Respiratory Chain Enzyme Activities Research Articles

Renoprotective effect of hyperin against CdCl2 prompted renal damage by activation of Nrf-2/Keap-1 ARE pathway in male mice

Objectives This study explored the mitigating properties of hyperin (HYP) on renotoxicity induced by cadmium chloride (CdCl2). Methods Four groups of seven male albino mice each were used in this experiment. Group 1 served as the control, receiving no treatment. Group 2 received daily oral gavage of CdCl2 at 0.3 mg/kg body weight for 28 d. Group 3 received both CdCl2 (0.3 mg/kg) and HYP (100 mg/kg) daily using the same administration method. Finally, Group 4 received only HYP (100 mg/kg) daily. Results Cd exposure significantly increased kidney dysfunction markers (blood urea nitrogen and creatinine) and oxidative stress (reactive oxygen species [ROS] and malondialdehyde [MDA]). Conversely, it decreased antioxidant enzyme activities (glutathione peroxidase (GPx] and catalase [CAT]) and glutathione (GSH) levels. Nuclear factor erythroid 2-related factor 2 (Nrf-2) and antioxidant gene expression decreased, while Kelch-like ECH-associated protein 1 expression increased. Additionally, Cd exposure increased inflammatory mediators (nuclear factor kappa B, tumor necrosis factor alpha [TNF-α], interleukin-1β [IL-1β], and cyclooxygenase-2) and apoptotic markers (Bax and caspase-3), alongside decreased Bcl-2 expression and renal tissue abnormalities. Mitochondrial dysfunction manifested with diminished activities of Krebs cycle and respiratory chain enzymes, and reduced mitochondrial membrane potential. Co-treatment with HYP significantly attenuated these detrimental effects through its anti-apoptotic, antioxidant, and anti-inflammatory properties. Conclusion HYP co-treatment significantly attenuated CdCl2-induced renal damage in mice, suggesting its potential as a protective agent against Cd-induced kidney toxicity.

ANALYSIS OF SCIENTIFIC RESEARCH ON CLINICAL APPLICATIONS OF L-CARNITINE IN PEDIATRIC PRACTICE

The L-carnitine molecule was discovered 115 years ago by two scientists, Prof. R. P. Krimber and Prof. V. S. Gulevich. In 1962, the role of carnitine as a carrier of long-chain fatty acids into the mitochondria through their internal, highly selective membrane was discovered. L-carnitine is a vital compound that plays a crucial role in fat metabolism and energy metabolism in the child's body. The purpose of the study is to analyze the literature data on the current features of the clinical and pharmacological substantiation of the use of L-carnitine in pediatric practice. The article discusses aspects of the use of L-carnitine in pediatric medicine. L-Carnitine, essential for fatty acid metabolism, is synthesized endogenously and obtained from dietary sources. Ninety-eight percent of it is accumulated in skeletal muscles. its critical role in primary deficiencies, such as systemic encephalomyopathies and isolated myopathies, is unequivocal. L-Carnitine modulates glucose metabolism and increases the activity of respiratory chain enzymes. In addition, it acts as an antioxidant, preventing oxidative damage and inhibiting apoptosis, a signal in response to oxidative stress. Studies show that L-carnitine may be beneficial for children with metabolic disorders, athletes, and other categories of patients. However, it is important to consider dosage, safety, and potential side effects. Thus, the accumulated clinical experience of L-carnitine use indicates various positive effects and allows us to consider it an effective preventive and therapeutic agent that can be used in the pediatric population. Its applications extend to scenarios requiring energy support during heightened mental, emotional, and physical stress, as an adjunct therapy for diverse somatic diseases, during post-illness rehabilitation, and for bolstering immune reserves.

Mitochondria, a Key Target in Amyotrophic Lateral Sclerosis Pathogenesis.

Mitochondrial dysfunction occurs in numerous neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS), where it contributes to motor neuron (MN) death. Of all the factors involved in ALS, mitochondria have been considered as a major player, as secondary mitochondrial dysfunction has been found in various models and patients. Abnormal mitochondrial morphology, defects in mitochondrial dynamics, altered activities of respiratory chain enzymes and increased production of reactive oxygen species have been described. Moreover, the identification of CHCHD10 variants in ALS patients was the first genetic evidence that a mitochondrial defect may be a primary cause of MN damage and directly links mitochondrial dysfunction to the pathogenesis of ALS. In this review, we focus on the role of mitochondria in ALS and highlight the pathogenic variants of ALS genes associated with impaired mitochondrial functions. The multiple pathways demonstrated in ALS pathogenesis suggest that all converge to a common endpoint leading to MN loss. This may explain the disappointing results obtained with treatments targeting a single pathological process. Fighting against mitochondrial dysfunction appears to be a promising avenue for developing combined therapies in the future.

Brain mitochondrial diversity and network organization predict anxiety-like behavior in male mice

The brain and behavior are under energetic constraints, limited by mitochondrial energy transformation capacity. However, the mitochondria-behavior relationship has not been systematically studied at a brain-wide scale. Here we examined the association between multiple features of mitochondrial respiratory chain capacity and stress-related behaviors in male mice with diverse behavioral phenotypes. Miniaturized assays of mitochondrial respiratory chain enzyme activities and mitochondrial DNA (mtDNA) content were deployed on 571 samples across 17 brain areas, defining specific patterns of mito-behavior associations. By applying multi-slice network analysis to our brain-wide mitochondrial dataset, we identified three large-scale networks of brain areas with shared mitochondrial signatures. A major network composed of cortico-striatal areas exhibited the strongest mitochondria-behavior correlations, accounting for up to 50% of animal-to-animal behavioral differences, suggesting that this mito-based network is functionally significant. The mito-based brain networks also overlapped with regional gene expression and structural connectivity, and exhibited distinct molecular mitochondrial phenotype signatures. This work provides convergent multimodal evidence anchored in enzyme activities, gene expression, and animal behavior that distinct, behaviorally-relevant mitochondrial phenotypes exist across the male mouse brain.

Development of muscle weakness in a mouse model of critical illness: does fibroblast growth factor 21 play a role?

BackgroundCritical illness is hallmarked by severe stress and organ damage. Fibroblast growth factor 21 (FGF21) has been shown to rise during critical illness. FGF21 is a pleiotropic hormone that mediates adaptive responses to tissue injury and repair in various chronic pathological conditions. Animal studies have suggested that the critical illness-induced rise in FGF21 may to a certain extent protect against acute lung, liver, kidney and brain injury. However, FGF21 has also been shown to mediate fasting-induced loss of muscle mass and force. Such loss of muscle mass and force is a frequent problem of critically ill patients, associated with adverse outcome. In the present study, we therefore investigated whether the critical illness-induced acute rise in FGF21 is muscle-protective or rather contributes to the pathophysiology of critical illness-induced muscle weakness.MethodsIn a catheterised mouse model of critical illness induced by surgery and sepsis, we first assessed the effects of genetic FGF21 inactivation, and hence the inability to acutely increase FGF21, on survival, body weight, muscle wasting and weakness, and markers of muscle cellular stress and dysfunction in acute (30 h) and prolonged (5 days) critical illness. Secondly, we assessed whether any effects were mirrored by supplementing an FGF21 analogue (LY2405319) in prolonged critical illness.ResultsFGF21 was not required for survival of sepsis. Genetic FGF21 inactivation aggravated the critical illness-induced body weight loss (p = 0.0003), loss of muscle force (p = 0.03) and shift to smaller myofibers. This was accompanied by a more pronounced rise in markers of endoplasmic reticulum stress in muscle, without effects on impairments in mitochondrial respiratory chain enzyme activities or autophagy activation. Supplementing critically ill mice with LY2405319 did not affect survival, muscle force or weight, or markers of muscle cellular stress/dysfunction.ConclusionsEndogenous FGF21 is not required for sepsis survival, but may partially protect muscle force and may reduce cellular stress in muscle. Exogenous FGF21 supplementation failed to improve muscle force or cellular stress, not supporting the clinical applicability of FGF21 supplementation to protect against muscle weakness during critical illness.

A case of combined oxidative phosphorylation deficiency 32 caused by MRPS34 gene variation and literature review

Objective: To investigate the clinical features and genetic features of combined oxidative phosphorylation deficiency 32 (COXPD32) caused by MRPS34 gene variation. Methods: The clinical data and genetic test of a child with COXPD32 hospitalized in the Department of Neurology, Children's Hospital, Capital Institute of Pediatrics in March 2021 were extracted and analyzed. A literature search was implemented using Wanfang, China biology medicine disc, China national knowledge infrastructure, ClinVar, human gene mutation database (HGMD) and Pubmed databases with the key words "MRPS34" "MRPS34 gene" and "combined oxidative phosphorylation deficiency 32" (up to February 2023). Clinical and genetic features of COXPD32 were summarized. Results: A boy aged 1 year and 9 months was admitted due to developmental delay. He showed mental and motor retardation, and was below the 3rd percentile for height, weight, and head circumference of children of the same age and gender. He had poor eye contact, esotropia, flat nasal bridge, limbs hypotonia, holding instability and tremors. In addition, Grade Ⅲ/6 systolic murmur were heard at left sternal border. Arterial blood gases suggested that severe metabolic acidosis with lactic acidosis. Brain magnetic resonance imaging (MRI) showed multiple symmetrical abnormal signals in the bilateral thalamus, midbrain, pons and medulla oblongata. Echocardiography showed atrial septal defect. Genetic testing identified the patient as a compound heterozygous variation of MRPS34 gene, c.580C>T (p.Gln194Ter) and c.94C>T (p.Gln32Ter), with c.580C>T being the first report and a diagnosis of COXPD32. His parents carried a heterozygous variant, respectively. The child improved after treatment with energy support, acidosis correction, and "cocktail" therapy (vitaminB1, vitaminB2, vitaminB6, vitaminC and coenzyme Q10). A total of 8 cases with COXPD32 were collected through 2 English literature reviews and this study. Among the 8 patients, 7 cases had onset during infancy and 1 was unknown, all had developmental delay or regression, 7 cases had feeding difficulty or dysphagia, followed by dystonia, lactic acidosis, ocular symptoms, microcephaly, constipation and dysmorphic facies(mild coarsening of facial features, small forehead, anterior hairline extending onto forehead,high and narrow palate, thick gums, short columella, and synophrys), 2 cases died of respiratory and circulatory failure, and 6 were still alive at the time of reporting, with an age range of 2 to 34 years. Blood and (or) cerebrospinal fluid lactate were elevated in all 8 patients. MRI in 7 cases manifested symmetrical abnormal signals in the brainstem, thalamus, and (or) basal ganglia. Urine organic acid test were all normal but 1 patient had alanine elevation. Five patients underwent respiratory chain enzyme activity testing, and all had varying degrees of enzyme activity reduction. Six variants were identified, 6 patients were homozygous variants, with c.322-10G>A was present in 4 patients from 2 families and 2 compound heterozygous variants. Conclusions: The clinical phenotype of COXPD32 is highly heterogenous and the severity of the disease varies from development delay, feeding difficulty, dystonia, high lactic acid, ocular symptoms and reduced mitochondrial respiratory chain enzyme activity in mild cases, which may survive into adulthood, to rapid death due to respiratory and circulatory failure in severe cases. COXPD32 needs to be considered in cases of unexplained acidosis, hyperlactatemia, feeding difficulties, development delay or regression, ocular symptoms, respiratory and circulatory failure, and symmetrical abnormal signals in the brainstem, thalamus, and (or) basal ganglia, and genetic testing can clarify the diagnosis.

Prolonged fluoride exposure induces spatial-memory deficit and hippocampal dysfunction by inhibiting small heat shock protein 22 in mice

Millions of residents in areas with high-fluoride drinking water supply ingest excessive levels of fluoride for long periods. This study investigated the mechanisms and impacts of lifelong exposure to naturally occurring moderate–high-fluoride drinking water on spatial-memory function by studying mice in controlled experiments. Spatial-memory deficits and disorders of hippocampal neuronal electrical activity were observed in mice exposed to 25-ppm or 50-ppm-fluoride drinking water for 56 weeks, but not in adult or old mice exposed to 50 ppm fluoride for 12 weeks. Ultrastructural analysis showed severely damaged hippocampal mitochondria, evidenced by reduced mitochondrial membrane potential and ATP content. Mitochondrial biogenesis was impaired in fluoride-exposed mice, manifesting as a significantly reduced mtDNA content, mtDNA-encoded subunits mtND6 and mtCO1, and respiratory complex activities. Fluoride reduced expression of Hsp22, a beneficial mediator of mitochondrial homeostasis, and decreased levels of signaling for the PGC-1α/TFAM pathway—which regulates mitochondrial biogenesis—and the NF-κβ/STAT3 pathway—which regulates mitochondrial respiratory chain enzyme activity. Hippocampus-specific Hsp22-overexpression improved fluoride-induced spatial-memory deficits by activating the PGC-1α/TFAM and STAT3 signaling pathways, while Hsp22-silencing aggravated the deficits by inhibiting both pathways. Downregulation of Hsp22 plays a vital role in fluoride-induced spatial-memory deficits by impacting mtDNA-encoding subsets and mitochondrial respiratory chain enzyme activity.

Cardiomyocyte mitochondrial respiration and microviscosity in rats of different ages in heart failure

Highlights. Heart failure in older age groups is an urgent medical and social issue. Mitochondrial dysfunction is a key link in the pathogenesis of heart failure. In this study we have demonstrated a decrease in mitochondrial respiratory function in old rats. In this age group, the development of heart failure is accompanied by a further decrease in respiratory control. An increase in the mitochondrial microviscosity in older animals may affect the activity of respiratory chain enzymes in heart failure.Aim. To study the respiratory activity and mitochondrial membrane microviscosity of cardiomyocyte of rats of different ages with heart failure.Methods. The study involved 22 2- and 15-month-old male Wistar rats. The animals were divided into 4 groups: 2 groups of intact animals of both ages (n = 12) and 2 groups of rats with isadrine model of heart failure (HF) (n = 10). HF was modeled by two subcutaneous injections of isoproterenol hydrochloride (170 mg/kg) at interval of 24 hours. Mitochondrial respiratory activity was assessed using respiratory control coefficient. The microviscosity of mitochondrial membranes was evaluated by eximerization coefficients of pyrene-based fluorescent probe in areas of protein-lipid and lipid-lipid contact. Comparative statistical analysis of independent groups was performed using the nonparametric Mann-Whitney test.Results. A decrease in mitochondrial respiratory control in older rats was shown in comparison with young animals. In the HF model, inter-age difference increases, but at the same time, in younger rats, the development of HF is not accompanied by significant changes in mitochondrial respiratory control. An age dependent decrease in the microviscosity of mitochondrial membranes in the area of protein-lipid and lipid-lipid interaction was revealed. In younger rats, the development of HF is characterized by a significant increase in microviscosity in the area of protein-lipid and lipid-lipid contact. In older rats, the development of the pathology is characterized by a significant decrease in microviscosity in the area of protein-lipid interaction.Conclusion. Multidirectional age-related changes in cardiomyocyte mitochondria of rats with heart failure were revealed. It was shown that mitochondria in younger rats retain their functional activity in the HF model unlike older rats.

De novo frameshift variant in MT-ND1 causes a mitochondrial complex I deficiency associated with MELAS syndrome

BackgroundThe variant m.3571_3572insC/MT-ND1 thus far only reported in oncocytic tumors of different tissues. However, the role of m.3571_3572insC in inherited mitochondrial diseases has yet to be elucidated. MethodsA patient diagnosed with MELAS syndrome was recruited, and detailed medical records were collected and reviewed. The muscle was biopsied for mitochondrial respiratory chain enzyme activity. Series of fibroblast clones bearing different m.3571_3572insC variant loads were generated from patient-derived fibroblasts and subjected to functional assays. ResultsComplex I deficiency was confirmed in the patient’s muscle via mitochondrial respiratory chain enzyme activity assay. The m.3571_3572insC was filtered for the candidate variant of the patient according to the guidelines for mitochondrial mRNA variants interpretation. Three cell clones with different m.3571_3572insC variant loads were generated to evaluate mitochondrial function. Blue native PAGE analysis revealed that m.3571_3572insC caused a deficiency in the mitochondrial complex I. Oxygen consumption rate, ATP production, and lactate assays found an impairment of cellular bioenergetic capacity due to m.3571_3572insC. Mitochondrial membrane potential was decreased, and mitochondrial reactive oxygen species production was increased with the variant of m.3571_3572insC. According to the competitive cell growth assay, the mutant cells had impaired cell growth capacity compared to wild type. ConclusionsA novel variant m.3571_3572insC was identified in a patient diagnosed with MELAS syndrome, and the variant impaired mitochondrial respiration by decreasing the activity of complex I. In conclusion, the genetic spectrum of mitochondrial diseases was expanded by including m.3571_3572insC/MT-ND1.

Neuromedin S Regulates Steroidogenesis through Maintaining Mitochondrial Morphology and Function via NMUR2 in Goat Ovarian Granulosa Cells.

Neuromedin S (NMS) plays various roles in reproductive regulation, while the mechanism by which NMS regulates ovarian steroidogenesis remains unclear. In the current study, we confirmed the enhancement role of NMS in steroidogenesis in goat ovarian granulosa cells (GCs). To further explore the specific mechanism, we conducted a knockdown of NMUR2 in GCs followed by treatment with NMS and determined the effects of NMS treatment on mitochondrial morphology and function. The results found that NMS treatment increased the production of estrogen and up-regulated the expression of STAR, CYP11A1, 3BHSD, and CYP19A1, while the effects of NMS treatment were blocked by the knockdown of NMUR2 in goat GCs. Moreover, NMS treatment enhanced the fusion of mitochondria and up-regulated the expression of OPA1, MFN1, and MFN2, and increased mitochondrial membrane potential, the activity of respiratory chain enzymes and ATP production by maintaining a low expression level of mitochondrial unfolded protein response markers. The effects of NMS treatment on mitochondria were reversed by NMUR2 knockdown and NMS cotreatment. The possible mechanism of the results above was revealed by NMS treatment activating the Hippo pathway effector YAP1 and then managing the expression of phosphorylation PPARGC1A (Ser571). Together, these data showed that NMS promoted the fusion of mitochondria and protected mitochondrial function from mitochondrial unfolded protein response possibly via the NMUR2/YAP1/PPARGC1A pathway, thereby affecting the steroidogenesis of goat GCs. By elaborating the potential mechanism of NMS in regulating estrogen production in goat GCs, our results can serve as the mechanism reference for follicular growth and development.

Dichloroacetate and thiamine improve survival and mitochondrial stress in a C. elegans model of dihydrolipoamide dehydrogenase deficiency.

Dihydrolipoamide dehydrogenase (DLD) deficiency is a recessive mitochondrial disorder caused by depletion of DLD from α-ketoacid dehydrogenase complexes. Caenorhabditis elegans animal models of DLD deficiency generated by graded feeding of dld-1(RNAi) revealed that full or partial reduction of DLD-1 expression recapitulated increased pyruvate levels typical of pyruvate dehydrogenase complex deficiency and significantly altered animal survival and health, with reductions in brood size, adult length, and neuromuscular function. DLD-1 deficiency dramatically increased mitochondrial unfolded protein stress response induction and adaptive mitochondrial proliferation. While ATP levels were reduced, respiratory chain enzyme activities and in vivo mitochondrial membrane potential were not significantly altered. DLD-1 depletion directly correlated with the induction of mitochondrial stress and impairment of worm growth and neuromuscular function. The safety and efficacy of dichloroacetate, thiamine, riboflavin, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), l-carnitine, and lipoic acid supplemental therapies empirically used for human DLD disease were objectively evaluated by life span and mitochondrial stress response studies. Only dichloroacetate and thiamine showed individual and synergistic therapeutic benefits. Collectively, these C. elegans dld-1(RNAi) animal model studies demonstrate the translational relevance of preclinical modeling of disease mechanisms and therapeutic candidates. Results suggest that clinical trials are warranted to evaluate the safety and efficacy of dichloroacetate and thiamine in human DLD disease.

Impact of measuring heteroplasmy of a pathogenic mitochondrial DNA variant at the single-cell level in individuals with mitochondrial disease.

Pathogenic mitochondrial DNA heteroplasmy has mainly been assessed with bulk sequencing in individuals with mitochondrial disease. However, the distribution of heteroplasmy at the single-cell level in skin fibroblasts obtained from individuals, together with detailed clinical and biochemical information, remains to be investigated. We used the mitochondrial DNA single-cell assay for the transposase-accessible chromatin sequencing method. Skin fibroblasts were obtained from six individuals with mitochondrial disease and pathogenic m.3243A>G variants of differing severity. Different distributions of heteroplasmy at the single-cell level were identified in skin fibroblasts from all six individuals. Four individuals with different outcomes showed similar averaged heteroplasmy rates with normal mitochondrial respiratory chain enzyme activity, while the distribution of single-cell heteroplasmy patterns differed among the individuals. This study showed different heteroplasmy distribution patterns at the single-cell level in individuals with the m.3243A>G variant, who had a similar averaged heteroplasmy rates with normal mitochondrial respiratory chain enzyme activity. Whether such different heteroplasmy distribution patterns explain the different clinical outcomes should be assessed further in future studies. Measuring heteroplasmy of pathogenic mitochondrial DNA variants at the single-cell level could be important in individuals with mitochondrial disease.

Prospective association between maternal allostatic load during pregnancy and child mitochondrial content and bioenergetic capacity

BackgroundMitochondria are multifunctional energy-producing and signaling organelles that support life and contribute to stress adaptation. There is a growing understanding of the dynamic relationship between stress exposure and mitochondrial biology; however, the influence of stress on key domains of mitochondrial biology during early-life, particularly the earliest phases of intra-uterine/prenatal period remains largely unknown. Thus, the goal of this study was to examine the impact of fetal exposure to stress (modeled as the biological construct allostatic load) upon mitochondrial biology in early childhood. MethodsIn n = 30 children (range: 3.5–6 years, 53% male), we quantified mitochondrial content via citrate synthase (CS) activity and mtDNA copy number (mtDNAcn), and measured mitochondrial bioenergetic capacity via respiratory chain enzyme activities (complexes I (CI), II (CII), and IV (CIV)) in platelet-depleted peripheral blood mononuclear cells (PBMCs). In a cohort of healthy pregnant women, maternal allostatic load was operationalized as a latent variable (sum of z-scores) representing an aggregation of early-, mid- and late-gestation measures of neuroendocrine (cortisol), immune (interleukin-6, C-reactive protein), metabolic (homeostasis model assessment of insulin resistance, free fatty acids), and cardiovascular (aggregate systolic and diastolic blood pressure) systems, as well as an anthropometric indicator (pre-pregnancy body mass index [BMI]). ResultsAn interquartile increase in maternal allostatic load during pregnancy was associated with higher mitochondrial content (24% and 15% higher CS and mtDNAcn), and a higher mitochondrial bioenergetic capacity (16%, 23%, and 25% higher CI, CII and CIV enzymatic activities) in child leukocytes. The positive association between maternal allostatic load during pregnancy and child mitochondrial content and bioenergetic capacity remained significant after accounting for the effects of key pre- and post-natal maternal and child covariates (p’s < 0.05, except CI p = 0.073). ConclusionWe report evidence that prenatal biological stress exposure, modeled as allostatic load, was associated with elevated child mitochondrial content and bioenergetic capacity in early childhood. This higher mitochondrial content and bioenergetic capacity (per leukocyte) may reflect increased energetic demands at the immune or organism level, and thus contribute to wear-and-tear and pathophysiology, and/or programmed pro-inflammatory phenotypes. These findings provide potential mechanistic insight into the cellular processes underlying developmental programming, and support the potential role that changes in mitochondrial content and bioenergetic functional capacity may play in altering life-long susceptibility for health and disease.

Multicentric Standardization of Protocols for the Diagnosis of Human Mitochondrial Respiratory Chain Defects.

The quantification of mitochondrial respiratory chain (MRC) enzymatic activities is essential for diagnosis of a wide range of mitochondrial diseases, ranging from inherited defects to secondary dysfunctions. MRC lesion is frequently linked to extended cell damage through the generation of proton leak or oxidative stress, threatening organ viability and patient health. However, the intrinsic challenge of a methodological setup and the high variability in measuring MRC enzymatic activities represents a major obstacle for comparative analysis amongst institutions. To improve experimental and statistical robustness, seven Spanish centers with extensive experience in mitochondrial research and diagnosis joined to standardize common protocols for spectrophotometric MRC enzymatic measurements using minimum amounts of sample. Herein, we present the detailed protocols, reference ranges, tips and troubleshooting methods for experimental and analytical setups in different sample preparations and tissues that will allow an international standardization of common protocols for the diagnosis of MRC defects. Methodological standardization is a crucial step to obtain comparable reference ranges and international standards for laboratory assays to set the path for further diagnosis and research in the field of mitochondrial diseases.

17β-estradiol rescues the damage of thiazolidinedione on chicken Sertoli cell proliferation via adiponectin

Thiazolidinedione (TZD) is an oral anti-diabetic drug that exhibits some side effects on the male reproductive system by interfering with the steroidogenesis and androgenic activity and also shows anti-proliferative effect on several cell types. This study investigated the effect of TZD on immature chicken Sertoli cell (SC) proliferation and the potential mechanism by which 17β-estradiol regulated this process. Chicken SC viability was investigated under different treatment concentration and time of TZD. 17β-estradiol (0.001 μM, 24 h) was added to analyze its effects on TZD-mediated cell viability, cell metabolic activity, cell growth, cell cycle progression, reactive oxygen species (ROS) level, antioxidant enzyme activity, mitochondria activity, oxygen consumption rate, adenosine triphosphate (ATP) level, and mitochondrial respiratory chain enzyme activity, adiponectin expression and several cell proliferation-related genes mRNA and protein levels. We performed the microRNA (miRNA) array to find TZD-induced differentially expressed miRNAs and validated whether miR-1577 can target on adiponectin via the dual luciferase reporter assay, as well as verified the effect of adiponectin addition with different concentrations on the SC viability. Further, SCs were transfected with miR-1577 agomir (a double-stranded synthetic miRNA mimic) in the presence or absence of TZD and antagomir (a single-stranded synthetic miRNA inhibitor) in the presence or absence of 17β-estradiol to analyze whether miR-1577 was involved in TZD-mediated SC proliferation and whether 17β-estradiol regulated this process. Results showed that TZD significantly inhibited SC viability, cell metabolic activity, cell growth, and cell cycle progression, while increased adiponectin level and ROS generation. TZD-treated SCs presented decreases of antioxidant enzyme activity, mitochondria activity, basal and maximal respiration, ATP production and level, mitochondrial respiratory chain enzyme activity, and mRNA and protein expressions of several cell proliferation-related genes, as well as the significant alteration of miRNA expressions (a total number of 55 miRNAs were up-regulated whereas 53 miRNAs down-regulated). Whereas, 17β-estradiol played a positive role in chicken SC proliferation and rescued the damage of TZD on SC proliferation by up-regulating miR-1577 expression whose target gene was validated to be the adiponectin. In addition, exogenous adiponectin (more than 1 μg/ml) treatment exhibited a significant inhibition on the SC viability. Transfection of miR-1577 agomir promoted the SC proliferation via down-expressed adiponectin, and increased the mitochondrial function and cell proliferation-related gene expression, while TZD weakened the positive effect of miR-1577 agomir on SCs. On the other hand, transfection of miR-1577 antagomir inhibited SC proliferation by producing the opposite effects on above parameters, while 17β-estradiol attenuated the negative effect of miR-1577 antagomir on SCs. These findings suggest down-expressed miR-1577 is involved in the regulation of TZD-inhibited SC proliferation through increasing adiponectin level, and this damage of TZD on the immature chicken SC proliferation can be ameliorated by appropriate dose of exogenous 17β-estradiol treatment. This study provides an insight into the cytoprotective effect of 17β-estradiol on TZD-damaged SC proliferation and may suggest a potential strategy for reducing the risk of SC dysfunction caused by the abuse of TZD.

Association of mitochondrial respiratory chain enzymes with the risk and mortality of sepsis among Chinese children

BackgroundSepsis is a leading cause of pediatric morbidity and mortality worldwide. The aim of this study was to explore the association of decreased mitochondrial respiratory chain enzyme activities with the risk for pediatric sepsis, and explore their association with mortality among affected children.MethodsA total of 50 incident cases with sepsis and 49 healthy controls participated in this study. The level of serum coenzyme Q10 was measured by high-performance liquid chromatography, and selected mitochondrial respiratory chain enzymes in WBC were measured using spectrophotometric. Logistic regression models were used to estimate odds ratio (OR) and 95% confidence interval (CI).ResultsThe levels of CoQ10, complex II, complex I + III and FoF1-ATPase were significantly higher in healthy controls than in children with sepsis (p < 0.001, = 0.004, < 0.001 and < 0.001, respectively). In children with sepsis, levels of CoQ10 and complex I + III were significantly higher in survived cases than in deceased cases (p < 0.001). Per 0.05 μmol/L, 50 nmol/min.mg and 100 nmol/min.mg increment in CoQ10, complex I + III and FoF1-ATPase were associated with significantly lowered risk of having sepsis, even after adjusting for confounding factors (OR = 0.85, 0.68 and 0.04, p = 0.001, < 0.001 and < 0.001, respectively). Per 0.05 μmol/L and 50 nmol/min.mg increment in CoQ10 and complex I + III was associated with significantly lowered risk of dying from sepsis during hospitalization, and significance retained after adjustment (OR = 0.73 and 0.76, 95% CI: 0.59 to 0.90 and 0.64 to 0.89, p = 0.004 and 0.001, respectively) in children with sepsis.ConclusionsOur findings indicate the promising predictive contribution of low serum CoQ10 and complex I + III to the risk of pediatric sepsis and its associated mortality during hospitalization among Chinese children.Trial registration The trial was registered with www.chictr.org.cn, number ChiCTR-IOR-15006446 on May 05, 2015. Retrospectively registered.

Sesamol Supplementation Mitigates Isoproterenol-Induced Cardiac Toxicity in Rats by Stabilizing Cardiac Mitochondrial and Lysosomal Enzymes

The current investigation was intended to evaluate the antimyocardial ischemic effects of sesamol on lactate dehydrogenase (LDH) isoenzymes, DNA damage, and mitochondrial and lysosomal enzyme activities in isoproterenol (ISO)-induced myocardial infarction (MI) in male albino Wistar strain rats. Rats that received ISO (85 mg/kg body weight (B.W) subcutaneously) for the first 2 consecutive days showed significant reduction in the activities of tricarboxylic acid (TCA) cycle enzymes (isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and succinate dehydrogenase) and respiratory chain enzymes (cytochrome c oxidase and nicotinamide adenine dinucleotide hydrogen (NADH) dehydrogenase) in the heart mitochondria. The activities of the lysosomal enzymes (α-and β-glucosidases, α and β-galactosidases, β-glucuronidase and β-N-acetyl glucosaminidase and cathepsin-B and cathepsin-D) were increased significantly in the heart homogenate of ISO-induced MI rats. ISO injection also increased the % of tail DNA, tail length, and tail moment and decreased the % of head DNA. Pretreatment with sesamol (50 mg/kg B.W) every day for a period of 9 days prevented the above abnormalities induced by ISO. In conclusion, it can be inferred that administration of sesamol has a potent beneficial role against ISO-induced damage to the mitochondria, lysosomes, and DNA, thereby preventing MI.

Blood-based mitochondrial respiratory chain function in major depression

Mitochondrial dysfunction has been implicated in major depressive disorder (MDD). A measure of mitochondrial respiratory chain (RC) enzymatic activity—the Mitochondrial Health Index (MHI)—has previously been found to correlate with stress and emotional states in caregivers. We here report mitochondrial RC activities, mitochondrial DNA copy number (mtDNAcn), and the composite MHI in unmedicated and somatically healthy subjects with MDD (n = 47) and healthy controls (HC) (n = 11). We also explore, in a subset of the MDD sample (n = 33), whether these markers are associated with response to 8 weeks of SSRI treatment. Mitochondrial RC complexes I, II, IV, citrate synthase (CS), mtDNAcn, and the MHI were assayed in peripheral blood mononuclear cells. Treatment response was defined as >50% decrease on the 25-item Hamilton Depression Rating Scale (HRDS-25). There were no significant differences in MHI or any of the mitochondrial markers between MDD subjects and HCs. Compared to SSRI nonresponders, SSRI responders had significantly higher baseline mitochondrial content markers CS (p = 0.02) and mtDNAcn (p = 0.02), and higher complex I activity (p = 0.01). Complex II activity increased significantly over treatment, irrespective of clinical response (p = 0.03). Complex I activity decreased in responders (n = 9), but increased in nonresponders (n = 18) (group x time interaction, p = 0.02). Absolute treatment-associated change in HDRS-25 scores correlated significantly with change in complex I activity between baseline and week 8 (r = 0.47, p = 0.01). Although mitochondrial markers did not distinguish MDD from controls, they did distinguish SSRI responders from nonresponders. If larger studies validate these mitochondrial differences, they may become useful biomarkers and identify new drug targets.

Mitochondrial respiratory chain and Krebs cycle enzyme function in human donor livers subjected to end-ischaemic hypothermic machine perfusion.

IntroductionMarginal human donor livers are highly susceptible to ischaemia reperfusion injury and mitochondrial dysfunction. Oxygenation during hypothermic machine perfusion (HMP) was proposed to protect the mitochondria but the mechanism is unclear. Additionally, the distribution and uptake of perfusate oxygen during HMP are unknown. This study aimed to examine the feasibility of mitochondrial function analysis during end-ischaemic HMP, assess potential mitochondrial viability biomarkers, and record oxygenation kinetics.MethodsThis was a randomised pilot study using human livers retrieved for transplant but not utilised. Livers (n = 38) were randomised at stage 1 into static cold storage (n = 6), hepatic artery HMP (n = 7), and non-oxygen supplemented portal vein HMP (n = 7) and at stage 2 into oxygen supplemented and non-oxygen supplemented portal vein HMP (n = 11 and 7, respectively). Mitochondrial parameters were compared between the groups and between low- and high-risk marginal livers based on donor history, organ steatosis and preservation period. The oxygen delivery efficiency was assessed in additional 6 livers using real-time measurements of perfusate and parenchymal oxygen.ResultsThe change in mitochondrial respiratory chain (complex I, II, III, IV) and Krebs cycle enzyme activity (aconitase, citrate synthase) before and after 4-hour preservation was not different between groups in both study stages (p > 0.05). Low-risk livers that could have been used clinically (n = 8) had lower complex II-III activities after 4-hour perfusion, compared with high-risk livers (73 nmol/mg/min vs. 113 nmol/mg/min, p = 0.01). Parenchymal pO2 was consistently lower than perfusate pO2 (p ≤ 0.001), stabilised in 28 minutes compared to 3 minutes in perfusate (p = 0.003), and decreased faster upon oxygen cessation (75 vs. 36 minutes, p = 0.003).ConclusionsActively oxygenated and air-equilibrated end-ischaemic HMP did not induce oxidative damage of aconitase, and respiratory chain complexes remained intact. Mitochondria likely respond to variable perfusate oxygen levels by adapting their respiratory function during end-ischaemic HMP. Complex II-III activities should be further investigated as viability biomarkers.

Mitochondrial phenotypes in purified human immune cell subtypes and cell mixtures.

Using a high-throughput mitochondrial phenotyping platform to quantify multiple mitochondrial features among molecularly defined immune cell subtypes, we quantify the natural variation in mitochondrial DNA copy number (mtDNAcn), citrate synthase, and respiratory chain enzymatic activities in human neutrophils, monocytes, B cells, and naïve and memory T lymphocyte subtypes. In mixed peripheral blood mononuclear cells (PBMCs) from the same individuals, we show to what extent mitochondrial measures are confounded by both cell type distributions and contaminating platelets. Cell subtype-specific measures among women and men spanning four decades of life indicate potential age- and sex-related differences, including an age-related elevation in mtDNAcn, which are masked or blunted in mixed PBMCs. Finally, a proof-of-concept, repeated-measures study in a single individual validates cell type differences and also reveals week-to-week changes in mitochondrial activities. Larger studies are required to validate and mechanistically extend these findings. These mitochondrial phenotyping data build upon established immunometabolic differences among leukocyte subpopulations, and provide foundational quantitative knowledge to develop interpretable blood-based assays of mitochondrial health.