Cardiac-specific overexpression of thioredoxin 1 attenuates mitochondrial and myocardial dysfunction in septic mice.

Sepsis-induced myocardial dysfunction is associated with increased oxidative stress and mitochondrial dysfunction. Current evidence suggests a protective role of thioredoxin-1 (Trx1) in the pathogenesis of cardiovascular diseases. However, it is unknown yet a putative role of Trx1 in sepsis-induced myocardial dysfunction, in which oxidative stress is an underlying cause. Transgenic male mice with Trx1 cardiac-specific overexpression (Trx1-Tg) and its wild-type control (wt) were subjected to cecal ligation and puncture or sham surgery. After 6, 18, and 24h, cardiac contractility, antioxidant enzymes, protein oxidation, and mitochondrial function were evaluated. Trx1 overexpression improved the average life expectancy (Trx1-Tg: 36, wt: 28h; p=0.0204). Sepsis induced a decrease in left ventricular developed pressure in both groups, while the contractile reserve, estimated as the response to ß-adrenergic stimulus, was higher in Trx1-Tg in relation to wt, after 6h of the procedure. Trx1 overexpression attenuated complex I inhibition, protein carbonylation, and loss of membrane potential, and preserved Mn superoxide dismutase activity at 24h. Ultrastructural alterations in mitochondrial cristae were accompanied by reduced optic atrophy 1 (OPA1) fusion protein, and activation of dynamin-related protein 1 (Drp1) (fission protein) in wt mice at 24h, suggesting mitochondrial fusion/fission imbalance. PGC-1α gene expression showed a 2.5-fold increase in Trx1-Tg at 24h, suggesting mitochondrial biogenesis induction. Autophagy, demonstrated by electron microscopy and increased LC3-II/LC3-I ratio, was observed earlier in Trx1-Tg. In conclusion, Trx1 overexpression extends antioxidant protection, attenuates mitochondrial damage, and activates mitochondrial turnover (mitophagy and biogenesis), preserves contractile reserve and prolongs survival during sepsis.

The pan-caspase inhibitor Emricasan (IDN-6556) decreases liver injury and fibrosis in a murine model of non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Hepatocyte apoptosis, the hallmark of non-alcoholic steatohepatitis (NASH) contributes to liver injury and fibrosis. Although, both the intrinsic and extrinsic apoptotic pathways are involved in the pathogenesis of NASH, the final common step of apoptosis is executed by a family of cysteine-proteases termed caspases. Thus, our aim was to ascertain if administration of Emricasan, a pan-caspase inhibitor, ameliorates liver injury and fibrosis in a murine model of NASH. METHODS: C57/BL6J-mice were fed regular chow or high fat diet (HFD) for 20 weeks. All mice were treated with vehicle or Emricasan. RESULTS: Mice fed a HFD diet demonstrate a five-fold increase in hepatocyte apoptosis by the TUNEL assay and a 1.5-fold and 1.3-fold increase in caspase-3 and-8 activities respectively; this increase in apoptosis was substantially attenuated in mice fed a HFD treated with Emricasan (HFD-Em). Likewise, liver injury and inflammation were reduced in mice fed HFD-Em as compare to HFD by measuring serum aspartate aminotransferase and alanine aminotransferase levels, NAS histological score and IL 1-ß, TNF-α, monocyte chemoattractant protein (MCP-1) and C-X-C chemokine ligand-2 (CXCL2) quantitative reverse-transcription polymerase chain reaction (qPCR). These differences could not be attributed to differences in hepatic steatosis as liver triglycerides content were similar in both HFD groups. Hepatic fibrosis was reduced by Emricasan in HFD animals by decreasing αSMA (a marker for hepatic stellate cell activation), fibrosis score, Sirius red staining, hydroxyproline liver content and profibrogenic cytokines by qPCR. CONCLUSION: In conclusion, these data demonstrate that in a murine model of NASH, liver injury and fibrosis are suppressed by inhibiting hepatocytes apoptosis and suggests that Emricasan may be an attractive antifibrotic therapy in NASH.

Calidad en el laboratorio de urgencias: determinación del estado ácido-base / Quality in the emergency laboratory: blood gas determination / Qualidade no laboratorio de urgências: determinaçâo do estado ácido-base

El procesamiento de una muestra en el laboratorio implica su paso a través de tres etapas: la preanalítica, la analítica y la postanalítica. En el presente trabajo se propuso analizar la etapa preanalítica para la determinación del estado ácido-base (EAB) a través de la comparación de muestras extraídas en diferentes tipos de jeringas y del efecto del tiempo de almacenamiento, estudiar el desempeño analítico y la concordancia entre dos analizadores gemelos y evaluar si los resultados permiten trabajar bajo las especificaciones de calidad requeridas. Para ello se utilizaron 2 microprocesadores automáticos de gases en sangre en los cuales se midieron pH, pCO2 y pO2, y se fijaron las especificaciones de calidad derivadas de CLIA (Clinical Laboratory Improvement Amendments) como requerimento a cumplir. La imprecisión intraensayo (CVi) y entreensayos (CVe) fue determinada con material de control comercial. Sobre un total de 239 muestras de pacientes, 20 se utilizaron para realizar la comparación entre muestras extraídas en jeringas con heparina sódica en solución y con heparina de litio liofilizada; 110 muestras se procesaron para evaluar el efecto del tiempo de almacenamiento y 109 para la comparación entre ambos instrumentos. Los CVi y CVe fueron aceptables para los 3 parámetros. Las diferencias halladas entre las muestras obtenidas con las diferentes jeringas no superaron los límites permitidos. No se observaron diferencias significativas en los resultados hasta los 120 minutos de procesamiento. Los coeficientes de correlación de las muestras procesadas en ambos instrumentos fueron 0,931 para pH, 0,985 para pCO2 y 0,950 para pO2. El sesgo fue aceptable así como también el porcentaje de equivalencia clínica para los tres parámetros. Se concluye que la evaluación de los resultados de instrumentos gemelos y la implementación de programas sistemáticos de conmutabilidad son fundamentales para conseguir la disminución progresiva del error y para poder utilizarlos indistintamente.

Measurement of blood gas, as another sample, involves three phases: preanalytical, analytical and postanalytical. The aim of this study is to evaluate the preanalytical phase comparing samples obtained from different syringes and the effect of storage time; to study the analytical performance; to compare the agreement between two instruments, and to analyse if the results were satisfactory taking into account CLIA specifications. Two microanalyzers were used, in which pH, pCO2 and pO2 were tested. Within-day imprecisions (CVi) and between-day imprecisions (CVe) were obtained with control materials. Two hundred and thirty-nine patient-samples were used; 20 were used for the comparison between samples obtained with heparinized syringes (sodium liquid heparin) and dry lithium heparin); 110 were processed to evaluate the effect of storage time, and 109 for the comparison between the analyzers. CVi and CVe were acceptable for the three parameters. The difference between the different syringes did not exceed the acceptable limits. No significant difference was noticed until 120 min of storage. The correlation coefficients for processed samples in both instruments were 0.931 for pH, 0.985 for PCO2 and 0.950 for PO2. The bias was acceptable as the clinical equivalence for the three parameters. Evaluating the results between identical instruments and introducing a systematic quality program has an important role in obtaining a progressive decrease of the total error between them, and using one or the other without distinction.

O processamento de uma amostra no laboratório envolve sua passagem através de três etapas: a pré-analítica, a analítica e a pós-analitica. No presente trabalho o objetivo foi analisar a etapa pré-analítica para a determinalo do estado ácido base (EAB) através da comparaçâo de amostras extraídas em diferentes tipos de seringas e do efeito do tempo de armazenamento, estudar o desempenho analítico e a concordancia entre dois analisadores idénticos e avaliar se os resultados permitem trabalhar sob as especificações de qualidade requeridas. Para isso foram utilizados 2 microprocessadores automáticos de gases em sangue nos quais se mediram pH, pCO2 e pO2, e se estabeleceram as especificagóes de qualidade decorrentes de CLIA (Clinical Laboratory Improvement Amendments) como requerimento a cumprir. A imprecisâo intraensaio (CVi) e entre ensaios (CVe) foi determinada com material de controle comercial. De um total de 239 amostras de pacientes, 20 foram utilizadas para realizar a comparaçâo entre amostras extraídas em seringas com heparina sódica em soluçâo e com heparina de litio liofilizada; 110 amostras foram processadas para avaliar o efeito do tempo de armazenamento e 109 para a comparaçâo entre ambos os instrumentos. Os CVi e CVe foram aceitáveis para os 3 parámetros. As diferengas encontradas entre as amostras obtidas com as diferentes seringas não superaram os limites permitidos. Nâo foram observadas diferengas significativas nos resultados atéos 120 minutos de processamento. Os coeficientes de correlaçâo das amostras processadas em ambos os instrumentos foram 0,931 para pH, 0,985 para pCO2 e 0,950 para pO2. O viés foi aceitável bem como também o percentual de equivaléncia clínica para os trés parámetros. A conclusâo é que a avaliaçâo dos resultados de instrumentos idénticos e a implementaçâo de programas sistemáticos de comutabilidade sâo fundamentais para conseguir a diminuiçâo progressiva do erro e para poder utilizálos indistintamente.

Defective leptin-AMP-dependent kinase pathway induces nitric oxide release and contributes to mitochondrial dysfunction and obesity in ob/ob mice.

AIMS: Obesity arises on defective neuroendocrine pathways that increase energy intake and reduce mitochondrial metabolism. In the metabolic syndrome, mitochondrial dysfunction accomplishes defects in fatty acid oxidation and reciprocal increase in triglyceride content with insulin resistance and hyperglycemia. Mitochondrial inhibition is attributed to reduced biogenesis, excessive fission, and low adipokine-AMP-activated protein kinase (AMPK) level, but lateness of the respiratory chain contributes to perturbations. Considering that nitric oxide (NO) binds cytochrome oxidase and inhibits respiration, we explored NO as a direct effector of mitochondrial dysfunction in the leptin-deficient ob/ob mice. RESULTS: A remarkable three- to fourfold increase in neuronal nitric oxide synthase (nNOS) expression and activity was detected by western blot, citrulline assay, electronic and confocal microscopy, flow cytometry, and NO electrode sensor in mitochondria from ob/ob mice. High NO reduced oxygen uptake in ob/ob mitochondria by inhibition of complex IV and nitration of complex I. Low metabolic status restricted ß-oxidation in obese mitochondria and displaced acetyl-CoA to fat synthesis; instead, small interference RNA nNOS caused a phenotype change with fat reduction in ob/ob adipocytes. INNOVATION: We evidenced that leptin increases mitochondrial respiration and fat utilization by potentially inhibiting NO release. Accordingly, leptin administration to ob/ob mice prevented nNOS overexpression and mitochondrial dysfunction in vivo and rescued leptin-dependent effects by matrix NO reduction, whereas leptin-Ob-Rb disruption increased the formation of mitochondrial NO in control adipocytes. We demonstrated that in ob/ob, hypoleptinemia is associated with critically low mitochondrial p-AMPK and that, oppositely to p-Akt2, p-AMPK is a negative modulator of nNOS. CONCLUSION: Thereby, defective leptin-AMPK pathway links mitochondrial NO to obesity with complex I syndrome and dysfunctional mitochondria.

Mitochondrial nitric oxide synthase: a masterpiece of metabolic adaptation, cell growth, transformation, and death.

Mitochondria are specialized organelles that control energy metabolism and also activate a multiplicity of pathways that modulate cell proliferation and mitochondrial biogenesis or, conversely, promote cell arrest and programmed cell death by a limited number of oxidative or nitrative reactions. Nitric oxide (NO) regulates oxygen uptake by reversible inhibition of cytochrome oxidase and the production of superoxide anion from the mitochondrial electron transfer chain. In this sense, NO produced by mtNOS will set the oxygen uptake level and contribute to oxidation-reduction reaction (redox)-dependent cell signaling. Modulation of translocation and activation of neuronal nitric oxide synthase (mtNOS activity) under different physiologic or pathologic conditions represents an adaptive response properly modulated to adjust mitochondria to different cell challenges.

Control of muscle mitochondria by insulin entails activation of Akt2-mtNOS pathway: implications for the metabolic syndrome.

BACKGROUND: In the metabolic syndrome with hyperinsulinemia, mitochondrial inhibition facilitates muscle fat and glycogen accumulation and accelerates its progression. In the last decade, nitric oxide (NO) emerged as a typical mitochondrial modulator by reversibly inhibiting citochrome oxidase and oxygen utilization. We wondered whether insulin-operated signaling pathways modulate mitochondrial respiration via NO, to alternatively release complete glucose oxidation to CO(2) and H(2)O or to drive glucose storage to glycogen. METHODOLOGY/PRINCIPAL FINDINGS: We illustrate here that NO produced by translocated nNOS (mtNOS) is the insulin-signaling molecule that controls mitochondrial oxygen utilization. We evoke a hyperinsulinemic-normoglycemic non-invasive clamp by subcutaneously injecting adult male rats with long-lasting human insulin glargine that remains stable in plasma by several hours. At a precise concentration, insulin increased phospho-Akt2 that translocates to mitochondria and determines in situ phosphorylation and substantial cooperative mtNOS activation (+4-8 fold, P<.05), high NO, and a lowering of mitochondrial oxygen uptake and resting metabolic rate (-25 to -60%, P<.05). Comparing in vivo insulin metabolic effects on gastrocnemius muscles by direct electroporation of siRNA nNOS or empty vector in the two legs of the same animal, confirmed that in the silenced muscles disrupted mtNOS allows higher oxygen uptake and complete (U-(14)C)-glucose utilization respect to normal mtNOS in the vector-treated ones (respectively 37+/-3 vs 10+/-1 micromolO(2)/h.g tissue and 13+/-1 vs 7.2+/-1 micromol (3)H(2)O/h.g tissue, P<.05), which reciprocally restricted glycogen-synthesis by a half. CONCLUSIONS/SIGNIFICANCE: These evidences show that after energy replenishment, insulin depresses mitochondrial respiration in skeletal muscle via NO which permits substrates to be deposited as macromolecules; at discrete hyperinsulinemia, persistent mtNOS activation could contribute to mitochondrial dysfunction with insulin resistance and obesity and therefore, to the progression of the metabolic syndrome.

The biological significance of mtNOS modulation.

In the last years, nitric oxide synthases (NOS) have been localized in mitochondria. At this site, NO yield directly regulates the activity of cytochrome oxidase, O(2) uptake and the production of reactive oxygen species. Recent studies showed that translocated neuronal nitric oxide synthase (nNOS) is posttranslationally modified including phosphorylation at Ser 1412 (in mice) and myristoylation in an internal residue. Different studies confirm that modified nNOS alpha is the main modulable isoform in mitochondria. Modulation of mtNOS was observed in different situations, like adaptation to reduced O(2) availability and hypoxia, adaptation to low environmental temperature, and processes linked to life and death by effects on kinases and transcription factors. We present here evidence about the role of mtNOS in the analyzed conditions.