Skeletal muscle dysfunction is associated with derangements in mitochondrial bioenergetics (but not UCP3) in a rodent model of sepsis

Muscle dysfunction is a common feature of severe sepsis and multi-organ failure. Recent evidence implicates bioenergetic dysfunction and oxidative damage as important underlying pathophysiological mechanisms. Increased abundance of uncoupling protein-3 (UCP-3) in sepsis suggests increased mitochondrial proton leak, which may reduce mitochondrial coupling efficiency but limit ROS production. Using a murine model, we examined metabolic, cardiovascular and skeletal muscle contractile changes following induction of peritoneal sepsis in wild-type and Ucp3(-/-) mice. Mitochondrial membrane potential (Δψm) was measured using two-photon microscopy in living diaphragm, and contractile function was measured in diaphragm muscle strips. The kinetic relationship between membrane potential and oxygen consumption was determined using a modular kinetic approach in isolated mitochondria. Sepsis was associated with significant whole body metabolic suppression, hypothermia and cardiovascular dysfunction. Maximal force generation was reduced and fatigue accelerated in ex vivo diaphragm muscle strips from septic mice. Mitochondrial membrane potential was lower in the isolated diaphragm from septic mice despite normal substrate oxidation kinetics and proton leak in skeletal muscle mitochondria. Even though wild-type mice exhibited an absolute 26 ± 6% higher UCP-3 protein abundance at 24 hours, no differences were seen in whole animal or diaphragm physiology, nor in survival rates, between wild-type and Ucp3(-/-) mice. In conclusion, this murine sepsis model shows a hypometabolic phenotype with evidence of significant cardiovascular and muscle dysfunction. This was associated with lower Δψm and alterations in mitochondrial ATP turnover and phosphorylation pathway. However, UCP-3 does not play an important functional role, despite its upregulation

Mitochondrial Activity and Skeletal Muscle Insulin Resistance in Kidney Disease

Insulin resistance is a key feature of the metabolic syndrome, a cluster of medical disorders that together increase the chance of developing type 2 diabetes and cardiovascular disease. In turn, type 2 diabetes may cause complications such as diabetic kidney disease (DKD). Obesity is a major risk factor for developing systemic insulin resistance, and skeletal muscle is the first tissue in susceptible individuals to lose its insulin responsiveness. Interestingly, lean individuals are not immune to insulin resistance either. Non-obese, non-diabetic subjects with chronic kidney disease (CKD), for example, exhibit insulin resistance at the very onset of CKD, even before clinical symptoms of renal failure are clear. This uraemic insulin resistance contributes to the muscle weakness and muscle wasting that many CKD patients face, especially during the later stages of the disease. Bioenergetic failure has been associated with the loss of skeletal muscle insulin sensitivity in obesity and uraemia, as well as in the development of kidney disease and its sarcopenic complications. In this mini review, we evaluate how mitochondrial activity of different renal cell types changes during DKD progression, and discuss the controversial role of oxidative stress and mitochondrial reactive oxygen species in DKD. We also compare the involvement of skeletal muscle mitochondria in uraemic and obesity-related muscle insulin resistance.</jats:p

A high-sensitivity electrochemiluminescence-based ELISA for the measurement of the oxidative stress biomarker, 3-nitrotyrosine, in human blood serum and cells

The generation of 3-nitrotyrosine, within proteins, is a post-translational modification resulting from oxidative or nitrative stress. It has been suggested that this modification could be used as a biomarker for inflammatory diseases. Despite the superiority of mass spectrometry-based determinations of nitrotyrosine, in a high-throughput clinical setting the measurement of nitrotyrosine by an enzyme-linked immunosorbent assay (ELISA) is likely to be more cost-effective. ELISAs offer an alternative means to detect nitrotyrosine, but many commercially available ELISAs are insufficiently sensitive to detect nitrotyrosine in healthy human serum. Here, we report the development, validation and clinical application of a novel electrochemiluminescence-based ELISA for nitrotyrosine which provides superior sensitivity (e.g. a 50-fold increase in sensitivity compared with one of the tested commercial colorimetric ELISAs). This nitrotyrosine ELISA has the following characteristics: a lower limit of quantitation of 0.04 nM nitrated albumin equivalents; intra- and inter-assay coefficients of variation of 6.5% and 11.3%, respectively; a mean recovery of 106 ± 3% and a mean linearity of 0.998 ± 0.001. Far higher nitration levels were measured in normal human blood cell populations when compared to plasma. Mass spectrometry was used to validate the new ELISA method. The analysis of the same set of chemically modified albumin samples using the ELISA method and mass spectrometry showed good agreement for the relative levels of nitration present in each sample. The assay was applied to serum samples from patients undergoing elective surgery which induces the human inflammatory response. Matched samples were collected before and one day after surgery. An increase in nitration was detected following surgery (median (IQR): 0.59 (0.00–1.34) and 0.97 (0.00–1.70) nitrotyrosine (fmol of nitrated albumin equivalents/mg protein) for pre- and post-surgery respectively. The reported assay is suitable for nitrotyrosine determination in patient serum samples, and may also be applicable as a means to determine oxidative stress in primary and cultured cell populations

Measurement of H₂O₂ within living drosophila during aging using a ratiometric mass spectrometry probe targeted to the mitochondrial matrix

Hydrogen peroxide (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; correlates with aging, it may not be causative

Regulation of the plant alternative oxidase by pyruvate

In addition to a conventional cytochrome pathway, plant mitochondria contain a second, nonprotonmotive route of electron transfer, comprised of a single ubiquinol:oxygen oxidoreductase, the alternative oxidase (AOX). This enzyme is regulated by two interrelated posttranslational mechanisms. Firstly, reduction of an intermolecular disulphide bond results in an ‘activated’ noncovalently linked dimer. Secondly, the reduced protein is further stimulated by a-keto acids, most notably pyruvate. This is thought to occur via a-keto acid interaction with a well-conserved cysteine residue. Previously, we have established a system to functionally express the Sauromatum guttatum alternative oxidase (Sg-AOX) in the fission yeast Schizosaccharomyces pombe. Interestingly, the resulting antimycin-resistant respiratory activity in isolated yeast mitochondria, does not appear to be stimulated by pyruvate. Here, we report on the expression of both Sg-AOX as well as a second plant isozyme, the Arabidopsis thaliana AOX1a protein (At-AOX1a), in the same yeast system. In contrast to Sg-AOX, At-AOX1a-dependent activity can be stimulated by pyruvate f1.4-fold in isolated yeast mitochondria and f4.5-fold in isolated mitochondrial membranes depleted of endogenous pyruvate. Whilst Sg AOX activity is confirmed to be completely independent of pyruvate, its dependence upon the Q-redox poise would suggest that it is in a constitutively active state, comparable to the ‘pyruvate-activated’ kinetic dependence of At AOX1a. These data indicate that Sg AOX is the first example of a plant enzyme that appears to function without a dependence on organic acids for full activity. This finding is of particular interest, given that both Sg AOX and At AOX1a conserve the cysteine residue believed to interact directly with pyruvate. As both proteins exhibit a very similar primary structure, we have been able to identify structural components, additional to the regulatory cysteine, that may account for the differences in the regulatory behaviour reported here. The implications of such findings are discussed in terms of the proposed structure of AOX

Increased growth in transgenic tobacco plants over-expressing the mtHSP70 homologue, PHSP1: Towards an understanding of the underlying mechanism

Matrix-located mitochondrial heat-shock proteins (mtHSP70s) have been identified in all eukaryotic systems studied to date and it is becoming clear that these chaperones have diverse functions. The yeast mtHSP70 is essential for the import and subsequent folding of newly synthesised proteins from the cytosol into the mitochondrial matrix. In addition to its role in mitochondrial protein import, it has been suggested that the plant mtHSP70 may also be involved in the phosphorylation of enzyme complexes, whilst a further role for the yeast protein is thought to be in the modulation of mitochondrial endonuclease activity. PHSP1 is a mtHSP70 homologue from pea leaf. It has been previously shown by our laboratory that overexpression of this protein in tobacco results in increased growth, relative to wild type, during seedling development. Levels of metabolites and maximum extractable activities of enzymes involved in carbon and nitrogen metabolism are presented here, together with respiratory and morphological analysis of leaf tissue from these plants. Data are discussed with respect to how these transgenic plants may aid our understanding of the metabolic roles that mitochondria play during plant development, with particular emphasis on carbon and nitrogen metabolis

Improved Survival in a Long-Term Rat Model of Sepsis Is Associated With Reduced Mitochondrial Calcium Uptake Despite Increased Energetic Demand

To investigate the relationship between prognosis, changes in mitochondrial calcium uptake, and bioenergetic status in the heart during sepsis

Early functional and transcriptomic changes in the myocardium predict outcome in a long-term rat model of sepsis

Myocardial function is depressed in sepsis and is an important prognosticator in the human condition. Using echocardiography in a long-term fluid-resuscitated Wistar rat model of faecal peritonitis we investigated whether depressed myocardial function could be detected at an early stage of sepsis and, if so, whether the degree of depression could predict eventual outcome. At 6 h post-insult, a stroke volume <0.17 ml prognosticated 3-day mortality with positive and negative predictive values of 93 and 80%, respectively. Subsequent fluid loading studies demonstrated intrinsic myocardial depression with poor-prognosis animals tolerating less fluid than either good-prognosis or sham-operated animals. Cardiac gene expression analysis at 6 h detected 527 transcripts significantly up- or down-regulated by the septic process, including genes related to inflammatory and cell cycle pathways. Predicted mortality was associated with significant differences in transcripts of genes expressing proteins related to the TLR2/MyD88 (Toll-like receptor 2/myeloid differentiation factor 88) and JAK/STAT (Janus kinase/signal transducer and activator of transcription) inflammatory pathways, β-adrenergic signalling and intracellular calcium cycling. Our findings highlight the presence of myocardial depression in early sepsis and its prognostic significance. Transcriptomic analysis in heart tissue identified changes in signalling pathways that correlated with clinical dysfunction. These pathways merit further study to both better understand and potentially modify the disease process