Homo sapiens
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ABSTRACT:
PROVIDER: PRJNA326399 | ENA |
REPOSITORIES: ENA
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Project description:DNA methylation on type-specific human skeletal muscle fibers
| PRJNA326906 | ENA
Project description:Purpose: Reduced Representation Bisulfite Sequencing (RRBS) DNA input requirements become a challenge when working with small pools of tissue-specific cell types. We describe an application of the RRBS method to assess DNA methylation on low-DNA input from human slow-twitch (MHC I) and fast-twitch (MHC IIa) skeletal muscle fibers. Methods : Fiber-type specific (MHC I and MHC IIa muscle fibers) total DNA was extracted from vastus lateralis muscle biopsies of 8 young physically active men (~25 yrs). A total of 16 DNA samples were generated : 8 DNA samples from pure MHC I and 8 DNA samples from pure MHC IIa muscle fibers. An equal quantity of DNA (4 ng) from each sample was combined to generate a "pooled" DNA sample representing all 8 subjects for each fiber type. Two fiber-type specific "pooled" samples of 32 ng of DNA were generated for library construction and sequencing, creating a Type 1 (MHC I muscle fibers) and Type 2a (MHC IIa muscle fibers) sample. Sequencing was performed using the HiSeq 2500 (Illumina) with 50 bp paired-end read parameters. Minimum sequencing read coverage of 5 (5x) was used as the cutoff for CpG-sites inclusion in the DNA methylation analysis. Fisher’s exact test was performed on CpG-sites that overlapped (i.e. identified in both samples) Type 1 and Type 2a samples to obtain p-values that indicate the likelihood of the site being a differentially methylated CpG-site (DMS). DMS with p<0.05 were classified as hypermethylated or hypomethylated if they were more or less methylated than the Type 1 sample, which was used as the reference sample. Results: The 32 ng of DNA from fiber-type specific muscle samples (Type 1 and 2a) used in this study ensured similar sequencing quality as compared to other studies using greater DNA input (>50 ng). Mapping ratios of ~47% and bisulfite conversion rates of ~97-98% were obtained.The unique and best alignment was successfully assessed for each of 17,376,728 CpG-sites in the Type 1 sample and 17,006,993 in the Type 2a sample, which represents ~30% of the total CpG number in the human genome. We identified 143,160 differentially methylated CpG-sites (DMS) across 14,046 genes among MHC I and MHC IIa muscle fibers. The analysis revealed that some genes predominantly expressed in MHC I were hypermethylated in MHC IIa muscle fibers. Conclusion: This study validates a low-DNA input RRBS method for human skeletal muscle samples to investigate the methylation patterns at a fiber-type specific level. These are the first fiber-type specific methylation data reported from human skeletal muscle. Considering the metabolic and structural differences between MHC I and MHC IIa muscle fibers, this technique could provide novel insights into the skeletal muscle methylation profile in relation to health, performance, disease or disuse.
2017-01-10 | GSE83741 | GEO
Project description:DNA methylation analysis of human skeletal muscle after endurance training
| PRJNA259207 | ENA
Project description:RNA-seq was performed to investigate the role of Rrm2b in skeletal muscle. Type II skeletal muscle fibers were collected from wild-type (C57BL/6) mice and two Rrm2b knockout models, the skeletal muscle-specific knockout (Rrm2b F/F;HSA-Cre, smKO) and satellite cell-specific knockout (Rrm2b F/F;Pax7-CreERT2, scKO).
2022-07-25 | E-MTAB-11318 | biostudies-arrayexpress
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs.
2011-08-25 | GSE23936 | GEO
Project description:Skeletal muscle plays an important role in the health-promoting effects of exercise training, yet the underlying mechanisms are not fully elucidated. Proteomics of skeletal muscle is challenging due to presence of non-muscle tissues and existence of different fiber types confounding the results. This can be circumvented by analysis of pure fibers; however this requires isolation of fibers from fresh tissues. We developed a workflow enabling proteomics analysis of isolated muscle fibers from freeze-dried muscle biopsies and identified >4000 proteins. We investigated effects of exercise training on the pool of slow and fast muscle fibers. Exercise altered expression of >500 proteins irrespective of fiber type covering several metabolic processes, mainly related to mitochondria. Furthermore, exercise training altered proteins involved in regulation of post-translational modifications, transcription, Ca++ signaling, fat, and glucose metabolism in a fiber type-specific manner. Our data serves as a valuable resource for elucidating molecular mechanisms underlying muscle performance and health. Finally, our workflow offers methodological advancement allowing proteomic analyses of already stored freeze-dried human muscle biopsies.
2020-12-21 | PXD012824 | Pride
Project description:Skeletal muscle is a heterogeneous tissue consisting of blood vessels, connective tissue, and muscle fibers. The last are highly adaptive and can change their molecular composition depending on external and internal factors, such as exercise, age, and disease. Thus, examination of the skeletal muscles at the fiber type level is essential to detect potential alterations. Therefore, we established a protocol in which myosin heavy chain isoform immunolabeled muscle fibers were laser microdissected and separately investigated by mass spectrometry to develop advanced proteomic profiles of all murine skeletal muscle fiber types. Our in-depth mass spectrometric analysis revealed unique fiber type protein profiles, confirming fiber type-specific metabolic properties and revealing a more versatile function of type IIx fibers. Furthermore, we found that multiple myopathy-associated proteins were enriched in type I and IIa fibers. To further optimize the assignment of fiber types based on the protein profile, we developed a hypothesis-free machine-learning approach (available at: https://github.com/mpc-bioinformatics/FiPSPi), identified a discriminative peptide panel, and confirmed our panel using a public data set.
2021-06-07 | PXD025359 | Pride
Project description:Skeletal muscle is a key tissue in human aging, which affects different muscle fiber types unequally. We developed a highly sensitive single muscle fiber proteomics workflow to study human aging and show that the senescence of slow and fast muscle fibers is characterized by diverging metabolic and protein quality control adaptations. Whereas mitochondrial content declines with aging in both fiber types, glycolysis and glycogen metabolism are upregulated in slow but downregulated in fast muscle fibers. Aging mitochondria decrease expression of the redox enzyme monoamine oxidase A. Slow fibers upregulate a subset of actin and myosin chaperones, whereas an opposite change happens in fast fibers. These changes in metabolism and sarcomere quality control may be related to the ability of slow, but not fast, muscle fibers to maintain their mass during aging. We conclude that single muscle fiber analysis by proteomics can elucidate pathophysiology in a sub-type specific manner.
2017-06-14 | PXD006182 | Pride
Project description:Skeletal muscles are composed of a heterogeneous collection of fiber types with different physiological adaption in response to a stimulus and disease-related conditions. Each fiber has a specific molecular expression of myosin heavy chain molecules (MyHC). So far MyHCs are currently the best marker proteins for characterization of individual fiber types and several proteome profiling studies have helped to dissect the molecular signature of whole muscles and individual fibers. Herein, we describe a mass spectrometric workflow to measure skeletal muscle fiber type-specific proteomes. To bypass the limited quantities of protein in single fibers, we developed a Proteomics high-throughput Fiber Typing (ProFiT) approach enabling profiling of MyHC in single fibers. Aliquots of protein extracts from separated muscle fibers were subjected to capillary LC-MS gradients to profile MyHC isoforms in a 96-well format. Muscle fibers with the same MyHC protein expression were pooled and subjected to proteomic, pulsed-SILAC and phosphoproteomic analysis. Our fiber type-specific quantitative proteome analysis confirmed the distribution of fiber types in the soleus muscle, substantiates metabolic adaptions in oxidative and glycolytic fibers, and highlighted significant differences between the proteomes of type IIb fibers from different muscle groups, including a differential expression of desmin and actinin-3. A detailed map of the Lys-6 incorporation rates in muscle fibers showed an increased turnover of slow fibers compared to fast fibers. In addition, labeling of mitochondrial respiratory chain complexes revealed a broad range of Lys-6 incorporation rates, depending on the localization of the subunits within distinct complexes.
2021-02-02 | PXD017755 | Pride
Project description:Genome wide DNA methylation changes with age in disease free human skeletal muscle
| PRJNA217793 | ENA