Changes In Mitochondrial Function Research Articles

Eukarion-134 Attenuates Endoplasmic Reticulum Stress-Induced Mitochondrial Dysfunction in Human Skeletal Muscle Cells.

Maladaptive endoplasmic reticulum (ER) stress is associated with modified reactive oxygen species (ROS) generation and mitochondrial abnormalities; and is postulated as a potential mechanism involved in muscle weakness in myositis, an acquired autoimmune neuromuscular disease. This study investigates the impact of ROS generation in an in vitro model of ER stress in skeletal muscle, using the ER stress inducer tunicamycin (24 h) in the presence or absence of a superoxide dismutase/catalase mimetic Eukarion (EUK)-134. Tunicamycin induced maladaptive ER stress, which was mitigated by EUK-134 at the transcriptional level. ER stress promoted mitochondrial dysfunction, described by substantial loss of mitochondrial membrane potential, as well as a reduction in respiratory control ratio, reserve capacity, phosphorylating respiration, and coupling efficiency, which was ameliorated by EUK-134. Tunicamycin induced ROS-mediated biogenesis and fusion of mitochondria, which, however, had high propensity of fragmentation, accompanied by upregulated mRNA levels of fission-related markers. Increased cellular ROS generation was observed under ER stress that was prevented by EUK-134, even though no changes in mitochondrial superoxide were noticeable. These findings suggest that targeting ROS generation using EUK-134 can amend aspects of ER stress-induced changes in mitochondrial dynamics and function, and therefore, in instances of chronic ER stress, such as in myositis, quenching ROS generation may be a promising therapy for muscle weakness and dysfunction.

Serum Coenzyme Q10 Levels are Decreased in Critically-Ill Septic Patients: Results From a Preliminary Study.

The increased oxidative stress resulting from the inflammatory responses in sepsis initiates changes in mitochondrial function which may result in organ damage, the most common cause of death in the intensive care unit (ICU). Deficiency of coenzyme Q10 (CoQ10), a key cofactor in the mitochondrial respiratory chain, could potentially disturb mitochondrial bioenergetics and oxidative stress, and may serve as a biomarker of mitochondrial dysfunction. Hence, we aimed to investigate in initially non-septic patients whether CoQ10 levels are decreased in sepsis and septic shock compared to ICU admission, and to evaluate its associations with severity scores, inflammatory biomarkers, and ICU outcomes. Observational retrospective analysis on 86 mechanically-ventilated, initially non-septic, ICU patients. CoQ10 was sequentially measured on ICU admission, sepsis, septic shock or at ICU discharge. CoQ10 was additionally measured in 25 healthy controls. Inflammatory biomarkers were determined at baseline and sepsis. On admission, ICU patients who developed sepsis had lower CoQ10 levels compared to healthy controls (0.89 vs. 1.04 µg/ml, p < 0.05), while at sepsis and septic shock CoQ10 levels decreased further (0.63 µg/ml; p < 0.001 and 0.42 µg/ml; p < 0.0001, respectively, from admission). In ICU patients who did not develop sepsis, admission CoQ10 levels were also lower than healthy subjects (0.81 µg/ml; p < 0.001) and were maintained at the same levels until discharge. CoQ10 levels in critically-ill patients are low on ICU admission compared to healthy controls and exhibit a further decrease in sepsis and septic shock. These results suggest that sepsis severity leads to CoQ10 depletion.

Negative Association between Caloric Intake and Estimated Glomerular Filtration Rate in a Chinese Population: Mediation Models Involving Mitochondrial Function

Background: With the shortening of leukocyte telomere length (LTL) and decrease in the copy number of mitochondrial DNA, mitochondrial dysfunction and oxidative stress are considered important drivers of the aging process. Although previous experimental studies report that caloric intake is associated with age-related renal dysfunction through the changes in mitochondrial function, there are insufficient epidemiological data to establish this association. Objective: We aimed to explore the association between caloric intake and renal function and to investigate whether mitochondrial DNA copy number (mtDNAcn) mediated this association by cross-sectional analysis. Methods: A total of 403 individuals from a Chinese rural cohort (women = 66.50%; mean age = 53.94 ± 10.27 years) with an estimated glomerular filtration rate (eGFR) ≥60 mL/min/1.73 m² and with differences in the glucose tolerance status (diabetes, n = 106; prediabetes, n = 125; normal glucose tolerance, n = 172) were included. Dietary data were obtained by a 24-h food recall, and caloric intake was normalized by ideal body weight. The mtDNAcn and LTL were detected using real-time PCR assay. The associations between caloric intake, aging markers, and renal function were analyzed by partial correlation analysis and multiple linear regression analysis. Mediation analysis was applied to examine the role of mtDNAcn in the association between caloric intake and eGFR. Results: Caloric intake was higher while age-adjusted mtDNAcn was lower in individuals with eGFR <90 mL/min/1.73 m² (n = 140) than in those with eGFR ≥90 mL/min/1.73 m² (n = 263). After adjusting for multiple factors, linear regression analysis revealed that caloric intake was negatively associated with eGFR and mtDNAcn, while mtDNAcn was positively associated with eGFR. Moreover, mediation analysis indicated that the indirect effect of caloric intake on eGFR through mtDNAcn was significant (β = –0.0505, 95% confidence interval –0.0931 to –0.0190). Conclusions: Caloric intake was negatively associated with eGFR in a Chinese population, and the association was partly mediated by decreased mtDNAcn.

Transcriptome analysis indicates dominant effects on ribosome and mitochondrial function of a premature termination codon mutation in the zebrafish gene psen2.

PRESENILIN 2 (PSEN2) is one of the genes mutated in early onset familial Alzheimer’s disease (EOfAD). PSEN2 shares significant amino acid sequence identity with another EOfAD-related gene PRESENILIN 1 (PSEN1), and partial functional redundancy is seen between these two genes. However, the complete range of functions of PSEN1 and PSEN2 is not yet understood. In this study, we performed targeted mutagenesis of the zebrafish psen2 gene to generate a premature termination codon close downstream of the translation start with the intention of creating a null mutation. Homozygotes for this mutation, psen2S4Ter, are viable and fertile, and adults do not show any gross psen2-dependent pigmentation defects, arguing against significant loss of γ-secretase activity. Also, assessment of the numbers of Dorsal Longitudinal Ascending (DoLA) interneurons that are responsive to psen2 but not psen1 activity during embryogenesis did not reveal decreased psen2 function. Transcripts containing the S4Ter mutation show no evidence of destabilization by nonsense-mediated decay. Forced expression in zebrafish embryos of fusions of psen2S4Ter 5’ mRNA sequences with sequence encoding enhanced green fluorescent protein (EGFP) indicated that the psen2S4Ter mutation permits utilization of cryptic, novel downstream translation start codons. These likely initiate translation of N-terminally truncated Psen2 proteins lacking late endosomal/lysosomal localization sequences and that obey the “reading frame preservation rule” of PRESENILIN EOfAD mutations. Transcriptome analysis of entire brains from a 6-month-old family of wild type, heterozygous and homozygous psen2S4Ter female siblings revealed profoundly dominant effects on gene expression likely indicating changes in ribosomal, mitochondrial, and anion transport functions.

Mitochondrial Dysfunction is a Key Pathway that Links Saturated Fat Intake to the Development and Progression of NAFLD.

Non‐Alcoholic fatty liver disease (NAFLD) is the most common form of liver disease and is characterized by fat accumulation in the liver. Hypercaloric diets generally increase hepatic fat accumulation, whereas hypocaloric diets decrease liver fat content. In addition, there is evidence to suggest that moderate amounts of unsaturated fatty acids seems to be protective for the development of a fatty liver, while consumption of saturated fatty acids (SFA) appears to predispose toward hepatic steatosis. Recent studies highlight a key role for mitochondrial dysfunction in the development and progression of NAFLD. It is proposed that changes in mitochondrial structure and function are key mechanisms by which SFA lead to the development and progression of NAFLD. In this review, it is described how SFA intake is associated with liver steatosis and decreases the efficiency of the respiratory transport chain. This results in the production of reactive oxygen species and damage to nearby structures, eventually leading to inflammation, apoptosis, and scarring of the liver. Furthermore, studies demonstrating that SFA intake affects the composition of mitochondrial membranes are presented, and this process accelerates the progression of NAFLD. It is likely that events are intertwined and reinforce each other, leading to a constant deterioration in health.

Molecular mechanisms of the effects of N-ethylmaleimide and 1,4-dithioerythritol on regulation of apoptosis in P19 cells under hypoxia

Relevance. Impairment of apoptosis regulation in P19 cells is correlated with generation of oxidative stress. Under hypoxia, changes in mitochondrial functions occur, which may exacerbate oxidative stress in the tumor cell. The aim of the study was to evaluate the effects of N-ethylmaleimide and 1,4-dithioerythritol on implementation and regulation of apoptosis in P19 cells under hypoxia in vitro . Materials and methods . P19 cells (mouse teratocarcinoma) cultured under hypoxia served as the material for the study. For redox status modulation, 5mM N-ethylmaleimide and 1,4-dithioerythritol in the final concentrations of 5 mM were used. The intracellular concentration of calcium ions, the transmembrane potential and the number of Annexin V, CD95 and CD120 positive cells were determined by flow cytometry. The levels of reduced, oxidized and protein-bound glutathione, protein SH groups, hydroxyl radical and protein carbonyl derivatives were measured by spectrophotometry. Results. The alteration in the redox status of the glutathione system under hypoxia, accompanied by oxidative modification of proteins (glutathionylation and carbonylation), influences the metabolism in the tumor cell on the whole. Under the effects of 1,4-dithioerythritol, an SH group protector, this alteration promotes formation of additional mechanisms to escape apoptosis, whereas under the effects of N-ethylmaleimide, an SH group blocker, it, on the contrary, promotes apoptosis activation. Conclusions . The changes in the redox homeostasis of the tumor cell and modulation of oxidative modification of proteins (glutathionylation and carbonylation) under hypoxia are one of the promising approaches to targeted regulation of cell death.

Potential role of mitochondria in synoviocytes.

Synoviocytes are located in the synovium lining layer, which is composed of macrophage-like synoviocytes (MLS) and fibroblast-like synoviocytes (FLS) with different characteristics. Mitochondria, which exist in most cells, are two membrane-covered organelles. In addition to providing the necessary ATP for synoviocytes, mitochondria are involved in the regulation of redox homeostasis and the integration of synoviocytes death signals. In recent years, mitochondrial dysfunction has been found in rheumatoid arthritis (RA) and osteoarthritis (OA). Interestingly, recent studies have started uncovering that mitochondria that were previously reported to play a role in chondrocytes or immune cells, but not known to have pronounced roles in synoviocytes, can actually play crucial roles in the regulation of the pathological properties of the synoviocytes. The purpose of this review is to summarize our current understanding of the key role of mitochondria in synoviocytes, including mitochondrial dysfunction in synoviocytes can induce and aggravate inflammatory responses and changes in mitochondrial structure and function with the involvement of multiple cytokines, signal pathway, and hypoxic state of synovial tissue alter the response of synoviocytes to apoptotic stimulation. Also, mitochondrial abnormalities in synoviocytes promote the synoviocytes invasion and proliferation.

β-Hydroxybutyrate Increases Exercise Capacity Associated with Changes in Mitochondrial Function in Skeletal Muscle.

β-hydroxybutyrate is the main ketone body generated by the liver under starvation. Under these conditions, it can sustain ATP levels by its oxidation in mitochondria. As mitochondria can modify its shape and function under different nutritional challenges, we study the chronic effects of β-hydroxybutyrate supplementation on mitochondrial morphology and function, and its relation to exercise capacity. Male C57BL/6 mice were supplemented with β-hydroxybutyrate mineral salt (3.2%) or control (CT, NaCl/KCl) for six weeks and submitted to a weekly exercise performance test. We found an increase in distance, maximal speed, and time to exhaustion at two weeks of supplementation. Fatty acid metabolism and OXPHOS subunit proteins declined at two weeks in soleus but not in tibialis anterior muscles. Oxygen consumption rate on permeabilized fibers indicated a decrease in the presence of pyruvate in the short-term treatment. Both the tibialis anterior and soleus showed decreased levels of Mitofusin 2, while electron microscopy assessment revealed a significant reduction in mitochondrial cristae shape in the tibialis anterior, while a reduction in the mitochondrial number was observed only in soleus. These results suggest that short, but not long-term, β-hydroxybutyrate supplementation increases exercise capacity, associated with modifications in mitochondrial morphology and function in mouse skeletal muscle.

Mitochondrial oxidative injury: a key player in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) has become the most prevalent liver disease worldwide. NAFLD is tightly linked to the metabolic syndrome, insulin resistance, and oxidative stress. Globally, its inflammatory form, nonalcoholic steatohepatitis (NASH), has become the main cause of liver-related morbidity and mortality, mainly due to liver cirrhosis and primary liver cancer. One hallmark of NASH is the presence of changes in mitochondrial morphology and function that are accompanied by a blocked flow of electrons in the respiratory chain, which increases formation of mitochondrial reactive oxygen species in a self-perpetuating vicious cycle. Consequences are oxidation of DNA bases and mitochondrial DNA depletion that are coupled with genetic and acquired mitochondrial DNA mutations, all impairing the resynthesis of respiratory chain polypeptides. In general, several maladaptations of pathways that usually maintain energy homeostasis occur with the early and late excess metabolic stress in NAFLD and NASH. We discuss the interplay between hepatocyte mitochondrial stress and inflammatory responses, focusing primarily on events initiated and maintained by mitochondrial free radical-induced damage in NAFLD. Importantly, mitochondrial oxidative stress and dysfunction are modulated by key pharmacological targets that are related to excess production of reactive oxygen species, mitochondrial turnover and the mitochondrial unfolded protein response, mitophagy, and mitochondrial biogenesis. However, the efficacy of such interventions depends on NAFLD/NASH disease stage.

Hexokinase II dissociation alone cannot account for changes in heart mitochondrial function, morphology and sensitivity to permeability transition pore opening following ischemia.

We previously demonstrated that hexokinase II (HK2) dissociation from mitochondria during cardiac ischemia correlates with cytochrome c (cyt-c) loss, oxidative stress and subsequent reperfusion injury. However, whether HK2 release is the primary signal mediating this ischemia-induced mitochondrial dysfunction was not established. To investigate this, we studied the effects of dissociating HK2 from isolated heart mitochondria. Mitochondria isolated from Langendorff-perfused rat hearts before and after 30 min global ischemia ± ischemic preconditioning (IPC) were subject to in vitro dissociation of HK2 by incubation with glucose-6-phosphate at pH 6.3. Prior HK2 dissociation from pre- or end-ischemic heart mitochondria had no effect on their cyt-c release, respiration (± ADP) or mitochondrial permeability transition pore (mPTP) opening. Inner mitochondrial membrane morphology was assessed indirectly by monitoring changes in light scattering (LS) and confirmed by transmission electron microscopy. Although no major ultrastructure differences were detected between pre- and end-ischemia mitochondria, the amplitude of changes in LS was reduced in the latter. This was prevented by IPC but not mimicked in vitro by HK2 dissociation. We also observed more Drp1, a mitochondrial fission protein, in end-ischemia mitochondria. IPC failed to prevent this increase but did decrease mitochondrial-associated dynamin 2. In vitro HK2 dissociation alone cannot replicate ischemia-induced effects on mitochondrial function implying that in vivo dissociation of HK2 modulates end-ischemia mitochondrial function indirectly perhaps involving interaction with mitochondrial fission proteins. The resulting changes in mitochondrial morphology and cristae structure would destabilize outer / inner membrane interactions, increase cyt-c release and enhance mPTP sensitivity to [Ca2+].

Mitochondrial alterations triggered by repeated exposure to fine (PM2.5-0.18) and quasi-ultrafine (PM0.18) fractions of ambient particulate matter

Nowadays ambient particulate matter (PM) levels still regularly exceed the guideline values established by World Health Organization in most urban areas. Numerous experimental studies have already demonstrated the airway toxicity of the fine fraction of PM (FP), mainly triggered by oxidative stress-induced airway inflammation. However, only few studies have actually paid close attention to the ultrafine fraction of PM (UFP), which is likely to be more easily internalized in cells and more biologically reactive. Mitochondria are major endogenous sources of reactive oxygen species (ROS) through oxidative metabolism, and coordinate many critical cellular signaling processes. Mitochondria have been often studied in the context of PM toxicity and generally associated with apoptosis activation. However, little is known about the underlying adaptation mechanisms that could occur following exposure at sub-apoptotic doses of ambient PM. Here, normal human bronchial epithelial BEAS-2B cells were acutely or repeatedly exposed to relatively low doses (5 µg.cm−2) of FP (PM2.5-0.18) or quasi-UFP (Q-UFP; PM0.18) to better access the critical changes in mitochondrial morphology, functions, and dynamics. No significant cytotoxicity nor increase of apoptotic events were reported for any exposure. Mitochondrial membrane potential (ΔΨm) and intracellular ATP content were also not significantly impaired. After cell exposure to sub-apoptotic doses of FP and notably Q-UFP, oxidative phosphorylation was increased as well as mitochondrial mass, resulting in increased production of mitochondrial superoxide anion. Given this oxidative boost, the NRF2-ARE signaling pathway was significantly activated. However, mitochondrial dynamic alterations in favor of accentuated fission process were observed, in particular after Q-UFP vs FP, and repeated vs acute exposure. Taken together, these results supported mitochondrial quality control and metabolism dysfunction as an early lung underlying mechanism of toxicity, thereby leading to accumulation of defective mitochondria and enhanced endogenous ROS generation. Therefore, these features might play a key role in maintaining PM-induced oxidative stress and inflammation within lung cells, which could dramatically contribute to the exacerbation of inflammatory chronic lung diseases. The prospective findings of this work could also offer new insights into the physiopathology of lung toxicity, arguably initiate and/or exacerbate by acutely and rather repeated exposure to ambient FP and mostly Q-UFP.

Evolution of altered tubular metabolism and mitochondrial function in sepsis-associated acute kidney injury.

Sepsis-associated acute kidney injury (s-AKI) has a staggering impact in patients and lacks any treatment. Incomplete understanding of the pathogenesis of s-AKI is a major barrier to the development of effective therapies. We address the gaps in knowledge regarding renal oxygenation, tubular metabolism, and mitochondrial function in the pathogenesis of s-AKI using the cecal ligation and puncture (CLP) model in mice. At 24 h after CLP, renal oxygen delivery was reduced; however, fractional oxygen extraction was unchanged, suggesting inefficient renal oxygen utilization despite decreased glomerular filtration rate and filtered load. To investigate the underlying mechanisms, we examined temporal changes in mitochondrial function and metabolism at 4 and 24 h after CLP. At 4 h after CLP, markers of mitochondrial content and biogenesis were increased in CLP kidneys, but mitochondrial oxygen consumption rates were suppressed in proximal tubules. Interestingly, at 24 h, proximal tubular mitochondria displayed high respiratory capacity, but with decreased mitochondrial content, biogenesis, fusion, and ATP levels in CLP kidneys, suggesting decreased ATP synthesis efficiency. We further investigated metabolic reprogramming after CLP and observed reduced expression of fatty acid oxidation enzymes but increased expression of glycolytic enzymes at 24 h. However, assessment of functional glycolysis revealed lower glycolytic capacity, glycolytic reserve, and compensatory glycolysis in CLP proximal tubules, which may explain their susceptibility to injury. In conclusion, we demonstrated significant alterations in renal oxygenation, tubular mitochondrial function, and metabolic reprogramming in s-AKI, which may play an important role in the progression of injury and recovery from AKI in sepsis.

Mitochondrial light switches: optogenetic approaches to control metabolism.

Developing new technologies to study metabolism is increasingly important as metabolic disease prevalence increases. Mitochondria control cellular metabolism and dynamic changes in mitochondrial function are associated with metabolic abnormalities in cardiovascular disease, cancer, and obesity. However, a lack of precise and reversible methods to control mitochondrial function has prevented moving from association to causation. Recent advances in optogenetics have addressed this challenge, and mitochondrial function can now be precisely controlled invivo using light. A class of genetically encoded, light-activated membrane channels and pumps has addressed mechanistic questions that promise to provide new insights into how cellular metabolism downstream of mitochondrial function contributes to disease. Here, we highlight emerging reagents-mitochondria-targeted light-activated cation channels or proton pumps-to decrease or increase mitochondrial activity upon light exposure, a technique we refer to as mitochondrial light switches, or mtSWITCH . The mtSWITCH technique is broadly applicable, as energy availability and metabolic signaling are conserved aspects of cellular function and health. Here, we outline the use of these tools in diverse cellular models of disease. We review the molecular details of each optogenetic tool, summarize the results obtained with each, and outline best practices for using optogenetic approaches to control mitochondrial function and downstream metabolism.

Subphysiological temperature induces pervasive alternative splicing in Chinese hamster ovary cells.

RNA sequencing (RNASeq) has been widely used to associate alterations in Chinese hamster ovary (CHO) cell gene expression with bioprocess phenotypes; however, alternative messenger RNA (mRNA) splicing, has thus far, received little attention. In this study, we utilized RNASeq for transcriptomic analysis of a monoclonal antibody (mAb) producing CHO K1 cell line subjected to a temperature shift. More than 2,465 instances of differential splicing were observed 24 hr after the reduction of cell culture temperature. A total of 1,197 of these alternative splicing events were identified in genes where no changes in abundance were detected by standard differential expression analysis. Ten examples of alternative splicing were selected for independent validation using quantitative polymerase chain reaction in the mAb-producing CHO K1 cell line used for RNASeq and a further two CHO K1 cell lines. This analysis provided evidence that exon skipping and mutually exclusive splicing events occur in genes linked to the cellular response to changes in temperature and mitochondrial function. While further work is required to determine the impact of these changes in mRNA sequence on cellular phenotype, this study demonstrates that alternative splicing analysis can be utilized to gain a deeper understanding of post-transcriptional regulation in CHO cells during biopharmaceutical production.

371-OR: Reduced Hepatic Expression of the Mitochondrial Quality Control Factor PARKIN Hastens the Progression of NAFLD in Mice and Is Associated with NASH in Patients

Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of pathologies, including steatosis, steatohepatitis (NASH) and fibrosis and is strongly associated with type 2 diabetes. Changes in mitochondrial function are implicated in the pathogenesis of NAFLD. Mitophagy is a mitochondrial quality control mechanism that allows for selective removal of damaged mitochondria from the cell. Whether changes in mitophagy occur during and contribute to NAFLD is unknown. PARKIN is a ubiquitin E3 ligase that regulates mitophagy by generating a ubiquitin signaling motif on the outer membrane of damaged mitochondria, marking them for degradation. We undertook the studies described to determine effects of genetic inhibition of mitophagy via conditional deletion of PARKIN in liver on progression of NAFLD. Twelve-week old male liver-specific PARKIN knockout (LKO) and wild type (WT) mice were fed a diet previously demonstrated to induce NAFLD that consisted of high-fat (45%), cholesterol (0.15%) and high-fructose corn syrup drinking water (42g/L) for 8 weeks. There was no difference in body weight or composition between groups. Plasma ALT and AST levels were increased 2-fold (p=0.18) and 1.7-fold (p&amp;lt;0.05), respectively, in LKO compared with WT mice, while plasma cholesterol and insulin levels were not different and plasma fatty acid levels were reduced by 27% (p&amp;lt;0.05). NAFLD activity score was increased 2-fold (p&amp;lt;0.05) in LKO mice reflecting an increase in steatosis, ballooning and inflammation. Gene expression markers of NAFLD progression including markers of inflammation (Ccl2, Cd68, Il-1b, ll-6) and fibrosis (Col1a1, Col3a1) were significantly increased in LKO mice. Consistent with these mouse data, we found PARKIN protein expression to be reduced in liver biopsies from patients with NASH compared with healthy controls. These data suggest that loss of PARKIN-mediated mitophagy may contribute to the progression of NAFLD. Disclosure R. Undamatla: None. L.R. Edmunds: None. B. Xie: None. A. Mills: None. I.J. Sipula: None. S.P. Monga: None. M.J. Jurczak: None. Funding National Institutes of Health (DK114012)

1,25-dihydroxyvitamin D3 prevents deleterious effects of erythromycin on mitochondrial function in rat heart isolated mitochondria.

Erythromycin (ERY) is a risk factor for cardiotoxicity through the mitochondria pathway. In the current study, we tested the hypothesis that erythromycin could impair mitochondrial function and oxidative stress and 1,25-dihydroxivitamin D3 (calcitriol) treatment could prevent these effects in rat heart isolated mitochondria. Rat heart mitochondria were isolated with mechanical lysis and differential centrifugation. Then isolated mitochondria were first pretreated with three different concentrations of 1,25-dihydroxivitamin D3 (2.5, 5 and 10µmol/L) for 5minutes at 37°C, after which erythromycin (10µmol/L) was added to promote deleterious effects on mitochondria. During 1hour of incubation, using by flow cytometry and biochemical evaluations, the parameters of mitochondrial toxicity were evaluated, including: succinate dehydrogenase (SDH) activity, mitochondrial swelling, mitochondrial membrane potential (MMP) collapse, reactive oxygen species (ROS) formation and lipid peroxidation (LP). The results showed that erythromycin (10µmol/L) caused a significant change in mitochondrial function, ROS formation, mitochondrial swelling, MMP collapse, increasing lipid peroxidation and oxidative stress. 1,25-dihydroxivitamin D3 (10µmol/L) reverted the effect of erythromycin on the testedparameters . In this study, we showed that erythromycin impairs mitochondrial function and induces mitochondrial toxicity in rat heart isolated mitochondria, which were reverted by calcitriol. These findings suggest that 1,25-dihydroxivitamin D3 may be a preventive/therapeutic strategy for cardiotoxicity complications caused by erythromycin.

P0851THE DIFFERENTIAL IMPACT OF INTRAVENOUS IRON IN A MODEL OF EXPERIMENTAL URAEMIA ON TISSUE MARKERS OF OXIDATIVE STRESS

Abstract Background and Aims Uraemia leads to changes in cardiac structure, metabolic remodelling and anaemia, key factors in the development of heart failure in patients with chronic kidney disease (CKD). Previous studies have identified abnormalities in mitochondrial function in experimental uraemia, potentially impairing energy provision and enhancing oxidative stress. The aim of this study was to characterise oxidant status and changes in mitochondrial function in uraemia and the impact of iron therapy. Method Experimental uraemia was induced in male Sprague-Dawley rats via a subtotal nephrectomy and parenteral iron administration at 6 weeks post-surgery (10mg/Kg body weight). Pro-oxidant activities and antioxidant capacities (oxidised glutathione (GSSG) relative to reduced (GSH)) in both sham and uraemic animals and lipid peroxidation (thiobarbituric acid-reactive substances (TBARS) was measured. Mitochondrial function was assessed using the SEAHORSE analyser. Results Oxidative stress was increased in cardiac tissue (69% increase in GSSG/GSH ratio, 38% rise in TBARS and significantly (79%) upregulated NADPH oxidase activity. Glutathione peroxidase (GPx) and glutathione reductase (GR) activities were significantly increased by 30%. Iron reduced GSSG/GSH ratio and TBARS by 62% and 51% respectively. Iron therapy was also associated with significantly increased aconitase activity. Parenteral iron improved respiratory reserve in cardiac mitochondria and skeletal mitochondria suggests IV iron restores mitochondrial function. Conclusion Uraemia leads or oxidative stress and mitochondrial dysfunction in cardiac tissue. Iron therapy reduces oxidative stress and improves cardiac mitochondrial function. This might lead to reduced adverse cardiovascular outcomes in CKD.

Aging-Related Increase of cGMP Disrupts Mitochondrial Homeostasis in Leydig Cells.

Since mitochondria play an essential role in the testosterone biosynthesis, serve as power centers and are a source of oxidative stress, a possible mitochondrial dysfunction could be connected with decreased activity of Leydig cells and lowered testosterone production during aging. Here we chronologically analyzed age-related alterations of mitochondrial function in Leydig cells correlated by the progressive rise of cGMP signaling and with respect to testosterone synthesis. To target cGMP signaling in Leydig cells, acute or long-term in vivo or ex vivo treatments with sildenafil (phosphodiesterase 5 [PDE5] inhibitor) were performed. Aging-related accumulation of cGMP in the Leydig cells is associated with mitochondrial dysfunction illustrated by reduced ATP and steroid production, lowered O2 consumption, increased mitochondrial abundance and mtDNA copies number, decreased expression of genes that regulate mitochondrial biogenesis (Ppargc1a/PGC1a-Tfam-Nrf1/NRF1), mitophagy (Pink1), fusion (Mfn1, Opa1), and increased Nrf2/NRF2. Acute in vivo PDE5 inhibition overaccumulated cGMP and stimulated testosterone but reduced ATP production in Leydig cells from adult, middle-aged, and old rats. The increased ATP/O ratio observed in cells from old compared to adult rats was diminished after stimulation of cGMP signaling. Opposite, long-term PDE5 inhibition decreased cGMP signaling and improved mitochondrial function/dynamics in Leydig cells from old rats. Mitochondrial abundance in Leydig cells decreased while ATP levels increased. Chronic treatment elevated Tfam, Nrf1, Nrf2, Opa1, Mfn1, Drp1, and normalized Pink1 expression. Altogether, long-term PDE5 inhibition prevented age-related NO and cGMP elevation, improved mitochondrial dynamics/function, and testosterone production. The results pointed on cGMP signaling in Leydig cells as a target for pharmacological manipulation of aging-associated changes in mitochondrial function and testosterone production.

Fluoxetine administration in juvenile overfed rats improves hypothalamic mitochondrial respiration and REDOX status and induces mitochondrial biogenesis transcriptional expression

Nutritional imbalance in early life may disrupt the hypothalamic control of energy homeostasis and increase the risk of metabolic disease. The hypothalamic serotonin (5-hydroxytryptamine; 5-HT) system based in the hypothalamus plays an important role in the homeostatic control of energy balance, however the mechanisms underlying the regulation of energy metabolism by 5-HT remain poorly described. Several crucial mitochondrial functions are altered by mitochondrial stress. Adaptations to this stress include changes in mitochondrial multiplication (i.e, mitochondrial biogenesis). Due to the scarcity of evidence regarding the effects of serotonin reuptake inhibitors (SSRI) such as fluoxetine (FLX) on mitochondrial function, we sought to investigate the potential contribution of FLX on changes in mitochondrial function and biogenesis occurring in overfed rats. Using a neonatal overfeeding model, male Wistar rats were divided into 4 groups between 39 and 59 days of age based on nutrition and FLX administration: normofed + vehicle (NV), normofed + FLX (NF), overfed + vehicle (OV) and overfed + FLX (OF). We found that neonatal overfeeding impaired mitochondrial respiration and increased oxidative stress biomarkers in the hypothalamus. FLX administration in overfed rats reestablished mitochondrial oxygen consumption, increased mitochondrial uncoupling protein 2 (Ucp2) expression, reduced total reactive species (RS) production and oxidative stress biomarkers, and up-regulated mitochondrial biogenesis-related genes. Taken together our results suggest that FLX administration in overfed rats improves mitochondrial respiratory chain activity and oxidative balance and increases the transcription of genes employed in mitochondrial biogenesis favoring mitochondrial energy efficiency in response to early nutritional imbalance.

A novel approach to measure mitochondrial respiration in frozen biological samples

Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together. However, the requirement for freshly isolated mitochondria hinders the feasibility of respirometry in multi‐site clinical studies and retrospective studies. Here, we describe a novel respirometry approach suited for frozen samples by restoring electron transfer components lost during freeze/thaw and correcting for variable permeabilization of mitochondrial membranes. This approach preserves 90–95% of the maximal respiratory capacity in frozen samples and can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity. We find that primary changes in mitochondrial function, detected in fresh tissue, are preserved in frozen samples years after collection. This approach will enable analysis of the integrated function of mitochondrial Complexes I to IV in one measurement, collected at remote sites or retrospectively in samples residing in tissue biobanks.