Project description:Global microarray (HG U133 Plus 2.0) was used for the first time to investigate the effects of resistance exercise on the transcriptome in slow-twitch myosin heavy chain (MHC) I and fast-twitch MHC IIa muscle fibers of young and old women. Vastus lateralis muscle biopsies were obtained pre and 4hrs post resistance exercise in the beginning (untrained state) and at the end (trained state) of a 12 wk progressive resistance training program. A total of 14 females were included in this investigation. The participants included 8 young (23±2y) and 6 old (85±1y) females. All subjects participated in 12 wks of progressive resistance training consisting of bilateral knee extensions with 3x10 reps at 70% of 1-RM, and 3d/wk for a total of 36 training sessions. Vastus lateralis biopsies were obtained in conjunction with the 1st and 36th (last) training session and included a basal biopsy and another biopsy 4hrs post the resistance exercise session. From each biopsy sample, we isolated individual muscle fibers. After myosin isoform identification of isolated fibers (SDS-PAGE), RNA extraction of 20 MHC I and 20 MHC IIa muscle fibers per biopsy sample followed. Thus, each resulting sample contained total RNA from 20 muscle fibers of identical fiber type (MHC I or MHC IIa). A total of 70 samples were analyzed on separate microarray chips, and samples were not pooled between subjects. The study design allowed us to examine the acute effects of resistance exercise on the transcriptome in MHC I and MHC IIa muscle fibers in the untrained and trained state.
Project description:Global microarray (HG U133 Plus 2.0) was used for the first time to investigate the effects of resistance exercise on the transcriptome in slow-twitch myosin heavy chain (MHC) I and fast-twitch MHC IIa muscle fibers of young and old women. Vastus lateralis muscle biopsies were obtained pre and 4hrs post resistance exercise in the beginning (untrained state) and at the end (trained state) of a 12 wk progressive resistance training program.
Project description:RNA-Seq was used for the first time to investigate the effects of resistance exercise on transcriptome dynamics in slow myosin heavy chain (MHC) I and fast MHC IIa muscle fibers among 3 groups of men. Vastus lateralis muscle biopsies were obtained before and 4 hours after resistance exercise.
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.
Project description:Effects of lifelong exercise and age on transcriptome dynamics in MHC I and MHC IIa muscle fibers at baseline and in response to resistance exercise
Project description:Global microarray (HG U133 Plus 2.0) was used to investigate the effects of resistance exercise and resistance training on the skeletal muscle transcriptome profile of 28 young and old adults. Vastus lateralis muscle biopsies were obtained pre and 4hrs post resistance exercise in the beginning (untrained state) and at the end (trained state) of a 12 wk progressive resistance training program.
Project description:Skeletal muscle must perform a wide range of kinds of work, and different fiber types have evolved to accommodate these different tasks. The attributes of fibers are determined in large part by the coordinated regulation of oxidative capacity, as reflected by mitochondrial content, and the specific makeup of myofibrillar proteins. Adult muscle fibers contain four myosin heavy chain isotypes: I, IIa, IIx and IIb. Type I and IIa fibers have slower twitches and are rich in mitochondria, while type IIb fibers are fast-twitch and predominantly glycolytic. The intermediate IIx fibers are less well understood. Previous work had shown that the transcriptional coactivator PGC-1 alpha could drive the formation of type I and IIa muscle fibers. We show here that mice with transgenic expression of PGC-1 beta in skeletal muscle results in marked induction of IIx fibers. The fibers in transgenic mice are rich in mitochondria and are highly oxidative. As a result, PGC-1 beta transgenic animals can perform oxidative activity for longer and at higher work loads than wild type animals. In cell culture, PGC-1 beta coactivates the MEF2 family of transcription factors to stimulate the MHC IIx promoter. Together, these data indicate that PGC-1 beta is sufficient to drive the formation in vivo of highly oxidative fibers with type IIx characteristics.
Project description:Effects of lifelong exercise and age on transcriptome dynamics in MHC I and MHC IIa muscle fibers at baseline and in response to resistance exercise (all samples)
Project description:Global microarray (HG U133 Plus 2.0) was used to investigate the effects of resistance exercise and resistance training on the skeletal muscle transcriptome profile of 28 young and old adults. Vastus lateralis muscle biopsies were obtained pre and 4hrs post resistance exercise in the beginning (untrained state) and at the end (trained state) of a 12 wk progressive resistance training program. A total of 28 subjects were included in this investigation. The young (n=16, 24±1y) participants included 8 males and 8 females. The old (n=12, 84±1y) participants included 6 males and 6 females. All subjects participated in 12 wks of progressive resistance training consisting of bilateral knee extensions with 3x10 reps at 70% of 1-RM, and 3d/wk for a total of 36 training sessions. Vastus lateralis biopsies were obtained in conjunction with the 1st and 36th (last) training session and included a basal biopsy and another biopsy 4hrs post the resistance exercise session (for a total of 4 biopsies per subject). The RNA integrity for all samples was very good with an average RIN number of 7.6. All 110 samples were analyzed on a separate microarray chip, and samples were not pooled between subjects. The study design allowed us to examine the acute effects of resistance exercise on the skeletal muscle transcriptome in the untrained and trained state. The design also provided information on the effects of resistance training on basal level gene expression and the effects of age on basal level gene expression
Project description:With aging, skeletal muscle plasticity is attenuated in response to exercise. Here, we report that senescent cells, identified using senescence markers senescence-associated β-Galactosidase (SA β-Gal) and p21 are very infrequent in resting muscle but emerge approximately two-weeks after a bout of resistance exercise in humans. We hypothesized that these cells contribute to blunted hypertrophic potential in old age. Using synergist ablation-induced mechanical overload of the plantaris muscle to model resistance training in adult (5-6 month) and old (23-24 month) male C57BL/6J mice, we found increased senescent cells in both age groups during hypertrophy. Consistent with the human data, there were negligible senescent cells in adult and old sham controls, but old mice had significantly more senescent cells 7- and 14-days following overload relative to young. Old mice had blunted whole muscle hypertrophy when compared to adult mice, along with smaller muscle fibers, specifically glycolytic Type 2x+2b fibers. To ablate senescent cells using a hit-and-run approach, old mice were treated with vehicle or a senolytic cocktail consisting of 5 mg/kg dasatinib and 50 mg/kg quercetin (D+Q) on day 7 and 10 during 14-days of overload; control mice underwent sham surgery with or without senolytic treatment. Old mice given D+Q had larger muscles and muscle fibers after 14 days of overload, fewer senescent cells when compared to vehicle-treated old mice, and changes in the expression of genes (i.e., Igf1, Ddit4, Mmp14) that are associated with hypertrophic growth . Our data collectively show that senescent cells emerge in human and mouse skeletal muscle following a hypertrophic stimulus, and that D+Q improves muscle growth in old mice.