Skeletal Muscle Samples Research Articles

Biallelic variants in TAMM41 are associated with low muscle cardiolipin levels, leading to neonatal mitochondrial disease.

SummaryMitochondrial disorders are clinically and genetically heterogeneous, with variants in mitochondrial or nuclear genes leading to varied clinical phenotypes. TAMM41 encodes a mitochondrial protein with cytidine diphosphate-diacylglycerol synthase activity: an essential early step in the biosynthesis of phosphatidylglycerol and cardiolipin. Cardiolipin is a mitochondria-specific phospholipid that is important for many mitochondrial processes. We report three unrelated individuals with mitochondrial disease that share clinical features, including lethargy at birth, hypotonia, developmental delay, myopathy, and ptosis. Whole exome and genome sequencing identified compound heterozygous variants in TAMM41 in each proband. Western blot analysis in fibroblasts showed a mild oxidative phosphorylation (OXPHOS) defect in only one of the three affected individuals. In skeletal muscle samples, however, there was severe loss of subunits of complexes I–IV and a decrease in fully assembled OXPHOS complexes I–V in two subjects as well as decreased TAMM41 protein levels. Similar to the tissue-specific observations on OXPHOS, cardiolipin levels were unchanged in subject fibroblasts but significantly decreased in the skeletal muscle of affected individuals. To assess the functional impact of the TAMM41 missense variants, the equivalent mutations were modeled in yeast. All three mutants failed to rescue the growth defect of the Δtam41 strains on non-fermentable (respiratory) medium compared with wild-type TAM41, confirming the pathogenicity of the variants. We establish that TAMM41 is an additional gene involved in mitochondrial phospholipid biosynthesis and modification and that its deficiency results in a mitochondrial disorder, though unlike families with pathogenic AGK (Sengers syndrome) and TAFAZZIN (Barth syndrome) variants, there was no evidence of cardiomyopathy.

Exercise and Training Regulation of Autophagy Markers in Human and Rat Skeletal Muscle.

Autophagy is a key intracellular mechanism by which cells degrade old or dysfunctional proteins and organelles. In skeletal muscle, evidence suggests that exercise increases autophagosome content and autophagy flux. However, the exercise-induced response seems to differ between rodents and humans, and little is known about how different exercise prescription parameters may affect these results. The present study utilised skeletal muscle samples obtained from four different experimental studies using rats and humans. Here, we show that, following exercise, in the soleus muscle of Wistar rats, there is an increase in LC3B-I protein levels immediately after exercise (+109%), and a subsequent increase in LC3B-II protein levels 3 h into the recovery (+97%), despite no change in Map1lc3b mRNA levels. Conversely, in human skeletal muscle, there is an immediate exercise-induced decrease in LC3B-II protein levels (−24%), independent of whether exercise is performed below or above the maximal lactate steady state, which returns to baseline 3.5 h following recovery, while no change in LC3B-I protein levels or MAP1LC3B mRNA levels is observed. SQSTM1/p62 protein and mRNA levels did not change in either rats or humans following exercise. By employing an ex vivo autophagy flux assay previously used in rodents we demonstrate that the exercise-induced decrease in LC3B-II protein levels in humans does not reflect a decreased autophagy flux. Instead, effect size analyses suggest a modest-to-large increase in autophagy flux following exercise that lasts up to 24 h. Our findings suggest that exercise-induced changes in autophagosome content markers differ between rodents and humans, and that exercise-induced decreases in LC3B-II protein levels do not reflect autophagy flux level.

Short-Term Supplementation With Fermented Red Clover Extract Reduces Vascular Inflammation in Early Post-menopausal Women.

The decline in estrogen at menopause poses a critical challenge to cardiovascular and metabolic health. Recently, a growing interest in the role of phytoestrogens, with a particular focus on isoflavones, has emerged as they can bind to estrogen receptors and may mimic the roles of endogenous estrogen. Fermented red clover extract (RC) contains isoflavones with superior bioavailability compared to non-fermented isoflavones, however little is known regarding the impact of isoflavones on cardiovascular and metabolic health. We assessed markers of vascular health in plasma and skeletal muscle samples obtained from healthy but sedentary early post-menopausal women (n = 10; 54 ± 4 years) following 2 weeks of twice daily treatment with placebo (PLA) or RC (60 mg isoflavones per day). The two interventions were administered using a randomized, double-blind, crossover design with a two-week washout period. Plasma samples were utilized for assessment of markers of vascular inflammation. There was a statistically significant reduction (~5.4%) in vascular cell adhesion molecule 1 (VCAM-1) following 2 weeks of RC supplementation compared to PLA (p = 0.03). In contrast, there was no effect of RC supplementation compared to PLA on skeletal muscle estrogen receptor content and enzymes related to vascular function, and angiogenesis. Supplementation with RC reduces vascular inflammation in early post-menopausal women and future studies should address the long-term impact of daily supplementation with RC after menopause.

Skeletal Muscle Expression of Actinin-3 (ACTN3) in Relation to Feed Efficiency Phenotype of F2 Bos indicus - Bos taurus Steers.

In this study, actinin-3 (ACTN3) gene expression was investigated in relation to the feed efficiency phenotype in Bos indicus - Bos taurus crossbred steers. A measure of relative feed efficiency based on residual feed intake relative to predictions from the NRC beef cattle model was analyzed by the use of a mixed linear model that included sire and family nested within sire as fixed effects and age, animal type, sex, condition, and breed as random effects for 173 F2 Nellore-Angus steers. Based on these residual intake observations, individuals were ranked from most efficient to least efficient. Skeletal muscle samples were analyzed from 54 steers in three groups of 18 (high efficiency, low efficiency, and a statistically average group). ACTN3, which encodes a muscle-specific structural protein, was previously identified as a candidate gene from a microarray analysis of RNA extracted from muscle samples obtained from a subset of steers from each of these three efficiency groups. The expression of ACTN3 was evaluated by quantitative reverse transcriptase PCR analysis. The expression of ACTN3 in skeletal muscle was 1.6-fold greater in the inefficient steer group than in the efficient group (p = 0.007). In addition to expression measurements, blocks of SNP haplotypes were assessed for breed or parent of origin effects. A maternal effect was observed for ACTN3 inheritance, indicating that a maternal B. indicus block conferred improved residual feed efficiency relative to the B. taurus copy (p = 0.03). A SNP haplotype analysis was also conducted for m-calpain (CAPN2) and fibronectin 1 (FN1), and a significant breed effect was observed for both genes, with B. indicus and B. taurus alleles each conferring favorable efficiency when inherited maternally (p = 0.03 and p = 0.04). Because the ACTN3 structural protein is specific to fast-twitch (type II) muscle fibers and not present in slow-twitch muscle fibers (type I), muscle samples used for expression analysis were also assayed for fiber type ratio (type II/type I). Inefficient animals had a fast fiber type ratio 1.8-fold greater than the efficient animals (p = 0.027). Because these fiber-types exhibit different metabolic profiles, we hypothesize that animals with a greater proportion of fast-twitch muscle fibers are also less feed efficient.

Transcriptome analyses indicate that heat stress-induced inflammation in white adipose tissue and oxidative stress in skeletal muscle is partially moderated by zilpaterol supplementation in beef cattle.

Heat stress (HS) triggers oxidative stress, systemic inflammation, and disrupts growth efficiency of livestock. β-adrenergic agonists supplemented to ruminant livestock improve growth performance, increase skeletal muscle mass, and decrease carcass fat. The objective of this study was to understand the independent and interacting effects of HS and zilpaterol hydrochloride (ZH) supplementation on the transcriptome of subcutaneous white adipose tissue and the longissimus dorsi muscle in steers. Twenty-four Red Angus-based steers were assigned to thermoneutral (TN; Temperature Humidity Index [THI] = 68) or HS (THI = 73-85) conditions and were not supplemented or supplemented with ZH (8.33mg/kg/d) for 21 d in a 2×2 factorial. Steers in the TN condition were pair-fed to the average daily feed intake of HS steers. RNA was isolated from adipose tissue and skeletal muscle samples collected via biopsy on 3, 10, and 21 d and sequenced using 3' Tag-Seq to an achieved average depth of 3.6 million reads/sample. Transcripts, mapped to ARS-UCD1.2, were quantified. Differential expression (DE) analyses were performed in DESeq2 with a significance threshold for false discovery rate of 0.05. In adipose, 4 loci (MISP3, APOL6, SLC25A4, and S100A12) were DE due to ZH on day 3, and 2 (RRAD, ALB) were DE due to the interaction of HS and ZH on day 10 (Padj < 0.05). In muscle, 40 loci (including TENM4 and OAZ1) were DE due to ZH on day 10, and 6 loci (HIF1A, LOC101903734, PDZD9, HNRNPU, MTUS1, and TMCO6) were DE due to environment on day 21 (Padj < 0.05). To explore biological pathways altered by environment, supplement, and their interaction, loci with DE (Praw < 0.05) were evaluated in Ingenuity Pathway Analysis. In adipose, 509 pathways were predicted to be altered (P < 0.01): 202 due to HS, 126 due to ZH, and 181 due to the interaction; these included inflammatory pathways predicted to be upregulated due to HS but downregulated due to the interaction of HS and ZH. In muscle, 113 pathways were predicted to be altered (P < 0.01): 23 due to HS, 66 due to ZH, and 24 due to the interaction of HS and ZH. Loci and pathway data in muscle suggest HS induced oxidative stress and that the stress response was moderated by ZH. Metabolic pathways were predicted to be altered due to HS, ZH, and their interaction in both tissues. These data provide evidence that HS and ZH interact to alter expression of genes in metabolic and immune function pathways and that ZH moderates some adverse effects of HS.

Endoplasmic reticulum stress and the unfolded protein response in skeletal muscle of subjects suffering from peritoneal sepsis

We provide a descriptive characterization of the unfolded protein response (UPR) in skeletal muscle of human patients with peritoneal sepsis and a sepsis model of C57BL/6J mice. Patients undergoing open surgery were included in a cross-sectional study and blood and skeletal muscle samples were taken. Key markers of the UPR and cluster of differentiation 68 (CD68) as surrogate of inflammatory injury were evaluated by real-time PCR and histochemical staining. CD68 mRNA increased with sepsis in skeletal muscle of patients and animals (p < 0.05). Mainly the inositol-requiring enzyme 1α branch of the UPR was upregulated as shown by elevated X-box binding-protein 1 (XBP1u) and its spliced isoform (XBP1s) mRNA (p < 0.05, respectively). Increased expression of Gadd34 indicated activation of PRKR-Like Endoplasmic Reticulum Kinase (PERK) branch of the UPR, and was only observed in mice (p < 0.001) but not human study subjects. Selected cell death signals were upregulated in human and murine muscle, demonstrated by increased bcl-2 associated X protein mRNA and TUNEL staining (p < 0.05). In conclusion we provide a first characterization of the UPR in skeletal muscle in human sepsis.

Poluentes aquáticos estão associados a alterações reprodutivas e genotoxicidade em peixe estuarino (Sciades herzbergii - Bloch, 1794) da Costa Equatorial Amazônica

Estuaries are constantly subject to continuous environmental impacts of human activities, such as fisheries, port or industry, and domestic sewage, with fish being one of the most affected aquatic animals, reflecting the impacts directly on their bodies. Thus, the aim of this study was to carry out the biomonitoring of an estuary located on the Amazonian Equatorial Coast through analysis of PAHs (Polycyclic Aromatic Hydrocarbons) in the water, in addition to trace metals, histopathological alterations and analysis of erythrocyte micronuclei in Sciades herzbergii. S. herzbergii was used as a model species, due to its estuarine-resident behavior. Gonad and gill samples were subjected to histopathological evaluations. The quantification of trace metals was performed in samples of skeletal muscles of the animals collected, where concentrations of Lead (Pb), Copper (Cu), Zinc (Zn), Cadmium (Cd), Magnesium (Mg), Iron (Fe) and Aluminum (Al) were found. Except for Cadmium (Cd), all the concentrations were above the recommended limits. The PAHs analysis revealed the presence of Naphthalene and Acenaphthene in the water samples Histopathological and genotoxic analyses revealed of lesions in 100% of the study specimens. Thus, the histological and genotoxic alterations found in 100% of S. herzbergii specimens captured in São José Bay-MA are potentially associated with PAH concentrations present in the water. These results are potentially associated with the presence of PAH and trace metals, both in water and in animal tissues, inferring a general scenario of environmental contamination which directly implies a risk to the health and survival of the local biota. This study shows the relevance of continuous biomonitoring of estuarine ecosystems, in order to guide authorities regarding sewage management and ensure the evolutionary development of estuarine species, especially fishes of importance in the local cuisine, therefore related to human food security.

Endurance Training in Humans Modulates the Bacterial DNA Signature of Skeletal Muscle

Accumulating evidence supports the existence of a tissue microbiota, which may regulate the physiological function of tissues in normal and pathological states. To gain insight into the regulation of tissue-borne bacteria in physiological conditions, we quantified and sequenced the 16S rRNA gene in aseptically collected skeletal muscle and blood samples from eight healthy male individuals subjected to six weeks of endurance training. Potential contamination bias was evaluated and the taxa profiles of each tissue were established. We detected bacterial DNA in skeletal muscle and blood, with background noise levels of detected bacterial DNA considerably lower in control versus tissue samples. In both muscle and blood, Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes were the most prominent phyla. Endurance training changed the content of resident bacterial DNA in skeletal muscle but not in blood, with Pseudomonas being less abundant, and both Staphylococcus and Acinetobacter being more abundant in muscle after exercise. Our results provide evidence that endurance training specifically remodels the bacterial DNA profile of skeletal muscle in healthy young men. Future investigations may shed light on the physiological impact, if any, of training-induced changes in bacterial DNA in skeletal muscle.

Metabolomics Coupled with Pathway Analysis Provides Insights into Sarco-Osteoporosis Metabolic Alterations and Estrogen Therapeutic Effects in Mice.

Postmenopausal osteoporosis (PMOP) and sarcopenia are common diseases that predominantly affect postmenopausal women. In the occurrence and development of these two diseases, they are potentially pathologically connected with each other at various molecular levels. However, the application of metabolomics in sarco-osteoporosis and the metabolic rewiring happening throughout the estrogen loss-replenish process have not been reported. To investigate the metabolic alteration of sarco-osteoporosis and the possible therapeutical effects of estradiol, 24 mice were randomly divided into sham surgery, ovariectomy (OVX), and estradiol-treated groups. Three-dimensional reconstructions and histopathology examination showed significant bone loss after ovariectomy. Estrogen can well protect against OVX-induced bone loss deterioration. UHPLC-Q-TOF/MS was preformed to profile semi- polar metabolites of skeletal muscle samples from all groups. Metabolomics analysis revealed metabolic rewiring occurred in OVX group, most of which can be reversed by estrogen supplementation. In total, 65 differential metabolites were identified, and pathway analysis revealed that sarco-osteoporosis was related to the alterations in purine metabolism, glycerophospholipid metabolism, arginine biosynthesis, tryptophan metabolism, histidine metabolism, oxidative phosphorylation, and thermogenesis, which provided possible explanations for the metabolic mechanism of sarco-osteoporosis. This study indicates that an UHPLC-Q-TOF/MS-based metabolomics approach can elucidate the metabolic reprogramming mechanisms of sarco-osteoporosis and provide biological evidence of the therapeutical effects of estrogen on sarco-osteoporosis.

Immunocompetent Mice Infected by Two Lineages of Dengue Virus Type 2: Observations on the Pathology of the Lung, Heart and Skeletal Muscle.

Dengue virus (DENV) infection by one of the four serotypes (DENV-1 to 4) may result in a wide spectrum of clinical manifestations, with unpredictable evolution and organ involvement. Due to its association with severe epidemics and clinical manifestations, DENV-2 has been substantially investigated. In fact, the first emergence of a new lineage of the DENV-2 Asian/American genotype in Brazil (Lineage II) in 2008 was associated with severe cases and increased mortality related to organ involvement. A major challenge for dengue pathogenesis studies has been a suitable animal model, but the use of immune-competent mice, although sometimes controversial, has proven to be useful, as histological observations in infected animals reveal tissue alterations consistent to those observed in dengue human cases. Here, we aimed to investigate the outcomes caused by two distinct lineages of the DENV-2 Asian/American genotype in the lung, heart and skeletal muscle tissues of infected BALB/c mice. Tissues were submitted to histopathology, immunohistochemistry, histomorphometry and transmission electron microscopy (TEM) analysis. The viral genome was detected in heart and skeletal muscle samples. The viral antigen was detected in cardiomyocytes and endothelial cells of heart tissue. Heart and lung tissue samples presented morphological alterations comparable to those seen in dengue human cases. Creatine kinase serum levels were higher in mice infected with both lineages of DENV-2. Additionally, statistically significant differences, concerning alveolar septa thickening and heart weight, were observed between BALB/c mice infected with both DENV-2 lineages, which was demonstrated to be an appropriate experimental model for dengue pathogenesis studies on lung, heart and skeletal muscle tissues.

Combining laser-irradiation and glycerol immersion of skeletal muscles to improve their optical transparency

Optical diagnostic methods are still suffering from relatively low resolution due to low light penetration produced by the multiple scattering properties of biological tissues. Light scattering reduction can improve various biomedical diagnostic and therapeutic applications. Therefore, the optical clearing approach was introduced to reduce light scattering and improve its penetration in soft and hard tissues. Many optical clearing protocols could be applied depending on the physical properties of the inspected tissue. These protocols are classified into physical, chemical, photochemical/photothermal, and compression. In the present study, light penetration in skeletal muscle samples has been improved via a combination of physical and photothermal approaches. Bovine skeletal muscle samples have been exposed to IR laser radiation (at 1064 nm) before immersion in 99% glycerol, providing a reduction in the immersing time and increasing the efficacy of the clearing process.Additionally, spectrochemical changes in the studied samples were investigated via laser-induced fluorescence technique before and after laser exposure and glycerol immersion. Tissue collimated transmittance has been monitored at different immersion times. A significant increase shows up, especially after laser exposure revealing the improvement in light penetration. Moreover, optical microscopic images of the samples show contrast improvement due to the light penetration enhancement.

Membrane lipids and maximum lifespan in clownfish

The longevity-homeoviscous adaptation (LHA) theory of ageing states that lipid composition of cell membranes is linked to metabolic rate and lifespan, which has been widely shown in mammals and birds but not sufficiently in fish. In this study, two species of the genus Amphiprion (Amphiprion percula and Amphiprion clarkii, with estimated maximum lifespan potentials [MLSP] of 30 and 9–16 years, respectively) and the damselfish Chromis viridis (estimated MLSP of 1–2 years) were chosen to test the LHA theory of ageing in a potential model of exceptional longevity. Brain, livers and samples of skeletal muscle were collected for lipid analyses and integral part in the computation of membrane peroxidation indexes (PIn) from phospholipid (PL) fractions and PL fatty acid composition. When only the two Amphiprion species were compared, results pointed to the existence of a negative correlation between membrane PIn value and maximum lifespan, well in line with the predictions from the LHA theory of ageing. Nevertheless, contradictory data were obtained when the two Amphiprion species were compared to the shorter-lived C. viridis. These results along with those obtained in previous studies on fish denote that the magnitude (and sometimes the direction) of the differences observed in membrane lipid composition and peroxidation index with MLSP cannot explain alone the diversity in longevity found among fishes.

The Insulin-Sensitizer Pioglitazone Remodels Adipose Tissue Phospholipids in Humans.

The insulin-sensitizer pioglitazone exerts its cardiometabolic benefits in type 2 diabetes (T2D) through a redistribution of body fat, from ectopic and visceral areas to subcutaneous adipose depots. Whereas excessive weight gain and lipid storage in obesity promotes insulin resistance and chronic inflammation, the expansion of subcutaneous adipose by pioglitazone is associated with a reversal of these immunometabolic deficits. The precise events driving this beneficial remodeling of adipose tissue with pioglitazone remain unclear, and whether insulin-sensitizers alter the lipidomic composition of human adipose has not previously been investigated. Using shotgun lipidomics, we explored the molecular lipid responses in subcutaneous adipose tissue following 6months of pioglitazone treatment (45mg/day) in obese humans with T2D. Despite an expected increase in body weight following pioglitazone treatment, no robust effects were observed on the composition of storage lipids (i.e., triglycerides) or the content of lipotoxic lipid species (e.g., ceramides and diacylglycerides) in adipose tissue. Instead, pioglitazone caused a selective remodeling of the glycerophospholipid pool, characterized by a decrease in lipids enriched for arachidonic acid, such as plasmanylethanolamines and phosphatidylinositols. This contributed to a greater overall saturation and shortened chain length of fatty acyl groups within cell membrane lipids, changes that are consistent with the purported induction of adipogenesis by pioglitazone. The mechanism through which pioglitazone lowered adipose tissue arachidonic acid, a major modulator of inflammatory pathways, did not involve alterations in phospholipase gene expression but was associated with a reduction in its precursor linoleic acid, an effect that was also observed in skeletal muscle samples from the same subjects. These findings offer important insights into the biological mechanisms through which pioglitazone protects the immunometabolic health of adipocytes in the face of increased lipid storage.

In vivo treatment with varespladib, a phospholipase A2 inhibitor, prevents the peripheral neurotoxicity and systemic disorders induced by Micrurus corallinus (coral snake) venom in rats

In this study, we investigated the action of varespladib (VPL) alone or in combination with a coral snake antivenom (CAV) on the local and systemic effects induced by Micrurus corallinus venom in rats. Adult male Wistar rats were exposed to venom (1.5 mg/kg – i.m.) and immediately treated with CAV (antivenom:venom ratio 1:1.5 ‘v/w’ – i.p.), VPL (0.5 mg/kg – i.p.), or both of these treatments. The animals were monitored for 120 min and then anesthetized to collect blood samples used for haematological and serum biochemical analysis; after euthanasia, skeletal muscle, renal and hepatic tissue samples were collected for histopathological analysis. M. corallinus venom caused local oedema without subcutaneous haemorrhage or apparent necrosis formation, although there was accentuated muscle morphological damage; none of the treatments prevented oedema formation but the combination of CAV and VPL reduced venom-induced myonecrosis. Venom caused neuromuscular paralysis and respiratory impairment in approximately 60 min following envenomation; CAV alone did not prevent the neurotoxic action, whereas VPL alone prevented neurotoxic symptoms developing as did the combination of CAV and VPL. Venom induced significant increase of serum CK and AST release, mostly due to local and systemic myotoxicity, which was partially prevented by the combination of CAV and VPL. The release of hepatotoxic serum biomarkers (LDH and ALP) induced by M. corallinus venom was not prevented by CAV and VPL when individually administered; their combination effectively prevented ALP release. The venom-induced nephrotoxicity (increase in serum creatinine concentration) was prevented by all the treatments. VPL alone or in combination with CAV significantly prevented the venom-induced lymphocytosis. In conclusion, VPL shows to be effective at preventing the neurotoxic, nephrotoxic, and inflammatory activities of M. corallinus venom. In addition, VPL acts synergistically with antivenom to prevent a number of systemic effects caused by M. corallinus venom.

Swine behavioral and physiological response to increasing sodium nitrite oral drench administration and resulting tissue residues

Objectives: This study aimed to evaluate the physiological and behavioral responses of pigs administered sodium nitrite, determine an ideal dosing rate by oral drenching of sodium nitrite for depopulation events, and evaluate the nitrite residue present in the ocular fluid and skeletal muscle after sodium nitrite administration. Materials and methods: Four groups of 10 market weight pigs (40 market weight pigs total) and 1 group of 10 sows were used. Each group of market weight animals received a different oral drench dose of sodium nitrite solution (1× [400-441 mg/kg], 2× [800-882 mg/kg], 2.5× [1000-1102 mg/kg], and 3× [1200-1323 mg/kg]) and was observed for distress behaviors. Two market weight animals in each treatment group were implanted with a monitor to measure body temperature, heart rate, and activity levels. The dosing rate with apparent best behavioral and physiological response was applied to the 10 sows and the same behaviors monitored. After death was confirmed, ocular fluid and skeletal muscle samples were collected from the sows. Results: An increased dosage of sodium nitrite greatly reduced the time to distress with a significant linear relationship. A higher frequency of vocalizations and the most frequent spikes in activity levels were observed in the lowest dosing group. No correlation was found between ocular fluid nitrite and skeletal muscle sodium nitrite concentrations. Implications: Oral drenching of sodium nitrite is a viable method for swine depopulation events. Higher doses of sodium nitrite have better welfare associations. Ocular fluid nitrite anion concentrations do not correlate with sodium nitrite skeletal muscle concentrations.

Association Between Changes in Serum and Skeletal Muscle Metabolomics Profile With Maximum Power Output Gains in Response to Different Aerobic Training Programs: The Times Study.

Purpose: High heterogeneity of the response of cardiorespiratory fitness (CRF) to standardized exercise doses has been reported in different training programs, but the associated mechanisms are not widely known. This study investigated whether changes in the metabolic profile and pathways in blood serum and the skeletal muscle are associated with the inter-individual variability of CRF responses to 8-wk of continuous endurance training (ET) or high-intensity interval training (HIIT).Methods: Eighty men, young and sedentary, were randomized into three groups, of which 70 completed 8 wk of intervention (> 90% of sessions): ET, HIIT, or control. Blood and vastus lateralis muscle tissue samples, as well as the measurement of CRF [maximal power output (MPO)] were obtained before and after the intervention. Blood serum and skeletal muscle samples were analyzed by 600 MHz 1H-NMR spectroscopy (metabolomics). Associations between the pretraining to post-training changes in the metabolic profile and MPO gains were explored via three analytical approaches: (1) correlation between pretraining to post-training changes in metabolites' concentration levels and MPO gains; (2) significant differences between low and high MPO responders; and (3) metabolite contribution to significantly altered pathways related to MPO gains. After, metabolites within these three levels of evidence were analyzed by multiple stepwise linear regression. The significance level was set at 1%.Results: The metabolomics profile panel yielded 43 serum and 70 muscle metabolites. From the metabolites within the three levels of evidence (15 serum and 4 muscle metabolites for ET; 5 serum and 1 muscle metabolites for HIIT), the variance in MPO gains was explained: 77.4% by the intervention effects, 6.9, 2.3, 3.2, and 2.2% by changes in skeletal muscle pyruvate and valine, serum glutamine and creatine phosphate, respectively, in ET; and 80.9% by the intervention effects; 7.2, 2.2, and 1.2% by changes in skeletal muscle glycolate, serum creatine and creatine phosphate, respectively, in HIIT. The most changed and impacted pathways by these metabolites were: arginine and proline metabolism, glycine, serine and threonine metabolism, and glyoxylate and dicarboxylate metabolism for both ET and HIIT programs; and additional alanine, aspartate and glutamate metabolism, arginine biosynthesis, glycolysis/gluconeogenesis, and pyruvate metabolism for ET.Conclusion: These results suggest that regulating the metabolism of amino acids and carbohydrates may be a potential mechanism for understanding the inter-individual variability of CRF in responses to ET and HIIT programs.

Isolation of Live Leukocytes from Human Inflammatory Muscles.

In inflammatory myopathies, the self-reactive immune cells involved in muscle aggression have been studied mostly using histological assessment of muscle biopsy sections; this methodology provides the advantage of visualizing and identifying cells within the tissue, but it does not allow further investigation. To gain access to live and isolated cells, many studies utilized blood samples; however, in the absence of biological tools to discriminate the leukocytes associated with the autoimmune process from those that emerged from responses against pathogens, the information observed on circulating immune cells often lacks in specificity, and thus result interpretation may prove difficult. In order to selectively retrieve self-reactive immune cells, we developed a protocol to isolate live leukocytes from human muscle biopsies, which allows for further analysis using a large range of methodologies. The protocol uses enzymatic digestion to release live leukocytes from freshly collected skeletal muscle samples, followed by filtration and separation of the leukocytes from the myocytes by density gradient centrifugation. The isolated cells can be submitted immediately to various analysis strategies to characterize ex vivo the specific cellular and molecular mechanisms responsible for self-directed immune muscle aggression or may be placed in culture for expansion.

Molecular Profiling of DNA Methylation and Alternative Splicing of Genes in Skeletal Muscle of Obese Rabbits.

DNA methylation and the alternative splicing of precursor messenger RNAs (pre-mRNAs) are two important genetic modification mechanisms. However, both are currently uncharacterized in the muscle metabolism of rabbits. Thus, we constructed the Tianfu black rabbit obesity model (obese rabbits fed with a 10% high-fat diet and control rabbits from 35 days to 70 days) and collected the skeletal muscle samples from the two groups for Genome methylation sequencing and RNA sequencing. DNA methylation data showed that the promoter regions of 599 genes and gene body region of 2522 genes had significantly differential methylation rates between the two groups, of which 288 genes had differential methylation rates in promoter and gene body regions. Analysis of alternative splicing showed 555 genes involved in exon skipping (ES) patterns, and 15 genes existed in differential methylation regions. Network analysis showed that 20 hub genes were associated with ubiquitinated protein degradation, muscle development pathways, and skeletal muscle energy metabolism. Our findings suggest that the two types of genetic modification have potential regulatory effects on skeletal muscle development and provide a basis for further mechanistic studies in the rabbit.

PSV-2 Maternal nutrient restriction and re-alimentation influences liver and muscle tissue development and gene expression

Abstract To determine the effects of maternal nutrient restriction and re-alimentation on fetal liver and muscle development, 48 pregnant ewes with singletons, were fed a control diet [100% National Research Council (NRC) requirements (CON)] starting at the beginning of gestation. On day 50 of gestation, ewes (n = 7) were euthanized and fetal liver and skeletal muscle samples were collected. The remaining animals were fed either CON or 60% NRC requirements (RES), a subset were euthanized at day 90 of gestation (n = 7/treatment), and fetal samples obtained. Remaining ewes were maintained on the current diet (CON-CON, n = 6; RES-RES, n = 7) or switched to alternative diet (CON-RES, RES-CON; n = 7/treatment). On day 130 of gestation, remaining ewes were euthanized, and fetal samples collected. Fetal liver was analyzed for general tissue morphology, and fetal skeletal muscles were analyzed for lipid accumulation. mRNA expression of growth and metabolic factors were quantified in liver and muscle tissues. Hepatocellular vacuolation was increased in RES-CON and RES-RES compared with CON-CON and CON-RES (P &amp;lt; 0.01). In semitendinosus and triceps brachii, intramyocellular lipid content increased 19% and 15%, respectively, in RES-CON and RES-RES compared with CON-CON and CON-RES (P£0.02) and in longissimus dorsi, lipid content was decreased 7% in CON-RES and RES-RES compared with CON-CON and RES-CON (P=0.01). In liver, insulin-like growth factor binding protein-1, glycogen synthase 2, and pyruvate dehydrogenase kinase 1 expression increased 1.92-fold, 1.45-fold, and 1.47-fold, respectively (P£0.03) in CON-RES and RES-RES compared with RES-CON and CON-CON. In LD, IGF1-R expression increased 3.19-fold in CON-RES and RES-RES compared with RES-CON and CON-CON (P = 0.05). These results demonstrate that maternal nutrient restriction followed by re-alimentation restores liver and muscle gene expression of growth and metabolic factors while negatively impacting liver composition and muscle lipid content potentially leading to altered tissue function and metabolism later in life. Supported by USDA-AFRI grants 2016-67016-24884 and 2017-67016-26568.

PSII-18 The effects of fish oil supplementation on markers of inflammation and skeletal muscle and adipose growth in pigs

Abstract Omega-3 fatty acids have immunomodulatory and anti-inflammatory effects. The objective of this project was to determine the effects of fish oil, a source of omega-3 fatty acids, on genes involved in inflammation and growth of skeletal muscle tissue after an LPS challenge. Male Landrace-New Hampshire weaned piglets (BW 8.21±0.83 kg) were used in a randomized complete block design and assigned to two treatments: 1) basal diet (n=7) and 2) basal diet plus 3% fish oil added (n = 7). Treatments were fed for 35 d. On d 34, an LPS challenge was performed and 24 h later, piglets were euthanized and skeletal muscle samples were collected from the longissimus lumborum and biceps femoris. Total mRNA was isolated and markers of inflammation [cyclophilin (Cyclo), nuclear factor kappa beta subunit-1 (NF-kB), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6)], skeletal muscle growth [paired box transcription factor-7 (Pax7), myogenic factor-5 (Myf5), myoblast determination factor-1 (MyoD), myogenin (MyoG)] and adipose growth (peroxisome proliferator activated receptor (PPARy), leptin, and adiponectin) were analyzed. Cyclophilin abundance was increased (P = 0.03) in fish-oil piglets compared to control piglets. Other markers of inflammation (TNF-α, IL-6, NF-kB) were not affected (P &amp;gt; 0.05) by fish-oil supplementation. Abundance of Myf5 was lower (P = 0.03) in fish oil piglets than control piglets. Other myogenic regulatory factors (Pax7, MyoD, MyoG) were not (P &amp;gt; 0.05) altered by treatment. Abundance of PPARy, leptin or adiponectin was not affected (P &amp;gt; 0.05) by fish-oil supplementation. Muscle location influenced (P &amp;lt; 0.01) abundance of leptin and adiponectin, with abundance being higher in the biceps femoris than in the longissimus lumborum. No other genes analyzed were impacted by muscle location (P &amp;gt; 0.05). Our findings suggest that supplementation of omega-3 fatty acids via fish-oil may affect the inflammatory response and skeletal muscle growth. Further research is needed to evaluate the impact of these results on animal production.