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DNA methylation assessment from human slow- and fast-twitch skeletal muscle fibers.

ABSTRACT: A new application of the reduced representation bisulfite sequencing method was developed using low-DNA input to investigate the epigenetic profile of human slow- and fast-twitch skeletal muscle fibers. Successful library construction was completed with as little as 15 ng of DNA, and high-quality sequencing data were obtained with 32 ng of DNA. Analysis identified 143,160 differentially methylated CpG sites across 14,046 genes. In both fiber types, selected genes predominantly expressed in slow or fast fibers were hypomethylated, which was supported by the RNA-sequencing analysis. These are the first fiber type-specific methylation data from human skeletal muscle and provide a unique platform for future research.NEW & NOTEWORTHY This study validates a low-DNA input reduced representation bisulfite sequencing method for human muscle biopsy 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 and thus provide initial insight into basal state differences in myosin heavy chain I and IIa muscle fibers among young, healthy men.

SUBMITTER: Begue G

PROVIDER: S-EPMC5407195 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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DNA methylation assessment from human slow- and fast-twitch skeletal muscle fibers.

Begue Gwénaëlle G Raue Ulrika U Jemiolo Bozena B Trappe Scott S

Journal of applied physiology (Bethesda, Md. : 1985) 20170105 4

A new application of the reduced representation bisulfite sequencing method was developed using low-DNA input to investigate the epigenetic profile of human slow- and fast-twitch skeletal muscle fibers. Successful library construction was completed with as little as 15 ng of DNA, and high-quality sequencing data were obtained with 32 ng of DNA. Analysis identified 143,160 differentially methylated CpG sites across 14,046 genes. In both fiber types, selected genes predominantly expressed in slow ...[more]

PMID: 28057818

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Project description:The contractile properties of fast-twitch and slow-twitch skeletal muscles are primarily determined by the myosin isoform content and modulated by a variety of sarcomere proteins. X-ray diffraction studies of regulatory mechanisms in muscle contraction have focused predominately on fast- or mixed-fibre muscle with slow muscle being much less studied. Here, we used time-resolved X-ray diffraction to investigate the dynamic behaviour of the myofilament proteins in relatively pure slow-twitch-fibre rat soleus (SOL) and pure fast-twitch-fibre rat extensor digitorum longus (EDL) muscle during twitch and tetanic contractions at optimal length. During twitch contractions the diffraction signatures indicating a transition in the myosin heads from ordered OFF states, where heads are held close to the thick filament backbone, to disordered ON states, where heads are free to bind to thin filaments, were found in EDL and not in SOL muscle. During tetanic contraction, changes in the disposition of myosin heads as active tension develops is a quasi-stepwise process in EDL muscle whereas in SOL muscle this relationship appears to be linear. The observed reduced extensibility of the thick filaments in SOL muscle as compared to EDL muscles indicates a molecular basis for this behaviour. These data indicate that for the EDL, thick filament activation is a cooperative strain-induced mechano-sensing mechanism, whereas for the SOL, thick filament activation has a more graded response. These different approaches to thick filament regulation in fast- and slow-twitch muscles may be adaptations for short-duration, strong contractions versus sustained, finely controlled contractions, respectively. KEY POINTS: Fast-twitch muscle and slow-twitch muscle are optimized for strong, short-duration contractions and for tonic postural activity, respectively. Structural events (OFF to ON transitions) in the myosin-containing thick filaments in fast muscle help determine the timing and strength of contractions, but these have not been studied in slow-twitch muscle. The X-ray diffraction signatures of structural OFF to ON transitions are different in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle, being completely absent during twitches in soleus muscle and blunted during tetanic contractions SOL as compared to EDL Quasi-stepwise thick filament structural OFF to ON transitions in fast twitch muscle may be an adaptation for rapid, ballistic movements, whereas more graded OFF to ON structural transitions in slow-twitch muscle may be an adaptation for slower, finer motions.

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.

Dataset Information

DNA methylation assessment from human slow- and fast-twitch skeletal muscle fibers.

Publications

DNA methylation assessment from human slow- and fast-twitch skeletal muscle fibers.

Similar Datasets

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