Skeletal Muscle Homogenates Research Articles

Assessment of Na+/K+ ATPase Activity in Small Rodent and Human Skeletal Muscle Samples.

In skeletal muscle, the Na/K ATPase (NKA) plays essential roles in processes linked to muscle contraction, fatigue, and energy metabolism; however, very little information exists regarding the regulation of NKA activity. The scarcity of information regarding NKA function in skeletal muscle likely stems from methodological constraints, as NKA contributes minimally to total cellular ATP utilization, and therefore contamination from other ATPases prevents the assessment of NKA activity in muscle homogenates. Here we introduce a method that improves accuracy and feasibility for the determination of NKA activity in small rodent muscle samples (5-10 mg) and in human skeletal muscle. Skeletal muscle homogenates from mice (n = 6) and humans (n = 3) were used to measure NKA and sarcoplasmic reticulum Ca ATPase (SERCA) activities with the addition of specific ATPase inhibitors to minimize "background noise." We observed that myosin ATPase activity was the major interfering factor for estimation of NKA activity in skeletal muscle homogenates, as the addition of 25 μM of blebbistatin, a specific myosin ATPase inhibitor, considerably minimized "background noise" (threefold) and enabled the determination of NKA maximal activity with values three times higher than previously reported. The specificity of the assay was demonstrated after the addition of 2 mM ouabain, which completely inhibited NKA. On the other hand, the addition of blebbistatin did not affect the ability to measure SERCA function. The coefficient of variation for NKA and SERCA assays were 6.2% and 4.4%, respectively. The present study has improved the methodology to determine NKA activity. We further show the feasibility of measuring NKA and SERCA activities from a common muscle homogenate. This methodology is expected to aid in our long-term understanding of how NKA affects skeletal muscle metabolic homeostasis and contractile function in diverse situations.

Adropin reduces blood glucose levels in mice by limiting hepatic glucose production.

Adropin is a liver‐ and brain‐secreted peptide hormone with striking effects on fuel metabolism regulation in a number of tissues. Previous studies demonstrated that adropin secretion is decreased in obese mice subjected to a long‐term high‐fat diet (HFD), and that whole‐body loss of adropin expression resulted in systemic insulin resistance. Treatment of obese mice with adropin improves glucose tolerance, which has been linked to increased glucose oxidation and inhibition of fatty acid utilization in isolated skeletal muscle homogenates. In this study, we used in vivo physiological measurements to determine how treatment of obese mice with adropin affects whole‐body glucose metabolism. Treatment with adropin reduced fasting blood glucose and, as shown previously, increased glucose tolerance in HFD mice during standard glucose tolerance tests. Under hyperinsulinemic‐euglycemic clamp conditions, adropin treatment led to a nonsignificant increase in whole‐body insulin sensitivity, and a significant reduction in whole‐body glucose uptake. Finally, we show that adropin treatment suppressed hepatic glucose production and improved hepatic insulin sensitivity. This correlated with reduced expression of fatty acid import proteins and gluconeogenic regulatory enzymes in the liver, suggesting that adropin treatment may impact the pathways that drive vital aspects of hepatic glucose metabolism.

Adropin treatment restores cardiac glucose oxidation in pre-diabetic obese mice

Exposure to a high fat (HF) diet promotes increased fatty acid uptake, fatty acid oxidation and lipid accumulation in the heart. These maladaptive changes impact cellular energy metabolism and may promote the development of cardiac dysfunction. Attempts to increase cardiac glucose utilization have been proposed as a way to reverse cardiomyopathy in obese and diabetic individuals. Adropin is a nutrient-regulated metabolic hormone shown to promote glucose oxidation over fatty acid oxidation in skeletal muscle homogenates in vitro. The focus of the current study was to investigate whether adropin can regulate substrate metabolism in the heart following prolonged exposure to a HF diet in vivo. Mice on a long-term HF diet received serial intraperitoneal injections of vehicle or adropin over three days. Cardiac glucose oxidation was significantly reduced in HF animals, which was rescued by acute adropin treatment. Significant decreases in cardiac pyruvate dehydrogenase activity were observed in HF animals, which were also reversed by adropin treatment. In contrast to previous studies, this change was unrelated to Pdk4 expression, which remained elevated in both vehicle- and adropin-treated HF mice. Instead, we show that adropin modulated the expression of the mitochondrial acetyltransferase enzyme GCN5L1, which altered the acetylation status and activity of fuel metabolism enzymes to favor glucose utilization. Our findings indicate that adropin exposure leads to increased cardiac glucose oxidation under HF conditions, and may provide a future therapeutic avenue in the treatment of diabetic cardiomyopathy.

Abstracts of the 11th International Conference on Cachexia, Sarcopenia and Muscle Wasting, Maastricht, The Netherlands, 7–9 December 2018 (Part 2)

Abstracts of the 11^th International Conference on Cachexia, Sarcopenia and Muscle Wasting, Maastricht, The Netherlands, 7–9 December 2018 (Part 2)

Potential adverse effects of botanical supplementation in high-fat-fed female mice

BackgroundInsulin resistance underlies metabolic syndrome and is associated with excess adiposity and visceral fat accumulation, which is more frequently observed in males than females. However, in young females, the prevalence of metabolic syndrome is rising, mainly driven by accumulation of abdominal visceral fat. The degree to which sex-related differences could influence the development of insulin resistance remains unclear, and studies of potential therapeutic strategies to combat metabolic syndrome using rodent models have focused predominantly on males. We therefore evaluated the effects of two nutritional supplements derived from botanical sources, an extract of Artemisia dracunculus L. (termed PMI5011) and Momordica charantia (commonly known as bitter melon), on female mice challenged with a high-fat diet in order to determine if dietary intake of these supplements could ameliorate obesity-induced insulin resistance and metabolic inflexibility in skeletal muscle.MethodsBody composition, physical activity and energy expenditure, fatty acid oxidation, insulin signaling, and gene and protein expression of factors controlling lipid metabolism and ectopic lipid accumulation were evaluated in female mice fed a high-fat diet supplemented with either PMI5011 or bitter melon. Statistical significance was assessed by unpaired two-tailed t test and repeated measures ANOVA.ResultsPMI5011 supplementation resulted in increased body weight and adiposity, while bitter melon did not induce changes in these parameters. Pyruvate tolerance testing indicated that both supplements increased hepatic glucose production. Both supplements induced a significant suppression in fatty acid oxidation in skeletal muscle homogenates treated with pyruvate, indicating enhanced metabolic flexibility. PMI5011 reduced lipid accumulation in skeletal muscle, while bitter melon induced a downward trend in lipid accumulation in the skeletal muscle and liver. This was accompanied by transcriptional regulation of autophagic genes by bitter melon in the liver.ConclusionsData from the current study indicates that dietary supplementation with PMI5011 and bitter melon evokes a divergent, and generally less favorable, set of metabolic responses in female mice compared to effects previously observed in males. Our findings underscore the importance of considering sex-related variations in responses to dietary supplementation aimed at combating metabolic syndrome.

ACTOPROTECTIVE ACTIVITY OF THE COMPLEX PLANT REMEDY

Background. Intensive physical exertion is one of the stress-factors leading to the decrease of non-specific resistance of the body, significant increase in oxygen consumption followed by activation of the lipid peroxidation and decrease of endogenic antioxidant system efficacy.The aim of the present study was to evaluate the influence of the complex plant remedy on exercise tolerance of laboratory animals and to reveal mechanisms increasing physical endurance.Materials and methods. The experiments were carried out on white Wistar rats weighing 190-220 g. The physical endurance of the animals was estimated by recording time of weighted swimming to complete exhaustion in the pool. The weighting was 7 % of the body mass. The water solution of the dry extract derived from five medicinal plants was introduced intragastrically to the animals of the experimental group at the dose of 100 mg/kg in the volume of 10 ml/kg for 7 days (once a day), an hour before testing. The content of ATP, lactic acid and pyruvic acid were determined in the skeletal muscle homogenate; glycogen was determined in the liver homogenate; in the blood serum there was estimated concentration of malonic dialdehyde, the activity of catalase; the concentration of reduced glutathione was determined in the whole blood and the activity of superoxide dismutase was estimated in erythrocytes.Conclusion. The course introduction of the complex plant remedy to rats results in resilience to intensive physical exertion due to optimization of energy metabolism, inhibition of free-radical biomacromolecule oxidation processes and increase in endogenic antioxidant system activity.

Kvβ subunit interacts with NEDD4 leading to decreased mouse skeletal muscle size.

BackgroundThe voltage‐gated potassium channel subunits 1–3 play significant physiological roles in excitable and non‐excitable tissues. However, the precise nature of its expression and functional identify in skeletal muscle remains unknown.HypothesisTherefore we hypothesized that Kvβ subunits play a significant physiological role in the skeletal muscle.Methods and ResultsIn the present study we identified that the voltage gated potassium channel subunit Kvβ interacts with NEDD4 in skeletal muscle. Previous research indicated a possible interaction with Kvβ and NEDD4 through pathway analysis. Utilizing immunoprecipitate pulldown technique, wild type skeletal muscle homogenates were incubated with Kvβ2 coated magnetic beads and run on SDS‐PAGE and immunoblotted with NEDD4 antibody demonstrating a visible band. Using a genetic deletion of kcnab we identified that the size of the major hind limb muscle is significantly decreased in Kvβ KO mice compared with wild type mice. Therefore we explored the underlying causes for this phenotype since Kvβ is ubiquitously expressed in skeletal muscle. Based on our investigation Kvβ plays a major role in muscle differentiation and overall size, however the proliferation aspect in skeletal muscle at least in the in vitro level remains unaffected. We utilized targeted siRNA knockdown approached to confirm these findings. In addition, tissue analysis reveals that the MHCI fiber type is significantly increased in Kvβ KO group compared with wild type.ConclusionOverall, this is the first study demonstrating that Kvβ2 plays a significant role in determining the size of the skeletal muscle and phenotype.Support or Funding InformationThis Research work was funded by National Institutes of Health (NIH) Grant R01‐HL‐102171 (S. M. Tipparaju).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transendothelial movement of adiponectin is restricted by glucocorticoids.

Altered permeability of the endothelial barrier in a variety of tissues has implications both in disease pathogenesis and treatment. Glucocorticoids are potent mediators of endothelial permeability, and this forms the basis for their heavily prescribed use as medications to treat ocular disease. However, the effect of glucocorticoids on endothelial barriers elsewhere in the body is less well studied. Here, we investigated glucocorticoid-mediated changes in endothelial flux of Adiponectin (Ad), a hormone with a critical role in diabetes. First, we used monolayers of endothelial cells in vitro and found that the glucocorticoid dexamethasone increased transendothelial electrical resistance and reduced permeability of polyethylene glycol (PEG, molecular weight 4000 Da). Dexamethasone reduced flux of Ad from the apical to basolateral side, measured both by ELISA and Western blotting. We then examined a diabetic rat model induced by treatment with exogenous corticosterone, which was characterized by glucose intolerance and hyperinsulinemia. There was no change in circulating Ad but less Ad protein in skeletal muscle homogenates, despite slightly higher mRNA levels, in diabetic vs control muscles. Dexamethasone-induced changes in Ad flux across endothelial monolayers were associated with alterations in the abundance of select claudin tight junction (TJ) proteins. shRNA-mediated knockdown of one such gene, claudin-7, in HUVEC resulted in decreased TEER and increased adiponectin flux, confirming the functional significance of Dex-induced changes in its expression. In conclusion, our study identifies glucocorticoid-mediated reductions in flux of Ad across endothelial monolayers in vivo and in vitro This suggests that impaired Ad action in target tissues, as a consequence of reduced transendothelial flux, may contribute to the glucocorticoid-induced diabetic phenotype.

Oxidative Balance in Rats during Adaptation to Swimming Load.

The main parameters of free radical oxidation and antioxidant defense in the blood plasma, erythrocytes, and homogenates of skeletal muscles, heart, liver, lungs, and kidneys were studied in adult outbred albino male rats with different degree of adaptation to moderate exposure to swimming. The rats were trained to swim regularly over 1 month. Changes in oxidative balance varied in organs and tissues and depended on the level of training. Malonic dialdehyde content in the erythrocytes after swimming increased by 13.8% in non-trained animals, but decreased by 19.2% in trained rats. Parameters of blood plasma reflect the general oxidative balance of organs and tissues.

Pathogenic variants in HTRA2 cause an early-onset mitochondrial syndrome associated with 3-methylglutaconic aciduria

Mitochondrial diseases collectively represent one of the most heterogeneous group of metabolic disorders. Symptoms can manifest at any age, presenting with isolated or multiple-organ involvement. Advances in next-generation sequencing strategies have greatly enhanced the diagnosis of patients with mitochondrial disease, particularly where a mitochondrial aetiology is strongly suspected yet OXPHOS activities in biopsied tissue samples appear normal. We used whole exome sequencing (WES) to identify the molecular basis of an early-onset mitochondrial syndrome—pathogenic biallelic variants in the HTRA2 gene, encoding a mitochondria-localised serine protease—in five subjects from two unrelated families characterised by seizures, neutropenia, hypotonia and cardio-respiratory problems. A unifying feature in all affected children was 3-methylglutaconic aciduria (3-MGA-uria), a common biochemical marker observed in some patients with mitochondrial dysfunction. Although functional studies of HTRA2 subjects’ fibroblasts and skeletal muscle homogenates showed severely decreased levels of mutant HTRA2 protein, the structural subunits and complexes of the mitochondrial respiratory chain appeared normal. We did detect a profound defect in OPA1 processing in HTRA2-deficient fibroblasts, suggesting a role for HTRA2 in the regulation of mitochondrial dynamics and OPA1 proteolysis. In addition, investigated subject fibroblasts were more susceptible to apoptotic insults. Our data support recent studies that described important functions for HTRA2 in programmed cell death and confirm that patients with genetically-unresolved 3-MGA-uria should be screened by WES with pathogenic variants in the HTRA2 gene prioritised for further analysis.

Thermal Acclimatization in Overwintering Tadpoles of the Green Frog, Lithobates clamitans (Latreille, 1801)

Seasonal acclimatization permits organisms to maintain function in the face of environmental change. Tadpoles of the green frog (Lithobates clamitans) overwinter as tadpoles in much of their range. Because they are active in winter, we hypothesized that green frog tadpoles would display acclimatization of metabolic and locomotor function. We collected tadpoles in Sewanee, Tennessee (35.2°N) in winter and summer. Tadpoles collected during each season were tested at both winter (8°C) and summer (26°C) temperatures. Winter tadpoles were able to maintain swimming performance at both temperatures, whereas swimming performance decreased at cold temperatures in summer tadpoles. There was no evidence for seasonal acclimatization of whole-animal metabolic rate. Although whole-animal metabolic acclimatization was not observed, the activities of cytochrome c oxidase, citrate synthase, and lactate dehydrogenase measured in skeletal muscle homogenates showed higher activity in winter-acclimatized tadpoles indicating compensation for temperature. Further, the composition of muscle membranes of winter tadpoles had less saturated and more monounsaturated fatty acids and a higher ω-3 balance, unsaturation index, and peroxidation index than summer tadpoles. These data indicate that reversible phenotypic plasticity of thermal physiology occurs in larval green frog tadpoles. They appear to compensate for colder temperatures to maintain burst-swimming velocity and the ability to escape predators without the cost of maintaining a constant, higher standard metabolic rate in the winter.

Local Aldosterone Synthesis in Skeletal Muscle

OBJECTIVETo characterize mineralocorticoid receptor (MR) regulation in normal vs. dystrophic skeletal muscle and elucidate the mechanism behind the efficacy of MR antagonists in dystrophic muscle.BACKGROUNDOur lab has recently shown MR is present in skeletal muscle fibers and represents a novel therapeutic target for treating both the skeletal and cardiac muscle pathologies of Duchenne muscular dystrophy (DMD). However, the underlying mechanisms and functional role of MR activation in skeletal muscle is still unclear. Excessive MR activation is known to contribute to the pathogenesis of heart failure, presumably due to increased levels of its natural ligand aldosterone. However, clinical trials have shown that addition of an MR antagonist to standard‐of‐care regimens (ACE inhibitors like lisinopril) improves morbidity and mortality in patients, even when circulating aldosterone levels are within the physiological range. There is now a growing body of evidence that shows aldosterone, which was previously thought to only be synthesized in the adrenal cortex can also be synthesized and metabolized locally in extra‐adrenal tissues, including the heart, particularly in pathological states.DESIGN/METHODSAldosterone synthase (CYP11B2) and other key enzymes in the aldosterone synthesis pathway were tested at the protein level in mouse skeletal muscles and myogenic cultures from normal and dystrophic models, and in normal human myogenic cultures using western blot analysis and immunofluorescence. Sequencing analysis of reverse transcribed PCR (RT‐PCR) of RNA from mouse skeletal muscle and human myotubes was used to confirm expression.RESULTSCYP11B2 is present in mouse skeletal muscle homogenates and up‐regulated in muscles from dystrophic mice (Figure 1). CYP11B2 does not appear to be coming from the skeletal muscle itself; immunofluorescence of dystrophic mouse muscles suggests this enzyme may be present in a subset of immune cells, which are present at much higher levels in dystrophic muscles (Figure 1). We have also found type 2 11‐beta hydroxysteroid dehydrogenase (11β‐HSD2), a key regulator of MR specificity, is present in normal human myotubes and is up‐regulated in dystrophic muscle (Figure 2).CONCLUSIONSCYP11B2 and 11β‐HSD2 levels are significantly increased in dystrophic muscle, suggesting MR activation is in excess of the physiological need. 11β‐HSD2 is expressed within the muscle fibers but CYP11B2 appears to be coming from a population of immune cells up‐regulated in dystrophic tissue due to increased inflammation and muscle degeneration.Support or Funding InformationNational Institutes of Health [R01 NS082868]

LRPPRC mutations cause early-onset multisystem mitochondrial disease outside of the French-Canadian population.

Mitochondrial Complex IV [cytochrome c oxidase (COX)] deficiency is one of the most common respiratory chain defects in humans. The clinical phenotypes associated with COX deficiency include liver disease, cardiomyopathy and Leigh syndrome, a neurodegenerative disorder characterized by bilateral high signal lesions in the brainstem and basal ganglia. COX deficiency can result from mutations affecting many different mitochondrial proteins. The French-Canadian variant of COX-deficient Leigh syndrome is unique to the Saguenay-Lac-Saint-Jean region of Québec and is caused by a founder mutation in the LRPPRC gene. This encodes the leucine-rich pentatricopeptide repeat domain protein (LRPPRC), which is involved in post-transcriptional regulation of mitochondrial gene expression. Here, we present the clinical and molecular characterization of novel, recessive LRPPRC gene mutations, identified using whole exome and candidate gene sequencing. The 10 patients come from seven unrelated families of UK-Caucasian, UK-Pakistani, UK-Indian, Turkish and Iraqi origin. They resemble the French-Canadian Leigh syndrome patients in having intermittent severe lactic acidosis and early-onset neurodevelopmental problems with episodes of deterioration. In addition, many of our patients have had neonatal cardiomyopathy or congenital malformations, most commonly affecting the heart and the brain. All patients who were tested had isolated COX deficiency in skeletal muscle. Functional characterization of patients' fibroblasts and skeletal muscle homogenates showed decreased levels of mutant LRPPRC protein and impaired Complex IV enzyme activity, associated with abnormal COX assembly and reduced steady-state levels of numerous oxidative phosphorylation subunits. We also identified a Complex I assembly defect in skeletal muscle, indicating different roles for LRPPRC in post-transcriptional regulation of mitochondrial mRNAs between tissues. Patient fibroblasts showed decreased steady-state levels of mitochondrial mRNAs, although the length of poly(A) tails of mitochondrial transcripts were unaffected. Our study identifies LRPPRC as an important disease-causing gene in an early-onset, multisystem and neurological mitochondrial disease, which should be considered as a cause of COX deficiency even in patients originating outside of the French-Canadian population.

LABORATORY VALIDATION OF A POINT-OF-CARE CARDIAC TROPONIN I ASSAY FOR USE IN WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS).

The VetScan® i-STAT® 1 Handheld Analyzer and cardiac troponin I (cTnI) cartridges (i-STAT cTnI assay) measured greater median cTnI concentration [cTnI] in free-ranging white-tailed deer (Odocoileus virginianus) hand-injected with anesthetic drugs after physical restraint in Clover traps than in those ground-darted with the same drugs. This suggested that Clover trapping induces myocardial damage, bringing the use of this capture method under scrutiny. The purpose of this study was to confirm the validity of the i-STAT cTnI assay in deer before recommending changes in capture methods. Median [cTnI] measured by the i-STAT cTnI assay ([cTnI]i) in heparinized whole blood collected from 52 healthy, reproductively mature, female deer physically restrained in a chute was 0.01 ng/ml (10-90% percentiles: 0.00-0.03 ng/ml; minimum, maximum: 0.00, 0.07 ng/ml); [cTnI]i was 0.00 ng/ml in 42% of the deer. There was no association between [cTnI]i and either clotting or hemolytic index. [cTnI]i was 0.00 ng/ml when deer skeletal muscle homogenate was added to deer blood with [cTnI]i of 0.00 ng/ml, confirming the i-STAT cTnI assay does not detect skeletal muscle troponins. When deer cardiac muscle homogenate was serially diluted with 1) deer blood, 2) deer plasma, and 3) cow blood, [cTnI]i was directly proportional (Y intercept=-0.09, 0.7, and -0.08 ng/ml, respectively; r2≥0.97) to the fraction of homogenate in each sample. Deer cardiac muscle homogenate was diluted with deer blood to produce three samples with low, intermediate, and high [cTnI]i; serial measurements (n=10) performed on each sample yielded coefficients of variation (CVs) of 8, 20, and 11%, respectively. Corresponding CVs when plasma was used as diluent were 13, 9, and 7%, respectively. [cTnI]i increased when plasma with a low [cTnI]i was stored at 20-24°C for 9 days. Three freeze-thaw cycles caused no systematic change in plasma [cTnI]i.

Amplitude-integrated EEG abnormalities in two neonates with encephalopathy due to mitochondrial dysfunction (respiratory chain complex I deficiency)

Amplitude-integrated electroencephalography (aEEG) findings in hypoxic-ischemic encephalopathy characterized by secondary energy failure have been described in detail. In contrast, reports of use of aEEG in patients with encephalopathy due to primary energy failure as seen in inherited metabolic disorders are sparse. We report two cases of lethal mitochondrial encephalopathy due to mitochondrial respiratory chain complex I deficiency. We present clinical course, laboratory evaluations, aEEG, conventional electroencephalography, magnetic resonance imaging and magnetic resonance spectroscopy findings of two cases identified with mitochondrial encephalopathy between 2002 and 2007. Both infants were born at term. Both presented with intractable seizures and mild hypotonia within the first days of life, in the absence of any evidence suggestive of hypoxic- ischemic encephalopathy. Both cases were treated with multiple anticonvulsants and in neither case were seizures fully controlled. Magnetic resonance imaging in both cases revealed a structurally normal brain and case one showed subtle diffuse deep white matter signal abnormality. Magnetic resonance spectroscopy revealed no elevation of lactate. In both cases, aEEG tracings were markedly abnormal, confirmed by electroencephalography. Case one showed status epilepticus on an abnormally high amplitude background and case two presented with a mainly discontinuous background pattern with intermittent burst-suppression pattern activity. Complex I activity in skeletal muscle homogenate was abnormally low in both patients. The use of aEEG as a valuable assessment and monitoring tool in patients with metabolic encephalopathy should be further promoted.

PP03.5 – 2756: Multiple symmetrical lipomatosis: An uncommon presentation of a mitochondrial disease

Objective To evaluate the possibility of mitochondrial involvement in a patient with multiple symmetrical lipomatosis (MSL) by testing mitochondrial DNA (mtDNA) in lymphocytes, skeletal muscle and adipose tissue, and by assaying OXPHOS complex activities in skeletal muscle. Methods Several tissues (lymphocytes, skeletal muscle, adipose tissue) from one patient originating from a family affected by MSL on three generations has been assayed using biochemical and molecular tests to detect mtDNA alterations and OXPHOS deficiencies as potential cause of MSL. Results mtDNA sequencing in patient's lymphocytes revealed the presence of the classic MERRF mutation (m.8344A> G) in heteroplasmic state (40%) which was even higher in skeletal muscle (68%) and adipose tissue (94%). Spectrophotometric analysis of OXPHOS activities in skeletal muscle homogenate revealed a decreased but not deficient activity of complex IV. Analysis in isolated skeletal muscle mitochondria using BN-PAGE followed by in–gel activity staining showed overall low activities of complexes I, III and IV which are the complexes containing subunits that are encoded by mtDNA and additionally revealed presence of subcomplexes of complex V. This latter is a hallmark of a defect in intramitochondrial translation. Conclusion MSL also known as Ekbom's syndrome, Madelung's disease and Launois-Bensaude syndrome is an unusual disorder characterized by the presence of lipomas with axial symmetrical distribution. Currently the pathogenic mechanisms are not known. In our opinion, assays of the OXPHOS complexes and mtDNA analysis are indicated in a patient presenting multiple symmetrical lipomatosis, especially with positive familial history. As this clinical feature may be the first sign of a mitochondrial disease extensive clinical evaluation and follow-up of these patients is mandatory.

Assessing the function of mitochondria in cytosolic context in human skeletal muscle: Adopting high-resolution respirometry to homogenate of needle biopsy tissue samples

Using skeletal muscle homogenates for respirometry has many advantages, but the main challenge is avoiding the damage to outer mitochondrial membrane (OMM) and complex I. By optimising the amount of muscle and careful titration of substrates and inhibitors we developed a new protocol and compared it to isolated mitochondria. We found acceptable damage to OMM (~10–15% increment of oxygen flux after addition of cytochrome c) and to complex I (~70% of electron flux). Homogenate retained ~90% of phosphorylation capacity of isolated mitochondria. The use of fresh homogenate was crucial as mitochondrial function declined rapidly after 2–3h of cold storage.

The role of mitochondrial dysfunction in the pathophysiology of ICU-acquired weakness

Methods Muscle biopsies were obtained from critically ill patients with severe ICUAW (defined as MRC score < 24 on two separate tests) and from otherwise healthy controls undergoing elective hip replacement. 50-100mg of the native sample was homogenized by a technique which perserves mitochondria in their cytosolic context [3]. Oxygen consumption was measured by high-resolution respirometry (Oxygraph-2Km Oroboros) under two occasions: 1. homogenate enriched with substrates was sequentially treated with an ATPase inhibitor (oligomycine), an uncoupler (FCCP) and KCN. This allowed us to measure oxidative phosphorylation, respiratory chain capacity, proton leak through inner mitochondrial membrane and non-mitochondrial O2 consumption. 2. skeletal muscle homogenate enriched with abundant ADP was treated with sequential addition of substrates and inhibitors of complexes I, II, III, and IV, respectively. Integrity of outer membrane was assessed by measuring the response to addition of cytochrome c. Oxygen consumption rate was normalized to citrate-synthase activity and total protein content.

Methylated Medium‐ and Long‐Chain Fatty Acids Provide Novel Sources of Anaplerotic Carbon for Fasting or Exercising Mice (LB438)

We hypothesized that methylated fatty acids (e.g. 2‐methylpentanoic acid (2MeP), phytanic or pristanic acids) would provide anaplerotic carbon to the tricarboxylic acid (TCA) cycle and thereby enhance fatty acid oxidation, especially under conditions when TCA cycle intermediates are depleted. The optimal dose of 2MeP and pristanic acid for increasing in vitro [1‐14C]‐oleic acid oxidation in liver or skeletal muscle homogenates from fasted mice (n=4) was assessed using doses of 0, 0.25, 0.5, 0.75, or 1.0 mM. The minimum dose (0.25 mM) maximally stimulated liver tissue oxidation of [1‐14C]‐oleate to 14CO2 by 2‐fold (P &lt; 0.05), but no stimulation was detected in muscle. Similar incubations with 0.25 mM 2MeP, hexanoate, palmitate, phytanic acid, or pristanic acid or 0.1 mM malate or propionyl‐CoA were conducted with liver and skeletal muscle homogenates from acutely exercised or sedentary mice. Regardless of exercise, incubation of liver homogenates with 2MeP increased [1‐14C]‐oleate oxidation to 14CO2 by 60% over control incubations containing hexanoate (P &lt; 0.05). Phytanic acid treatment increased [14C]‐acid‐soluble product accumulation in liver tissue as compared to palmitate (P &lt; 0.05). Anaplerotic stimulation was unaffected by exercised state (P &gt; 0.05) and not evidenced in muscle homogenates. Results were consistent with our hypothesis that methyl‐branched fatty acids may provide anaplerotic carbon and thereby stimulate in vitro fatty acid oxidation.Grant Funding Source: North Carolina Agricultural Research Service and by USDA‐AFRI training grant # 2010‐65200‐20354

Co-Expression of SERCA Isoforms, Phospholamban and Sarcolipin in Human Skeletal Muscle Fibers

Sarcolipin (SLN) and phospholamban (PLN) inhibit the activity of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) by reducing their apparent affinity for Ca2+. A ternary complex between SLN, PLN, and SERCAs results in super-inhibition of SERCA activity. Analysis of skeletal muscle homogenate has limited our current understanding of whether SLN and PLN regulate SERCA1a, SERCA2a, or both in skeletal muscle and whether SLN and PLN are co-expressed in skeletal muscle fibers. Biopsies from human vastus lateralis were analyzed through single fiber Western blotting and immunohisto/fluorescence staining to circumvent this limitation. With a newly generated SLN antibody, we report for the first time that SLN protein is present in human skeletal muscle. Addition of the SLN antibody (50 µg) to vastus lateralis homogenates increased the apparent Ca2+ affinity of SERCA (K Ca, pCa units) (-Ab, 5.85 ± 0.02 vs. +Ab, 5.95 ± 0.02) and maximal SERCA activity (μmol/g protein/min) (-Ab, 122 ± 6.4 vs. +Ab, 159 ± 11) demonstrating a functional interaction between SLN and SERCAs in human vastus lateralis. Specifically, our results suggest that although SLN and PLN may preferentially regulate SERCA1a, and SERCA2a, respectively, physiologically they both may regulate either SERCA isoform. Furthermore, we show that SLN and PLN co-immunoprecipitate in human vastus lateralis homogenate and are simultaneously expressed in 81% of the fibers analyzed with Western blotting which implies that super-inhibition of SERCA may exist in human skeletal muscle. Finally, we demonstrate unequivocally that mouse soleus contains PLN protein suggesting that super-inhibition of SERCA may also be important physiologically in rodent skeletal muscle.