctions in a subpopulation of RBD mitochondria, which was also demonstrated in the 3D analysis of FIB-SEM tomography. In conclusion, chronic undernutrition impairs mitochondrial functions in renal proximal tubules, with profound alterations in the matrix and internal membrane ultrastructure that culminate with the compromise of ATP supply for transport processes.

Abstract Mo001: Inhibition of TAOK1-mediated Cardiomyocyte Death Attenuates Doxorubicin-Induced Cardiotoxicity

Background and Objective: Doxorubicin (Dox)-induced cardiotoxicity (DIC) is one of the most serious side effects of cancer treatments. However, no effective treatment is currently available. In this study, we aimed to identify novel therapeutic targets using CRISPR/Cas9-based screening of human cardiomyocytes derived from pluripotent stem cells (hPSC-CMs). Methods and Results: We performed a kinase-focused CRISPR gene knockout screen in hPSC-CMs and identified thousand and one amino acid protein kinase 1 (TAOK1 ) as a potential regulator of DOX-induced cardiomyocyte death. We generated TAOK1-knockout (KO) hPSC-CMs using CRISPR/Cas9 and found that TAOK1-KO hPSC-CMs showed higher cell viability than control KO cells after 1μM DOX treatment (49.3% vs. 30.5%, P = 0.03). TAOK1-KO decreased DOX-induced ROS production (-58.8%, P = 0.01) and DNA damage (-28.8%, P &lt; 0.01) in hPSC-CMs. Transcriptome analysis using RNA-seq of TAOK1-KO hPSC-CMs identified 483 differentially expressed genes, and Hallmark pathway analysis revealed upregulation of the oxidative phosphorylation pathway (FDR &lt; 0.01) in TAOK1-KO hPSC-CMs compared to the control. Extracellular flux analysis demonstrated higher basal and maximal respiratory capacity in TAOK1-KO hPSC-CMs compared to control after DOX treatment (1.2 vs. 0.6, and 3.8 vs. 2.0 pmol/min/μg, respectively, P &lt; 0.01 for all). Next, we overexpressed TAOK1 in hPSC-CMs using adenovirus vector and found that TAOK1 overexpression (OE) augmented DOX-induced cell viability reduction in hPSC-CMs compared to control after 0.5μM DOX treatment (-63.4 % vs. -19.2%, P &lt; 0.01). Western blot analysis showed that DOX-induced p38 phosphorylation was suppressed by TAOK1-KO (P &lt; 0.01) and enhanced (P = 0.01) by TAOK1-OE in hPSC-CMs. Augmented DOX-induced cardiomyocyte death in TAOK1-OE hPSC-CMs was partially rescued by inhibitors of p38 MAPK or apoptosis. Lastly, we demonstrated that TAOK1 suppression using AAV-mediated gene silencing mitigated the decline in the left ventricular ejection fraction in a mouse model of DIC (-30.0% vs. -17.7%, P &lt; 0.01). Histological analysis of heart tissue revealed that TAOK1 suppression reduced myocardial fibrosis and apoptosis of cardiac cells in a mouse model of DIC (6.6% vs. 4.3%, P = 0.02, and 0.31% vs. 0.18%, P&lt; 0.01, respectively). Conclusion: Our results suggest that TAOK1 regulates DOX-induced cardiomyocyte death; thus, its inhibition could be a potential therapeutic approach for DIC.

Abstract Tu117: Unraveling the Role of Mitochondrial Ribosomal Protein L7/L12 (MRPL12) in Diabetic Cardiomyopathy

Diabetic cardiomyopathy (DMCM) is a major cause of death in diabetic patients. Emerging evidence shows that mitochondrial dysfunction contributes to heart failure in diabetes. However, the molecular mechanisms of mitochondrial dysfunction mediating heart failure in diabetes are still poorly understood. The current study aimed to investigate the role of mitochondrial ribosomal protein L7/L12 (MRPL12) in human and mouse models of type II diabetes (db/db mice, and high-fat diet fed (HFD) mice) with or without induction of myocardial infarction (MI). Mitochondrial respiration was measured by using sea horse and mitochondrial membrane potential was measured by using confocal microscopy. Data was analyzed by using the mean of the groups was compared using a student t-test (for 2 groups) and ANOVA. We found increased MRPL12 levels in diabetic patients with ischemic heart disease compared to non-diabetic patients. Further we found elevated MRPL12 levels in the LV tissue of HFD fed mice with MI compared to low-fat diet-fed mice with MI. With the overexpression of MRPL12 under hyperglycemic conditions, the level of oxidative phosphorylation (OXPHOS) was found downregulated, but cellular ATP and human cardiomyocyte cell death remained unchanged, However, there was notable impairment in mitochondrial membrane potential (MMP) in hyperglycemia condition, along with changes in basal respiration oxygen consumption rate (OCR) and maximal respiratory capacity OCR. Overall, our results suggest MRPL12 may have a compensatory role in the diabetic hearts with ischemic heart disease. Therefore, our finding proposes new insights into the role of MRPL12 in the pathophysiology of MI in diabetes.

Role of mitochondrial ribosomal protein L7/L12 (MRPL12) in diabetic ischemic heart disease

BackgroundMyocardial infarction (MI) is a significant cause of death in diabetic patients. Growing evidence suggests that mitochondrial dysfunction contributes to heart failure in diabetes. However, the molecular mechanisms of mitochondrial dysfunction mediating heart failure in diabetes are still poorly understood. MethodsWe examined MRPL12 levels in right atrial appendage tissues from diabetic patients undergoing coronary artery bypass graft (CABG) surgery. Using AC-16 cells overexpressing MRPL12 under normal and hyperglycemic conditions we performed mitochondrial functional assays OXPHOS, bioenergetics, mitochondrial membrane potential, ATP production and cell death. ResultsWe observed elevated MRPL12 levels in heart tissue samples from diabetic patients with ischemic heart disease compared to non-diabetic patients. Overexpression of MRPL12 under hyperglycemic conditions did not affect oxidative phosphorylation (OXPHOS) levels, cellular ATP levels, or cardiomyocyte cell death. However, notable impairment in mitochondrial membrane potential (MMP) was observed under hyperglycemic conditions, along with alterations in both basal respiration oxygen consumption rate (OCR) and maximal respiratory capacity OCR. ConclusionsOverall, our results suggest that MRPL12 may have a compensatory role in the diabetic myocardium with ischemic heart disease, suggesting that MRPL12 may implicate in the pathophysiology of MI in diabetes.

O-134 Down regulation of Clock gene in testicle may affect energy metabolism of sertoli cell

Abstract Study question Whether decrease expression of Clock gene in the testis could affect the energy metabolism of Sertoli cells and what is the possible mechanism? Summary answer Down-regulating of Clock gene could regulate the expression of PIK3CB affect AMPK signaling pathways, significantly weakening the energy supply of sertoli cells in mouse testicular. What is known already The causes of Nonobstructive azoospermia (NOA) and other male infertility diseases are diverse and complex, including spermatogenesis and germ cell maturation at different stages of meiosis, the expression level of Clock gene maybe is one reason of which. In the testis of NOA patients, expression level of Clock gene was significantly decreased compared with Obstructive azoospermia (OA), meanwhile, the decrease of Clock gene expression can result in the decrease of male mouse fetal number. The decrease of Clock gene expression cause some kind of male infertility problem is confirmed, but the process and mechanism is not clear. Study design, size, duration Differential expression genes related to low expression of Clock and pathways related to spermatogenesis were screened out through transcriptome analysis on testicular tissue samples from patients with NOA(n = 24) and OA(n = 17). The model was established by transfecting mouse testicular Sertoli cell line (TM4 cells) with lentivirus containing Clock knock-down gene and energy metabolism effects were detected by Seahorse tests. Adifluorescein reporter gene was constructed to detect the binding of CLOCK and candidated differential expressed gene. Participants/materials, setting, methods Normal testis sequencing data were obtained from GTEX database as control. Combined with sequencing data, the differentially expressed genes were obtained as candidate gene, which expression was verified by RT-qPCR, Western-blotting. Energy metabolism were detected by Seahorse mitochondrial-stress test, glycolysis-rate test. Adifluorescein reporter gene for the promoter regions of candidate gene was constructed and transfected into TM4 cells to detect whether CLOCK could regulate candidate gene by binding to the E-box of the promoter region. Main results and the role of chance After GO(GeneOntology) and KEGG(Kyoto Encyclopedia of Genes and Genomes) enrichment analysis, PIK3CB which involved in AMPK signaling pathway was selected for further verification and molecular mechanism study. In Seahorse tests, the basal respiratory capacity, maximal respiratory capacity OCR, spare respiratory capacity and ATP-related respiratory capacity of TM4 cells were significantly decreased after Clock gene knockdown, but there were no significant differences in glycolytic rate, basal glycolysis, compensatory glycolysis level and glycolytic rate. RT-qPCR and Western-blotting confirmed that after knock down of Clock gene, the expression level of Pik3cb gene was down-regulated. That imply when Clock gene is decreased, it regulates the expression of PIK3CB affects AMPK signaling pathways, weakening the ability of mouse testicular sertoli cells to supply energy to various levels of spermatogenic cells and sperm cells, which may be one reason for the occurrence of nonobstructive azoospermia. During this process, there were significant changes in the mitochondrial energy supply of testicular supporting cells, but no significant changes in glycolysis. Finally, the dual luciferin report showed that the Clock gene could bind to the promoter regions of Pik3cb to regulate the expression of PIK3CB, and that the promoter of PIK3CB was activated by the transcription factor CLOCK. Limitations, reasons for caution Although we identified that Clock gene could bind to the promoter regions of Pik3cb to regulate the expression of PIK3CB and affect AMPK signaling pathways, weakening the ability of mouse testicular sertoli cells to supply energy, the available expression level and whether it will be work in vivo is unknown. Wider implications of the findings The results imply that the main energy changes affect the occurrence and development of spermatogonia and primary spermatocytes near the base of the seminiferous tubules are related to the influence of Clock on genes involved in regulating mitochondrial energy supply. It need more further studies. Trial registration number not applicable

Comprehensive characterization of mitochondrial bioenergetics at different larval stages reveals novel insights about the developmental metabolism of Caenorhabditis elegans.

Mitochondrial bioenergetic processes are fundamental to development, stress responses, and health. Caenorhabditis elegans is widely used to study developmental biology, mitochondrial disease, and mitochondrial toxicity. Oxidative phosphorylation generally increases during development in many species, and genetic and environmental factors may alter this normal trajectory. Altered mitochondrial function during development can lead to both drastic, short-term responses including arrested development and death, and subtle consequences that may persist throughout life and into subsequent generations. Understanding normal and altered developmental mitochondrial biology in C. elegans is currently constrained by incomplete and conflicting reports on how mitochondrial bioenergetic parameters change during development in this species. We used a Seahorse XFe24 Extracellular Flux (XF) Analyzer to carry out a comprehensive analysis of mitochondrial and non-mitochondrial oxygen consumption rates (OCR) throughout larval development in C. elegans. We optimized and describe conditions for analysis of basal OCR, basal mitochondrial OCR, ATP-linked OCR, spare and maximal respiratory capacity, proton leak, and non-mitochondrial OCR. A key consideration is normalization, and we present and discuss results as normalized per individual worm, protein content, worm volume, mitochondrial DNA (mtDNA) count, nuclear DNA (ncDNA) count, and mtDNA:ncDNA ratio. Which normalization process is best depends on the question being asked, and differences in normalization explain some of the discrepancies in previously reported developmental changes in OCR in C. elegans. Broadly, when normalized to worm number, our results agree with previous reports in showing dramatic increases in OCR throughout development. However, when normalized to total protein, worm volume, or ncDNA or mtDNA count, after a significant 2-3-fold increase from L1 to L2 stages, we found small or no changes in most OCR parameters from the L2 to the L4 stage, other than a marginal increase at L3 in spare and maximal respiratory capacity. Overall, our results indicate an earlier cellular shift to oxidative metabolism than suggested in most previous literature.

Free essential amino acid feeding improves endurance during resistance training via DRP1-dependent mitochondrial remodelling.

Loss of muscle strength and endurance with aging or in various conditions negatively affects quality of life. Resistance exercise training (RET) is the most powerful means to improve muscle mass and strength, but it does not generally lead to improvements in endurance capacity. Free essential amino acids (EAAs) act as precursors and stimuli for synthesis of both mitochondrial and myofibrillar proteins that could potentially confer endurance and strength gains. Thus, we hypothesized that daily consumption of a dietary supplement of nine free EAAs with RET improves endurance in addition to the strength gains by RET. Male C57BL6J mice (9weeks old) were assigned to control (CON), EAA, RET (ladder climbing, 3 times a week), or combined treatment of EAA and RET (EAA+RET) groups. Physical functions focusing on strength or endurance were assessed before and after the interventions. Several analyses were performed to gain better insight into the mechanisms by which muscle function was improved. We determined cumulative rates of myofibrillar and mitochondrial protein synthesis using 2H2O labelling and mass spectrometry; assessed ex vivo contractile properties and in vitro mitochondrial function, evaluated neuromuscular junction (NMJ) stability, and assessed implicated molecular singling pathways. Furthermore, whole-body and muscle insulin sensitivity along with glucose metabolism, were evaluated using a hyperinsulinaemic-euglycaemic clamp. EAA+RET increased muscle mass (10%, P<0.05) and strength (6%, P<0.05) more than RET alone, due to an enhanced rate of integrated muscle protein synthesis (19%, P<0.05) with concomitant activation of Akt1/mTORC1 signalling. Muscle quality (muscle strength normalized to mass) was improved by RET (i.e., RET and EAA+RET) compared with sedentary groups (10%, P<0.05), which was associated with increased AchR cluster size and MuSK activation (P<0.05). EAA+RET also increased endurance capacity more than RET alone (26%, P<0.05) by increasing both mitochondrial protein synthesis (53%, P<0.05) and DRP1 activation (P<0.05). Maximal respiratory capacity increased (P<0.05) through activation of the mTORC1-DRP1 signalling axis. These favourable effects were accompanied by an improvement in basal glucose metabolism (i.e., blood glucose concentrations and endogenous glucose production vs. CON, P<0.05). Combined treatment with balanced free EAAs and RET may effectively promote endurance capacity as well as muscle strength through increased muscle protein synthesis, improved NMJ stability, and enhanced mitochondrial dynamics via mTORC1-DRP1 axis activation, ultimately leading to improved basal glucose metabolism.

Mitochondrial Dysfunction in Sporadic Amyotrophic Lateral Sclerosis Patients: Insights from High-Resolution Respirometry.

Amyotrophic lateral sclerosis is a severe neurodegenerative disease whose exact cause is still unclear. Currently, research attention is turning to the mitochondrion as a critical organelle of energy metabolism. Current knowledge is sufficient to confirm the involvement of the mitochondria in the pathophysiology of the disease, since the mitochondria are involved in many processes in the cell; however, the exact mechanism of involvement is still unclear. We used peripheral blood mononuclear cells isolated from whole fresh blood from patients with amyotrophic lateral sclerosis for measurement and matched an age- and sex-matched set of healthy subjects. The group of patients consisted of patients examined and diagnosed at the neurological clinic of the University Hospital Martin. The set of controls consisted of healthy individuals who were actively searched, and controls were selected on the basis of age and sex. The group consisted of 26 patients with sporadic forms of ALS (13 women, 13 men), diagnosed based on the definitive criteria of El Escorial. The average age of patients was 54 years, and the average age of healthy controls was 56 years. We used a high-resolution O2K respirometry method, Oxygraph-2k, to measure mitochondrial respiration. Basal respiration was lower in patients by 29.48%, pyruvate-stimulated respiration (respiratory chain complex I) was lower by 29.26%, and maximal respiratory capacity was lower by 28.15%. The decrease in succinate-stimulated respiration (respiratory chain complex II) was 26.91%. Our data confirm changes in mitochondrial respiration in ALS patients, manifested by the reduced function of complex I and complex II of the respiratory chain. These defects are severe enough to confirm this disease's hypothesized mitochondrial damage. Therefore, research interest in the future should be directed towards a deeper understanding of the involvement of mitochondria and respiratory complexes in the pathophysiology of the disease. This understanding could develop new biomarkers in diagnostics and subsequent therapeutic interventions.

Cilia-deficient renal tubule cells are primed for injury with mitochondrial defects and aberrant tryptophan metabolism.

The exocyst and Ift88 are necessary for primary ciliogenesis. Overexpression of Exoc5 (OE), a central exocyst component, resulted in longer cilia and enhanced injury recovery. Mitochondria are involved in acute kidney injury (AKI). To investigate cilia and mitochondria, basal respiration and mitochondrial maximal and spare respiratory capacity were measured in Exoc5 OE, Exoc5 knockdown (KD), Exoc5 ciliary targeting sequence mutant (CTS-mut), control Madin-Darby canine kidney (MDCK), Ift88 knockout (KO), and Ift88 rescue cells. In Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells, these parameters were decreased. In Exoc5 OE and Ift88 rescue cells they were increased. Reactive oxygen species were higher in Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells compared with Exoc5 OE, control, and Ift88 rescue cells. By electron microscopy, mitochondria appeared abnormal in Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells. A metabolomics screen of control, Exoc5 KD, Exoc5 CTS-mut, Exoc5 OE, Ift88 KO, and Ift88 rescue cells showed a marked increase in tryptophan levels in Exoc5 CTS-mut (113-fold) and Exoc5 KD (58-fold) compared with control cells. A 21% increase was seen in Ift88 KO compared with rescue cells. In Exoc5 OE compared with control cells, tryptophan was decreased 59%. To determine the effects of ciliary loss on AKI, we generated proximal tubule-specific Exoc5 and Ift88 KO mice. These mice had loss of primary cilia, decreased mitochondrial ATP synthase, and increased tryptophan in proximal tubules with greater injury following ischemia-reperfusion. These data indicate that cilia-deficient renal tubule cells are primed for injury with mitochondrial defects in tryptophan metabolism.NEW & NOTEWORTHY Mitochondria are centrally involved in acute kidney injury (AKI). Here, we show that cilia-deficient renal tubule cells both in vitro in cell culture and in vivo in mice are primed for injury with mitochondrial defects and aberrant tryptophan metabolism. These data suggest therapeutic strategies such as enhancing ciliogenesis or improving mitochondrial function to protect patients at risk for AKI.

Hypoxia Exposure Fine-Tunes Mitochondrial Function in Primary Dermal Fibroblasts Derived from Loggerhead Sea Turtles

Sea turtles are constantly exposed to changes in oxygen tension derived from extended breath-hold diving, but the cellular-level response to such changes is not completely understood. Most studies in hypoxia-tolerant reptiles have focused on the freshwater turtle Trachemys scripta, which responds to anoxia by inducing metabolic rewiring and preserving mitochondrial integrity, facilitating effcient mitochondrial respiration during reoxygenation. Here, we derived primary dermal fibroblasts from loggerhead sea turtles and western fence lizards to study the bioenergetic cellular response to hypoxia exposure in diving and non-diving reptiles. Cells from both species stained positive for the fibroblast markers vimentin and PDGFRb and responded to hypoxia exposure (0.1% O2) by accumulating HIF-1α. We measured oxygen consumption rates (OCR) in intact cells using Seahorse technology. We found that basal, maximal, and spare respiratory capacity was lower in lizard than in loggerhead cells at 26oC under normoxic (room air) conditions (p &lt; 0.0001). Exposure to short (1h) and long-term hypoxia (24h) altered mitochondrial function in both species. Maximal and spare respiratory capacity were lower in loggerhead than in lizard cells (p &lt; 0.01) after 1h hypoxia followed by 1h reoxygenation. In contrast, basal and maximal respiratory capacity was higher in loggerhead compared to lizard cells after 24h hypoxia (p &lt; 0.04). Nonetheless, the overall OCR after 24h hypoxia in both species was lower than that observed after 1h hypoxia. Overall, there was a significant reduction in cellular respiration in both species from short-term to long-term hypoxia exposure. In lizard cells, basal OCR decreased by a factor of three while maximal OCR decreased by a factor of 4.5. In loggerhead cells, both the basal and maximal OCR decreased by a factor of two. Our results demonstrate species-specific regulation of cellular respiration after hypoxia exposure. Remarkably, loggerhead cells show a steady downregulation of cellular respiration in response to hypoxia exposure and faster recovery after extended hypoxia (24h) than lizard cells, aligning with the remarkable hypoxic tolerance exhibited by sea turtles, which can endure up to 7 hours of breath-holding underwater. Understanding the cellular mechanisms that drive hypoxic tolerance in diving animals can provide insights for treating human conditions characterized by hypoxia signaling. Ford Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cardiac Sodium Channel Nav1.5 Arg222Gln Variant Induces a Sex-Specific Arrhythmia and Alters Mitochondrial Function

Introduction: Encoded by the SCN5A gene, cardiac Nav1.5 sodium channels initiate the action potential to trigger a heartbeat. As the predominant sodium channel in the heart, variants can have devastating consequences, leading to potentially lethal arrhythmias. With the gain of function arginine to glutamine (Arg222Gln) variant in Nav1.5, patients develop intermittent arrhythmias and dilated cardiomyopathy. The stress induced by chronic arrhythmias can trigger adverse metabolic reprogramming, exacerbating the underlying condition. We have generated a murine model harboring the human Arg222Gln variant where we observe that only males produce a clear observable arrhythmia. We hypothesize that the maladaptive metabolic response to the arrhythmia caused by the Arg222Gln variant maybe be a potential link to the sex differences. Methods: The human exon containing the Arg222Gln mutation was knocked into a murine model under the endogenous SCN5A promoter. Cellular metabolism (oxygen consumption) was measured using primary adult mouse cardiomyocytes (CMs) with the Seahorse XFe analyzer. Mitochondrial membrane potential was measured using ratiometric JC-1 dye and imaged on a scanning confocal microscope. Heart tissues were diced into 1mm3 cubes and fixed to perform serial block face electron microscopy to obtain 3D structure of mitochondria and mitochondrial morphology. Western blots were used to detect protein levels. Results: Basal respiration of CMs were unchanged, however the maximal respiratory capacity of Arg222Gln males was significantly lower (116.9±13.5pmol O2/min, p&lt;0.05) compared to WT males (407.3±16.4pmol O2/min). There were no differences between WT females (451.4±22.4pmol O2/min) and Arg222Gln females (463.2±33.7pmol O2/min). JC-1 dye revealed that the Arg222Gln males (0.6213±0.0172 red/green ratio, p&lt;0.001) have significantly lowered mitochondrial polarization compared to WT males (0.7550 ±0.0257). There were no differences between WT females (0.6236±0.0154) and Arg222Gln females (0.6466±0.0139). Arg222Gln males have reduced mitochondria area (1.155±0.052μm2, p&lt;0.05) compared to WT males (1.332±0.060μm2) and are more fragmented (p&lt;0.05). Electron transport chain complex protein levels and mitochondrial biomass measured by voltage dependent anion channel protein levels remain unchanged between WT males and Arg222Gln males (P&gt;0.05). The ratio of phosphorylated dynamin related (DRP) / total DRP protein levels were significantly higher in Arg222Gln males compared to WT males, indicative of mitochondrial fission (p&lt;0.05). Conclusion: We demonstrate evidence of a mitochondrial dysfunction in a murine model of arrhythmia caused by a gain of function Nav1.5 variant, adversely affecting overall cellular metabolism and mitochondrial quality. Our data suggests that the difference in metabolic reprogramming could play a role in the cardioprotection against arrhythmia in Arg222Gln females or explain why Arg222Gln males are severely affected by the arrhythmia. Future experiments will aim to elucidate how the difference in sex affects cardiac metabolism in the context of Arg222Gln Nav1.5 variant. This work was supported by a Canadian Institutes of Health Research Project Grant, the SickKids Foundation through the Curtis Joseph and Harold Groves Chair in Anesthesia and Pain Medicine (JTM) and the Department of Anesthesiology and Pain Medicine, University of Toronto through a Merit Award (JTM). TYTL is supported by the CIHR Canadian Graduate Scholarship — Doctoral Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mitochondrial Health, Physical Functioning, and Daily Affect: Bioenergetic Mechanisms of Dementia Caregiver Well-Being.

Chronic stress adversely affects mental and physical well-being. However, health outcomes vary among people experiencing the same stressor. Individual differences in physical and emotional well-being may depend on mitochondrial biology, as energy production is crucial for stress regulation. This study investigated whether mitochondrial respiratory capacity corresponds to individual differences in dementia spousal caregivers' mental and physical health. Spousal caregivers of individuals with Alzheimer's disease and related dementias ( N = 102, mean age = 71, 78% female, 83% White) provided peripheral blood samples and completed self-report questionnaires on quality of life, caregiver burden, and a 7-day affect scale. Multiple and mixed linear regressions were used to test the relationship between mitochondrial biology and well-being. Spare respiratory capacity ( b = 12.76, confidence interval [CI] = 5.23-20.28, p = .001), maximum respiratory capacity ( b = 8.45, CI = 4.54-12.35, p < .0001), and ATP-linked respiration ( b = 10.11, CI = 5.05-15.18, p = .0001) were positively associated with physical functioning. At average ( b = -2.23, CI = -3.64 to -0.82, p = .002) and below average ( b = -4.96, CI = -7.22 to 2.70, p < .0001) levels of spare respiratory capacity, caregiver burden was negatively associated with daily positive affect. At above average levels of spare respiratory capacity, caregiver burden was not associated with positive affect ( p = .65). Findings suggest that higher mitochondrial respiratory capacity is associated with better psychological and physical health-a pattern consistent with related research. These findings provide some of the earliest evidence that cellular bioenergetics are related to well-being.

Hypoxia exposure blunts angiogenic signaling and upregulates the antioxidant system in endothelial cells derived from elephant seals

BackgroundElephant seals exhibit extreme hypoxemic tolerance derived from repetitive hypoxia/reoxygenation episodes they experience during diving bouts. Real-time assessment of the molecular changes underlying protection against hypoxic injury in seals remains restricted by their at-sea inaccessibility. Hence, we developed a proliferative arterial endothelial cell culture model from elephant seals and used RNA-seq, functional assays, and confocal microscopy to assess the molecular response to prolonged hypoxia.ResultsSeal and human endothelial cells exposed to 1% O2 for up to 6 h respond differently to acute and prolonged hypoxia. Seal cells decouple stabilization of the hypoxia-sensitive transcriptional regulator HIF-1α from angiogenic signaling. Rapid upregulation of genes involved in glutathione (GSH) metabolism supports the maintenance of GSH pools, and intracellular succinate increases in seal but not human cells. High maximal and spare respiratory capacity in seal cells after hypoxia exposure occurs in concert with increasing mitochondrial branch length and independent from major changes in extracellular acidification rate, suggesting that seal cells recover oxidative metabolism without significant glycolytic dependency after hypoxia exposure.ConclusionsWe found that the glutathione antioxidant system is upregulated in seal endothelial cells during hypoxia, while this system remains static in comparable human cells. Furthermore, we found that in contrast to human cells, hypoxia exposure rapidly activates HIF-1 in seal cells, but this response is decoupled from the canonical angiogenesis pathway. These results highlight the unique mechanisms that confer extraordinary tolerance to limited oxygen availability in a champion diving mammal.

Aberrant expression of thyroidal hormone receptor α exasperating mitochondrial dysfunction induced sarcopenia in aged mice.

Disrupted mitochondrial dynamics and mitophagy contribute to functional deterioration of skeletal muscle (SM) during aging, but the regulatory mechanisms are poorly understood. Our previous study demonstrated that the expression of thyroid hormone receptor α (TRα) decreased significantly in aged mice, suggesting that the alteration of thyroidal elements, especially the decreased TRα, might attenuate local THs action thus to cause the degeneration of SM with aging, while the underlying mechanism remains to be further explored. In this study, decreased expression of myogenic regulators Myf5, MyoD1, mitophagy markers Pink1, LC3II/I, p62, as well as mitochondrial dynamic factors Mfn1 and Opa1, accompanied by increased reactive oxygen species (ROS), showed concomitant changes with reduced TRα expression in aged mice. Further TRα loss- and gain-of-function studies in C2C12 revealed that silencing of TRα not only down-regulated the expression of above-mentioned myogenic regulators, mitophagy markers and mitochondrial dynamic factors, but also led to a significant decrease in mitochondrial activity and maximum respiratory capacity, as well as more mitochondrial ROS and damaged mitochondria. Notedly, overexpression of TRα could up-regulate the expression of those myogenic regulators, mitophagy markers and mitochondrial dynamic factors, meanwhile also led to an increase in mitochondrial activity and number. These results confirmed that TRα could concertedly regulate mitochondrial dynamics, autophagy, and activity, and myogenic regulators rhythmically altered with TRα expression. Summarily, these results suggested that the decline of TRα might cause the degeneration of SM with aging by regulating mitochondrial dynamics, mitophagy and myogenesis.

β-hydroxybutyrate exposure restores mitochondrial function in skeletal muscle satellite cells of critically ill patients

Background and AimDysfunction of skeletal muscle satellite cells might impair muscle regeneration and prolong ICU-acquired weakness, a condition associated with disability and delayed death. This study aimed to elucidate the distinct metabolic effects of critical illness and β-OHB on satellite cells isolated from these patients. MethodsSatellite cells were extracted from vastus lateralis muscle biopsies of patients with ICU-acquired weakness (n = 10) and control group of healthy volunteers or patients undergoing elective hip replacement surgery (n = 10). The cells were exposed to standard culture media supplemented with β-OH-butyrate to assess its influence on cell proliferation by ELISA, mitochondrial functions by extracellular flux analysis, electron transport chain complexes by high resolution respirometry, and ROS production by confocal microscopy. ResultsCritical illness led to a decline in maximal respiratory capacity, ATP production and glycolytic capacity and increased ROS production in ICU patients’ cells. Notably, the function of complex II was impaired due to critical illness but restored to normal levels upon exposure to β-OH-butyrate. While β-OH-butyrate significantly reduced ROS production in both control and ICU groups, it had no significant impact on global mitochondrial functions. ConclusionCritical illness induces measurable bioenergetic dysfunction of skeletal muscle satellite cells. β-OH-butyrate displayed a potential in rectifying complex II dysfunction caused by critical illness and this warrants further exploration.