scholarly journals Leucine Supplementation Decreases HDAC4 Expression and Nuclear Localization in Skeletal Muscle Fiber of Rats Submitted to Hindlimb Immobilization

Cells ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 2582
Author(s):  
Paula K. N. Alves ◽  
André Cruz ◽  
William J. Silva ◽  
Siegfried Labeit ◽  
Anselmo S. Moriscot
Keyword(s):  
Skeletal Muscle ◽  
Target Genes ◽  
Marked Decrease ◽  
Mrna Levels ◽  
Rna Seq ◽  
Rat Skeletal Muscle ◽  
Leucine Supplementation ◽  
Male Wistar Rats ◽  
Histone Deacetylase 4 ◽  
The Impact

In this study we surveyed a rat skeletal muscle RNA-Seq for genes that are induced by hindlimb immobilization and, in turn, become attenuated by leucine supplementation. This approach, in search of leucine-atrophy protection mediating genes, identified histone deacetylase 4 (HDAC4) as highly responsive to both hindlimb immobilization and leucine supplementation. We then examined the impact of leucine on HDAC4 expression, tissue localization, and target genes. A total of 76 male Wistar rats (~280 g) were submitted to hindlimb immobilization and/or leucine supplementation for 3, 7 and 12 days. These animals were euthanized, and soleus muscle was removed for further analysis. RNA-Seq analysis of hindlimb immobilized rats indicated a sharp induction (log2 = 3.4) of HDAC4 expression which was attenuated by leucine supplementation (~50%). Real-time PCR and protein expression analysis by Western blot confirmed increased HDAC4 mRNA after 7 days of hindlimb immobilization and mitigation of induction by leucine supplementation. Regarding the HDAC4 localization, the proportion of positive nuclei was higher in the immobilized group and decreased after leucine supplementation. Also, we found a marked decrease of myogenin and MAFbx-atrogin-1 mRNA levels upon leucine supplementation, while CAMKII and DACH2 mRNA levels were increased by leucine supplementation. Our data suggest that HDAC4 inhibition might be involved in the anti-atrophic effects of leucine.

mRNA levels for α-subunit of prolyl 4-hydroxylase and fibrillar collagens in immobilized rat skeletal muscle

1999 ◽  
Vol 87 (1) ◽  
pp. 90-96 ◽  
Author(s):  
Xiao-Yan Han ◽  
Wei Wang ◽  
Raili Myllylä ◽  
Paula Virtanen ◽  
Jarmo Karpakka ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Tibialis Anterior ◽  
Collagen Synthesis ◽  
Plantar Flexion ◽  
Mrna Level ◽  
Type I ◽  
Mrna Levels ◽  
Rat Skeletal Muscle ◽  
Α Subunit ◽  
Fibrillar Collagens

There is evidence that immobilization causes a decrease in total collagen synthesis in skeletal muscle within a few days. In this study, early immobilization effects on the expression of prolyl 4-hydroxylase (PH) and the main fibrillar collagens at mRNA and protein levels were investigated in rat skeletal muscle. The right hindlimb was immobilized in full plantar flexion for 1, 3, and 7 days. Steady-state mRNAs for α- and β-subunits of PH and type I and III procollagen, PH activity, and collagen content were measured in gastrocnemius and plantaris muscles. Type I and III procollagen mRNAs were also measured in soleus and tibialis anterior muscles. The mRNA level for the PH α-subunit decreased by 49 and 55% ( P < 0.01) in gastrocnemius muscle and by 41 and 39% ( P < 0.05) in plantaris muscle after immobilization for 1 and 3 days, respectively. PH activity was decreased ( P < 0.05–0.01) in both muscles at days 3 and 7. The mRNA levels for type I and III procollagen were decreased by 26–56% ( P < 0.05–0.001) in soleus, tibialis anterior, and plantaris muscles at day 3. The present results thus suggest that pretranslational downregulation plays a key role in fibrillar collagen synthesis in the early phase of immobilization-induced muscle atrophy.

scholarly journals TWIST1 and TWIST2 regulate glycogen storage and inflammatory genes in skeletal muscle

2015 ◽  
Vol 224 (3) ◽  
pp. 303-313 ◽  
Author(s):  
Jonathan M Mudry ◽  
Julie Massart ◽  
Ferenc L M Szekeres ◽  
Anna Krook
Keyword(s):  
Skeletal Muscle ◽  
Metabolic Diseases ◽  
Glycogen Synthesis ◽  
C2c12 Cells ◽  
Acid Oxidation ◽  
Diabetic Patients ◽  
Mrna Levels ◽  
Intact Mouse ◽  
Mouse Muscle ◽  
The Impact

TWIST proteins are important for development of embryonic skeletal muscle and play a role in the metabolism of tumor and white adipose tissue. The impact of TWIST on metabolism in skeletal muscle is incompletely studied. Our aim was to assess the impact of TWIST1 and TWIST2 overexpression on glucose and lipid metabolism. In intact mouse muscle, overexpression of Twist reduced total glycogen content without altering glucose uptake. Expression of TWIST1 or TWIST2 reducedPdk4mRNA, while increasing mRNA levels ofIl6,Tnfα, andIl1β. Phosphorylation of AKT was increased and protein abundance of acetyl CoA carboxylase (ACC) was decreased in skeletal muscle overexpressing TWIST1 or TWIST2. Glycogen synthesis and fatty acid oxidation remained stable in C2C12 cells overexpressing TWIST1 or TWIST2. Finally, skeletal muscle mRNA levels remain unaltered inob/obmice, type 2 diabetic patients, or in healthy subjects before and after 3 months of exercise training. Collectively, our results indicate that TWIST1 and TWIST2 are expressed in skeletal muscle. Overexpression of these proteins impacts proteins in metabolic pathways and mRNA level of cytokines. However, skeletal muscle levels of TWIST transcripts are unaltered in metabolic diseases.

scholarly journals Thyroxine Induces Acute Relaxation of Rat Skeletal Muscle Arteries via Integrin αvβ3, ERK1/2 and Integrin-Linked Kinase

2021 ◽  
Vol 12 ◽  
Author(s):  
Ekaterina K. Selivanova ◽  
Dina K. Gaynullina ◽  
Olga S. Tarasova
Keyword(s):  
Skeletal Muscle ◽  
Kinase Inhibitors ◽  
Isometric Force ◽  
Rho Kinase ◽  
Integrin Αvβ3 ◽  
Acute Effects ◽  
Rat Skeletal Muscle ◽  
Subsequent Decrease ◽  
Adrenoceptor Agonist ◽  
Male Wistar Rats

Aim: Hyperthyroidism is associated with a decreased peripheral vascular resistance, which could be caused by the vasodilator genomic or non-genomic effects of thyroid hormones (TH). Non-genomic, or acute, effects develop within several minutes and involve a wide tissue-specific spectrum of molecular pathways poorly studied in vasculature. We aimed to investigate the mechanisms of acute effects of TH on rat skeletal muscle arteries.Methods: Sural arteries from male Wistar rats were used for isometric force recording (wire myography) and phosphorylated protein content measurement (Western blotting).Results: Both triiodothyronine (T3) and thyroxine (T4) reduced contractile response of sural arteries to α1-adrenoceptor agonist methoxamine. The effect of T4 was more prominent than T3 and not affected by iopanoic acid, an inhibitor of deiodinase 2. Endothelium denudation abolished the effect of T3, but not T4. Integrin αvβ3 inhibitor tetrac abolished the effect of T4 in endothelium-denuded arteries. T4 weakened methoxamine-induced elevation of phospho-MLC2 (Ser19) content in arterial samples. The effect of T4 in endothelium-denuded arteries was abolished by inhibiting ERK1/2 activation with U0126 as well as by ILK inhibitor Cpd22 but persisted in the presence of Src- or Rho-kinase inhibitors (PP2 and Y27632, respectively).Conclusion: Acute non-genomic relaxation of sural arteries induced by T3 is endothelium-dependent and that induced by T4 is endothelium-independent. The effect of T4 on α1-adrenergic contraction is stronger compared to T3 and involves the suppression of extracellular matrix signaling via integrin αvβ3, ERK1/2 and ILK with subsequent decrease of MLC2 (Ser19) phosphorylation.

scholarly journals Human and rat skeletal muscle single-nuclei multi-omic integrative analyses nominate causal cell types, regulatory elements, and SNPs for complex traits

2021 ◽  
Author(s):  
Peter Orchard ◽  
Nandini Manickam ◽  
Christa Ventresca ◽  
Swarooparani Vadlamudi ◽  
Arushi Varshney ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Cell ◽  
Muscle Fiber ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Skeletal Muscle Cell ◽  
Rna Seq ◽  
Rat Skeletal Muscle ◽  
Integrative Analyses ◽  
Single Nucleus

Skeletal muscle accounts for the largest proportion of human body mass, on average, and is a key tissue in complex diseases and mobility. It is composed of several different cell and muscle fiber types. Here, we optimize single-nucleus ATAC-seq (snATAC-seq) to map skeletal muscle cell–specific chromatin accessibility landscapes in frozen human and rat samples, and single-nucleus RNA-seq (snRNA-seq) to map cell-specific transcriptomes in human. We additionally perform multi-omics profiling (gene expression and chromatin accessibility) on human and rat muscle samples. We capture type I and type II muscle fiber signatures, which are generally missed by existing single-cell RNA-seq methods. We perform cross-modality and cross-species integrative analyses on 33,862 nuclei and identify seven cell types ranging in abundance from 59.6% to 1.0% of all nuclei. We introduce a regression-based approach to infer cell types by comparing transcription start site–distal ATAC-seq peaks to reference enhancer maps and show consistency with RNA-based marker gene cell type assignments. We find heterogeneity in enrichment of genetic variants linked to complex phenotypes from the UK Biobank and diabetes genome-wide association studies in cell-specific ATAC-seq peaks, with the most striking enrichment patterns in muscle mesenchymal stem cells (∼3.5% of nuclei). Finally, we overlay these chromatin accessibility maps on GWAS data to nominate causal cell types, SNPs, transcription factor motifs, and target genes for type 2 diabetes signals. These chromatin accessibility profiles for human and rat skeletal muscle cell types are a useful resource for nominating causal GWAS SNPs and cell types.

Cachectin/TNF or IL-1 alpha induces cachexia with redistribution of body proteins

1989 ◽  
Vol 256 (3) ◽  
pp. R659-R665 ◽  
Author(s):  
Y. Fong ◽  
L. L. Moldawer ◽  
M. Marano ◽  
H. Wei ◽  
A. Barber ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Content ◽  
Muscle Protein ◽  
Interleukin 1 ◽  
Metabolic Effects ◽  
Liver Protein ◽  
Mrna Levels ◽  
Skeletal Muscle Protein ◽  
Male Wistar Rats

Macrophage secretory products are suspected to participate in the severe lean tissue wasting related to chronic illness. The protein metabolic effects of chronic, 7-day cachectin/tumor necrosis factor (cachectin) or interleukin 1 alpha (IL-1 alpha) administration in vivo were studied in male Wistar rats that were 1) freely fed, 2) pair fed, 3) total protein and calorie starved, 4) twice daily lipopolysaccharide (LPS) administered, 5) twice daily cachectin administered, and 6) twice daily IL-1 alpha administered. LPS, cachectin, or IL-1 alpha administration produced anorexia; weight loss in these groups was comparable to respective pair-fed animals. However, LPS, cachectin, or IL-1 alpha accelerated peripheral protein wasting while preserving liver protein content, unlike the pattern in the pair-fed or starved animals in which loss of liver proteins and relative preservation of skeletal muscle protein were observed. The decrease in skeletal muscle protein content in LPS- or cytokine-treated animals was associated with coordinate decreases in muscle mRNA levels for the myofibrillar proteins myosin heavy chain, myosin light chain, actin, and in the 18S and 28S subunits of ribosomal RNA. We conclude that chronic exposure to the cytokines, IL-1 alpha or cachectin, can simulate those body and muscle protein changes seen in experimental LPS administration or chronic disease and markedly differ from the pattern of protein redistribution due to caloric restriction.

scholarly journals Reduced miR-26b Expression in Megakaryocytes and Platelets Contributes to Elevated Level of Platelet Activation Status in Sepsis

2020 ◽  
Vol 21 (3) ◽  
pp. 866 ◽  
Author(s):  
Bernadett Szilágyi ◽  
Zsolt Fejes ◽  
Szilárd Póliska ◽  
Marianna Pócsi ◽  
Zsolt Czimmerer ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Mirna Expression ◽  
Mrna Level ◽  
Severe Infection ◽  
Mrna Levels ◽  
Rna Seq ◽  
Calpain Inhibition ◽  
The Impact ◽  
Lps Treatment ◽  
Activation Status

In sepsis, platelets may become activated via toll-like receptors (TLRs), causing microvascular thrombosis. Megakaryocytes (MKs) also express these receptors; thus, severe infection may modulate thrombopoiesis. To explore the relevance of altered miRNAs in platelet activation upon sepsis, we first investigated sepsis-induced miRNA expression in platelets of septic patients. The effect of abnormal Dicer level on miRNA expression was also evaluated. miRNAs were profiled in septic vs. normal platelets using TaqMan Open Array. We validated platelet miR-26b with its target SELP (P-selectin) mRNA levels and correlated them with clinical outcomes. The impact of sepsis on MK transcriptome was analyzed in MEG-01 cells after lipopolysaccharide (LPS) treatment by RNA-seq. Sepsis-reduced miR-26b was further studied using Dicer1 siRNA and calpain inhibition in MEG-01 cells. Out of 390 platelet miRNAs detected, there were 121 significantly decreased, and 61 upregulated in sepsis vs. controls. Septic platelets showed attenuated miR-26b, which were associated with disease severity and mortality. SELP mRNA level was elevated in sepsis, especially in platelets with increased mean platelet volume, causing higher P-selectin expression. Downregulation of Dicer1 generated lower miR-26b with higher SELP mRNA, while calpeptin restored miR-26b in MEG-01 cells. In conclusion, decreased miR-26b in MKs and platelets contributes to an increased level of platelet activation status in sepsis.

Regulation of Erythropoiesis by Histone Methyltransferase EZH2 Inhibitor 3-Deazaneplanocin A (DZNep)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 982-982
Author(s):  
Tohru Fujiwara ◽  
Haruka Saitoh ◽  
Yoko Okitsu ◽  
Noriko Fukuhara ◽  
Yasushi Onishi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Wide Range ◽  
The Impact ◽  
Chip Analysis

Abstract Abstract 982 Background. EZH2, a core component of Polycomb repressive complex 2 (PRC2), plays a role in transcriptional repression through mediating trimethylation of histone H3 at lysine 27 (H3K27), and is involved in various biological processes, including hematopoiesis. Overexpression of EZH2 has been identified in a wide range of solid tumors as well as hematological malignancies. Recent studies indicated that 3-deazaneplanocin A (DZNep), an inhibitor of EZH2, preferentially induces apoptosis in cancer cells, including acute myeloid leukemia and myelodysplastic syndromes, implying that EZH2 may be a potential new target for epigenetic treatment. On the other hand, whereas PRC2 complex has been reported to participate in epigenetic silencing of a subset of GATA-1 target genes during erythroid differentiation (Yu et al. Mol Cell 2009; Ross et al. MCB 2012), the impact of DZNep on erythropoiesis has not been evaluated. Method. The K562 erythroid cell line was used for the analysis. The cells were treated with DZNep at doses of 0.2 and 1 microM for 72 h. Quantitative ChIP analysis was performed using antibodies to acetylated H3K9 and GATA-1 (Abcam). siRNA-mediated knockdown of EZH2 was conducted using Amaxa nucleofection technology™ (Amaxa Inc.). For transcription profiling, SurePrint G3 Human GE 8 × 60K (Agilent) and Human Oligo chip 25K (Toray) were used for DZNep-treated and EZH2 knockdown K562 cells, respectively. Gene Ontology was analyzed using the DAVID Bioinformatics Program (http://david.abcc.ncifcrf.gov/). Results. We first confirmed that DZNep treatment decreased EZH2 protein expression without significantly affecting EZH2 mRNA levels, suggesting that EZH2 was inhibited at the posttranscriptional level. We also confirmed that DZNep treatment significantly inhibited cell growth. Interestingly, the treatment significantly induced erythroid differentiation of K562 cells, as determined by benzidine staining. Transcriptional profiling with untreated and DZNep-treated K562 cells (1 microM) revealed that 789 and 698 genes were upregulated and downregulated (> 2-fold), respectively. The DZNep-induced gene ensemble included prototypical GATA-1 targets, such as SLC4A1, EPB42, ALAS2, HBA, HBG, and HBB. Concomitantly, DZNep treatment at both 0.2 and 1 microM upregulated GATA-1 protein level as determined by Western blotting, whereas the effect on its mRNA levels was weak (1.02- and 1.43-fold induction with 0.2 and 1 microM DZNep treatment, P = 0.73 and 0.026, respectively). Furthermore, analysis using cycloheximide treatment, which blocks protein synthesis, indicated that DZNep treatment could prolong the half-life of GATA-1 protein, suggesting that DZNep may stabilize GATA-1 protein, possibly by affecting proteolytic pathways. Quantitative ChIP analysis confirmed significantly increased GATA-1 occupancy as well as increased acetylated H3K9 levels at the regulatory regions of these target genes. Next, to examine whether the observed results of DZNep treatment were due to the direct inhibition of EZH2 or hitherto unrecognized effects of the compound, we conducted siRNA-mediated transient knockdown of EZH2 in K562 cells. Quantitative RT-PCR analysis demonstrated that siRNA-mediated EZH2 knockdown had no significant effect on the expression of GATA-1 as well as erythroid-lineage related genes. Furthermore, transcription profiles of the genes in the quantitative range of the array were quite similar between control and EZH2 siRNA-treated K562 cells, with a correlation efficient of 0.977. Based on our profiling results, we are currently exploring the molecular mechanisms by which DZNep promotes erythroid differentiation of K562 cells. Conclusion. DZNep promotes erythroid differentiation of K562 cells, presumably through a mechanism not directly related to EZH2 inhibition. Our microarray analysis of DZNep-treated K562 cells may provide a better understanding of the mechanism of action of DZNep. Disclosures: No relevant conflicts of interest to declare.

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