Biology subjects

Godwin, J. S.

Publications and source records attributed to Godwin, J. S..

12 recordsLinked to original sources

bioRxiv2026

Aging concomitantly reduces skeletal muscle proteome plasticity and hypertrophic responses to resistance training

Skeletal muscle mass and training adaptations decline with aging, yet the proteomic basis of these attenuated responses remains unclear. We hypothesized that aging is accompanied by diminished proteome plasticity in response to resistance training (RT). The soluble proteome of VL biopsies was profiled in 17 younger (21.9 {+/-} 2.5 yr) and 15 older (57.5 {+/-} 6.9 yr) untrained males before and after 10-12 weeks of supervised RT using data-independent acquisition mass spectrometry (2,113 quantified proteins). At baseline, we detected 196 differentially expressed proteins (DEPs) significantly differed between age groups by {Pi}-score (278 by FDR). A 5.6-fold difference in training-responsive was observed in younger vs. older adults (100 vs. 18 {Pi}-score DEPs; 134 vs. 0 FDR-significant). Despite this quantitative attenuation, 61.6% of proteins changed in the same direction in both age groups (Spearman {rho} = 0.284, p = 3.46 x 10-), indicating conserved but amplitude-compressed training responses (median |log2FC|: 0.13 young vs. 0.09 old). RT in older adults partially reversed the aging proteome in that directionally different changes were observed in 75.2% of aging- or training-significant proteins in aging and training contrasts, with ribosomal and translational machinery showing the strongest reversal (cytoplasmic translation NES: -2.90 with aging, +2.60 with training). Ten WGCNA co-expression modules were identified, with age emerging as the dominant organizing principle (Turquoise module r-equiv = +0.59, p < 0.001). Module eigengenes discriminated age groups at the univariate level (Turquoise/Lipid Catabolism AUC = 0.96, q < 0.012), and training-induced module changes correlated with hypertrophic outcomes. Aging markedly attenuates but does not qualitatively alter skeletal muscle proteome plasticity. RT partially reverses aging proteome signatures, with translational machinery being the most responsive and mitochondrial programs the least responsive. Baseline proteomic state constrains adaptive capacity, suggesting that the molecular features distinguishing aging muscle directly may limit its hypertrophic response to RT.

physiology↗

bioRxiv2025

Skeletal muscle mitochondrial responses to a single bout and six weeks of high load versus high volume resistance training in previously trained men

The effects of higher-load (HL) versus higher-volume (HV) resistance training (RT) on various molecular outcomes are similar. However, mitochondrial responses remain understudied. Therefore, the purpose of this study was to interrogate mitochondrial mRNA and protein responses to acute and chronic HL versus HV RT. Vastus lateralis biopsies from resistance trained males in two prior studies were assessed. In STUDY 1, 11 college-aged men completed an acute bout of either HL or HV RT exercises to failure. Biopsies were collected at PRE, 3 hours post-, and 6 hours post-exercise. In STUDY 2, 15 college-aged men participated in six weeks of supervised unilateral RT where each leg was assigned to either HL or HV RT. Biopsies were collected from both legs prior to and 72 hours following last training bout of the intervention. Biopsies from both studies were used to assess mitochondrial mRNAs, and STUDY 2 biopsies were assayed for mitochondrial proteins and CS activity. Results from both studies revealed several significant main effects of time but no significant interactions. Additionally, CS activity, a surrogate of mitochondrial content, decreased following chronic RT (p=0.016) but no interaction was observed between the HV and HL leg over time (p=0.882). In conclusion, while RT resulted in both acute mitochondrial mRNA as well as chronic CS activity and mitochondrial protein responses, there were no differences in the HL versus HV paradigms on these outcomes.

physiology↗

bioRxiv2024

Leg immobilization and subsequent recovery resistance training affect skeletal muscle angiogenesis related markers in young healthy adults regardless of prior resistance training experience

We recently reported that resistance trained (T, n=10) and untrained (UT, n=11) young adults experience vastus lateralis (VL) muscle atrophy following two weeks of disuse, and 8 weeks of recovery resistance training (RT) promotes VL hypertrophy in both participant cohorts. However, angiogenesis targets and muscle capillary number were not examined and currently no human studies that have sought to determine if disuse followed by recovery RT affects these outcomes. Thus, we examined whether disuse and/or recovery RT affected these outcomes. All participants underwent two weeks of left leg immobilization using locking leg braces and crutches followed by eight weeks (3d/week) of knee extensor focused progressive RT. VL biopsies were obtained at baseline (PRE), immediately after disuse (MID), and after RT (POST). Western blotting was used to assay angiogenesis markers and immunohistochemistry was performed in 16/21 participants to determine type I and II muscle fiber capillary number. Significant main effects of time (p<0.05) were observed for protein levels of VEGF (MID 0.100). Although disuse and recovery RT affect skeletal muscle angiogenesis-related protein targets, prior training history does not differentially affect these outcomes. NEW AND NOTEWORTHYThis is the first study to examine how limb immobilization and recovery resistance training affect molecular outcomes related to angiogenesis in younger adults with or without a prior training history. Regardless of resistance training history, the molecular responses are largely similar between participant cohorts and is suggestive of a reduced (pre-mid) and increased (mid-post) angiogenic response, with disuse and subsequent recovery resistance training.

physiology↗

bioRxiv2024

Skeletal muscle ribosome analysis: a comparison of common assay methods and utilization of a novel RiboAb antibody cocktail

Cellular and tissue total RNA concentrations have been widely reported to represent ribosome content, a metric that reflects the trophic state of skeletal muscle. Although various assays are used to assess total RNA concentrations, there is a need to homologize the various quantification approaches. Thus, we analyzed C2C12 myotubes and mouse skeletal muscle to determine if total RNA concentrations provided through UV-Vis spectroscopy (UV), fluorometry only (Fluor), and fluorometry-based microfluidic chip electrophoresis (MFGE) were representative of cellular and muscle tissue rRNA concentrations (i.e., MFGE 18S+28S rRNA, criterion metric of ribosome content). We also sought to determine whether a novel ribosomal protein antibody cocktail (termed RiboAb) corresponded with 18S+28S rRNA concentrations. Compared to non-treated C2C12 myotubes, 24-hour insulin-like growth factor-1 (IGF-1) treatments increased 18S+28S rRNA concentrations ([~]2.0-fold; p<0.001) and total RNA concentrations based on UV ([~]1.9-fold; p<0.001), Fluor ([~]2.3 fold; p=0.001), and MFGE ([~]2.1-fold, p<0.001). In C57BL/6 mice, 10 days of mechanical overload (MOV) via synergist ablation elevated plantaris 18S+28S rRNA concentrations ([~]1.7-fold; p=0.017) and total RNA concentrations according to UV ([~]1.5-fold; p=0.033), Fluor ([~]1.6-fold; p=0.001), and MFGE ([~]1.8-fold, p=0.017). In both experiments, total RNA concentration data yielded from all three techniques exhibited significant positive correlations to 18S+28S rRNA concentrations. Ribosome pelleting experiments indicated that the proteins assayed with the RiboAb cocktail (rps3/6 and rpl5/11) were exclusively associated with the ribosome pellet. Additionally, C2C12 myotube and mouse plantaris RiboAb levels were higher with IGF-1 treatments and MOV, respectively, relative to controls (1.3-fold and 1.7-fold, respectively, p<0.017), and values correlated with rRNA concentrations (r=0.637 and r=0.853, respectively, p<0.005). These data confirm that total RNA concentrations yielded from the UV, Fluor, MFGE techniques are valid surrogates of cell/tissue ribosome content. We also propose that the RiboAb cocktail may serve as a surrogate for changes in ribosome content in these models, although future research is needed to examine the feasibility of the RiboAb cocktail in humans as well as utility with other applications (e.g., immunohistochemistry and/or tissue fractionation experiments).

molecular biology↗

bioRxiv2024

Effects of leg immobilization and recovery resistance training on skeletal muscle-molecular markers in previously resistance trained versus untrained adults

We sought to examine how resistance training (RT) status in young healthy individuals, either well-trained (T, n=10 (8 males)) or untrained (UT, n=11 (8 males)), affected muscle size and molecular markers with leg immobilization followed by recovery RT. All participants underwent two weeks of left leg immobilization via the use of crutches and a locking leg brace. After this two-week period, all participants underwent eight weeks (3 d/week) of knee extensor focused progressive RT. Vastus lateralis (VL) ultrasound-derived thickness and muscle cross-sectional area were measured at baseline (PRE), immediately after disuse (MID), and after RT (POST) with VL muscle biopsies collected at these time points. T and UT presented lower ultrasound derived VL size (cross-sectional area and thickness) values at MID versus PRE (p[≤]0.001), and values increased in both groups from MID to POST (p<0.05); however, VL size increased from PRE to POST in UT only (p<0.001). Mean and type II myofiber cross-sectional area (fCSA) values demonstrated a main effect of time where PRE and POST were greater than MID (p<0.05) and main effect of training status where T was greater than UT (P[≤]0.012). In both groups, satellite cell number was not affected by leg immobilization but increased in response to RT (p[≤]0.014), with T being greater than UT across all time points (p=0.004). Additionally, ribosome content (total RNA) decreased (p=0.010) from PRE to MID while the endoplasmic reticulum stress proteins (BiP, Xbp1s, and CHOP) increased from MID to POST regardless of training status. Finally, the phosphorylation states of mechanistic target of rapamycin complex-1 signaling proteins were not significantly altered for either group throughout the intervention. In conclusion, immobilization-induced muscle atrophy and recovery RT hypertrophy outcomes are similar between UT and T participants, and the lack of molecular signature differences between groups supports these findings. However, these data are limited to younger adults undergoing non-complicated disuse. Thus, further investigation to determine the impact of training status on prolonged leg immobilization models mirroring current medical protocols (e.g., following orthopedic injury and surgery) is warranted.

physiology↗

bioRxiv2024

Resistance Exercise and Mechanical Overload Upregulate Vimentin for Skeletal Muscle Remodeling

Our laboratory has performed various experiments examining the proteomic alterations that occur with mechanical overload (MOV)-induced skeletal muscle hypertrophy. In the current study we first sought to determine how 10 weeks of resistance training in 15 college-aged females affected protein concentrations in different tissue fractions. Training, which promoted significantly lower body muscle- and fiber-level hypertrophy, notably increased sarcolemmal/membrane protein content (+10.1%, p<0.05). Sarcolemmal/membrane protein isolates were queried using mass spectrometry-based proteomics, [~]10% (38/387) of proteins associated with the sarcolemma were up-regulated (>1.5-fold, p<0.05), and one of these targets (the intermediate filament vimentin; VIM) warranted further mechanistic investigation. VIM expression was first examined in the plantaris muscles of 4-month-old C57BL/6J mice following 10- and 20-days of MOV via synergist ablation. Relative to Sham (control) mice, VIM mRNA and protein content was significantly higher in MOV mice and immunohistochemistry indicated that VIM was predominantly present in the extracellular matrix (ECM). The 10- and 20-day MOV experiments were replicated in Pax7-DTA (tamoxifen-induced, satellite cell depleted) mice, which reduced the presence of VIM in the ECM. Finally, a third set of 10- and 20-day MOV experiments were performed in C57BL/6 mice intramuscularly injected with either AAV9-scrambled (control) or AAV9-VIM shRNA. While VIM shRNA mice presented with lower VIM in the ECM ([~]50%), plantaris masses in response to MOV were similar between the injection groups. However, VIM shRNA mice presented with appreciably more MyHCemb-positive fibers with centrally located nuclei, indicating a regenerative phenotype. Using an integrative approach, we propose that skeletal muscle VIM is a mechanosensitive target predominantly localized to the ECM, and satellite cells are involved in its expression. Moreover, a disruption in VIM expression during MOV leads to dysfunctional skeletal muscle hypertrophy.

molecular biology↗

bioRxiv2024

Skeletal muscle myosin heavy chain protein fragmentation as a potential marker of protein degradation in response to resistance training and disuse atrophy

We sought to examine how resistance exercise (RE), cycling exercise, and disuse atrophy affect myosin heavy chain (MyHC) protein fragmentation in humans. In the first study (1boutRE), younger adult men (n=8; 5{+/-}2 years of RE experience) performed a lower body RE bout with vastus lateralis (VL) biopsies obtained immediately before, 3-, and 6-hours post-exercise. In the second study (10weekRT), VL biopsies were obtained in untrained younger adults (n=36, 18 men and 18 women) before and 24 hours (24h) after their first/naive RE bout. These participants also engaged in 10 weeks (24 sessions) of resistance training and donated VL biopsies before and 24h after their last RE bout. VL biopsies were also examined from a third acute cycling study (n=7) and a fourth study involving two weeks of leg immobilization (n=20, 15 men and 5 women) to determine how MyHC fragmentation was affected. In the 1boutRE study, the fragmentation of all MyHC isoforms (MyHCTotal) increased 3 hours post-RE ([~] +200%, p=0.018) and returned to pre-exercise levels by 6 hours post-RE. Immunoprecipitation of MyHCTotal revealed ubiquitination levels remained unaffected at the 3- and 6-hour post-RE time points. Interestingly, a greater increase in magnitude for MyHC type IIa versus I isoform fragmentation occurred 3-hours post-RE (8.6{+/-}6.3-fold versus 2.1{+/-}0.7-fold, p=0.018). In all 10weekRT participants, the first/naive and last RE bouts increased MyHCTotal fragmentation 24h post-RE (+65% and +36%, respectively; p<0.001); however, the last RE bout response was attenuated compared to the first bout (p=0.045). The first/naive bout response was significantly elevated in females only (p<0.001), albeit females also demonstrated a last bout attenuation response (p=0.002). Although an acute cycling bout did not alter MyHCTotal fragmentation, [~]8% VL atrophy with two weeks of leg immobilization led to robust MyHCTotal fragmentation (+108%, p<0.001), and no sex-based differences were observed. In summary, RE and disuse atrophy increase MyHC protein fragmentation. A dampened response with 10 weeks of resistance training, and more refined responses in well-trained men, suggest this is an adaptive process. Given the null polyubiquitination IP findings, more research is needed to determine how MyHC fragments are processed. Moreover, further research is needed to determine how aging and disease-associated muscle atrophy affect these outcomes, and whether MyHC fragmentation is a viable surrogate for muscle protein turnover rates.

molecular biology↗

bioRxiv2024

Relative rDNA copy number is not associated with resistance training-induced skeletal muscle hypertrophy and does not affect myotube anabolism in vitro

Ribosomal DNA (rDNA) copies are organized in tandem repeats across multiple chromosomes, and inter-individual variation in rDNA copy number has been speculated to be a modifier of the hypertrophic responses to resistance training. In the current study, 82 apparently healthy participants (n=53 males, 21{+/-}1 years old; n=29 females, 21{+/-}2 years old) performed 10-12 weeks of supervised full-body resistance training. Whole-body, mid-thigh, and histological skeletal muscle hypertrophy outcomes were determined, as was relative rDNA copy number from pre-intervention vastus lateralis (VL) biopsies. Pre- and post-intervention VL biopsy mRNA/rRNA markers of ribosome content and biogenesis were assayed in all participants, and these targets were also assayed in the 29 females 24 hours following their first workout bout. Across all 82 participants, no significant associations were evident between relative rDNA copy number and training-induced changes in whole body lean mass (r = -0.034, p=0.764), vastus lateralis thickness (r = 0.093, p=0.408), mean myofiber cross-sectional area (r = -0.128, p=0.259), or changes in muscle RNA concentrations (r = 0.026, p=0.818). Several significant, positive associations in females support ribosome biogenesis being linked to training-induced myofiber hypertrophy. Follow-up studies using LHCN-M2 myotubes demonstrate a reduction in relative rDNA copy number induced by bisphenol A (BPA). However, BPA did not significantly affect myotube diameter or prevent insulin-like-growth factor-induced hypertrophy. These findings provide strong evidence that relative rDNA copy number is not associated with myofiber anabolism and provide further mechanistic evidence for ribosome biogenesis being involved in this phenomenon.

molecular biology↗

bioRxiv2023

A Novel Imaging Method (FIM-ID) Reveals that Myofibrillogenesis Plays a Major Role in the Mechanically Induced Growth of Skeletal Muscle

An increase in mechanical loading, such as that which occurs during resistance exercise, induces radial growth of muscle fibers (i.e., an increase in cross-sectional area). Muscle fibers are largely composed of myofibrils, but whether radial growth is mediated by an increase in the size of the myofibrils (i.e., myofibril hypertrophy) and/or the number of myofibrils (i.e., myofibrillogenesis) is not known. Electron microscopy (EM) can provide images with the level of resolution that is needed to address this question, but the acquisition and subsequent analysis of EM images is a time- and cost-intensive process. To overcome this, we developed a novel method for visualizing myofibrils with a standard fluorescence microscope (FIM-ID). Images from FIM-ID have a high degree of resolution and contrast, and these properties enabled us to develop pipelines for automated measurements of myofibril size and number. After extensively validating the automated measurements, we used both mouse and human models of increased mechanical loading to discover that the radial growth of muscle fibers is largely mediated by myofibrillogenesis. Collectively, the outcomes of this study offer insight into a fundamentally important topic in the field of muscle growth and provide future investigators with a time- and cost-effective means to study it.

cell biology↗

bioRxiv2023

Resistance Training Diminishes Mitochondrial Adaptations to Subsequent Endurance Training

We investigated the effects of performing a period of resistance training (RT) on the performance and molecular adaptations to a subsequent period of endurance training (ET). Twenty-five young adults were divided into RT+ET (n=13), which underwent seven weeks of RT followed by seven weeks of ET, and ET-only (n=12), which performed seven weeks of ET. Body composition, endurance performance, and muscle biopsies were collected before RT (T1, baseline for RT+ET), before ET (T2, post RT for RT+ET and baseline for ET), and after ET (T3). Immunohistochemistry was performed to determine fiber cross-sectional area (fCSA), myonuclear content, myonuclear domain size, satellite cell number, and mitochondrial content. Western blots were used to quantify markers of mitochondrial remodeling. Citrate synthase activity and markers of ribosome content were also investigated. Resistance training improved body composition and strength, increased vastus lateralis thickness, mixed and type II fCSA, myonuclear number, markers of ribosome content, and satellite cell content (p<0.050). In response to ET, both groups similarly decreased body fat percentage and improved endurance performance (e.g., VO2max, and speed at which the onset of blood lactate accumulation occurred during the VO2max test). Levels of mitochondrial complexes I-IV in the ET-only group increased 32-66%, while the RT+ET group increased 1-11%. Additionally, mixed fiber relative mitochondrial content increased 15% in the ET-only group but decreased 13% in the RT+ET group. In conclusion, RT performed prior to ET had no additional benefits to ET adaptations. Moreover, prior RT seemed to impair mitochondrial adaptations to ET. KEY POINTS SUMMARYO_LIResistance training is largely underappreciated as a method to improve endurance performance, despite reports showing it may improve mitochondrial function. C_LIO_LIAlthough several concurrent training studies are available, in this study we investigated the effects of performing a period resistance training on the performance and molecular adaptations to subsequent endurance training. C_LIO_LIPrior resistance training did not improve endurance performance and impaired most mitochondrial adaptations to subsequent endurance training, but that seemed to be a result of detraining from resistance training. C_LI

molecular biology↗

bioRxiv2022

Skeletal muscle DNA methylation and mRNA responses to a bout of higher versus lower load resistance exercise in previously trained men

We sought to determine the skeletal muscle genome-wide DNA methylation and mRNA responses to one bout of lower-load (LL) versus higher-load (HL) resistance exercise. Trained college-aged males (n=11, 23{+/-}4 years old, 4{+/-}3 years self-reported training) performed LL or HL bouts to failure separated by one week. The HL bout (i.e., 80 Fail) consisted of four sets of back squats and four sets of leg extensions to failure using 80% of participants estimated one-repetition maximum (i.e., est. 1-RM). The LL bout (i.e., 30 Fail) implemented the same paradigm with 30% of est. 1-RM. Vastus lateralis muscle biopsies were collected before, 3 hours, and 6 hours after each bout. Muscle DNA and RNA were batch-isolated and analyzed using the 850k Illumina MethylationEPIC array and Clariom S mRNA microarray, respectively. Performed repetitions were significantly greater during the 30 Fail versus 80 Fail (p<0.001), although total training volume (sets x reps x load) was not significantly different between bouts (p=0.571). Regardless of bout, more CpG site methylation changes were observed at 3-versus 6-hours post exercise (239,951 versus 12,419, respectively; p<0.01), and nuclear global ten-eleven translocation (TET) activity, but not global DNA methyltransferase activity, increased 3- and 6-hours following exercise regardless of bout. The percentage of genes significantly altered at the mRNA level that demonstrated opposite DNA methylation patterns was greater 3- versus 6-hours following exercise (~75% versus ~15%, respectively). Moreover, high percentages of genes that were up- or downregulated 6 hours following exercise also demonstrated significantly inversed DNA methylation patterns across one or more CpG sites 3 hours following exercise (65% and 82%, respectively). While 30 Fail decreased DNA methylation across various promoter regions versus 80 Fail, transcriptome-wide mRNA and bioinformatics indicated that gene expression signatures were largely similar between bouts. Bioinformatics overlay of DNA methylation and mRNA expression data indicated that genes related to "Focal adhesion", "MAPK signaling", and "PI3K-Akt signaling" were significantly affected at the 3- and 6-hour time points, and again this was regardless of bout. In conclusion, extensive molecular profiling suggests that post-exercise alterations in the skeletal muscle DNA methylome and mRNA transcriptome elicited by LL and HL training bouts to failure are largely similar, and this could be related to equal volumes performed between bouts.

molecular biology↗

bioRxiv2021

Resistance training rejuvenates the mitochondrial methylome in aged human skeletal muscle

Resistance training (RT) alters skeletal muscle nuclear DNA methylation patterns (or the methylome). However, no study has examined if RT affects the mitochondrial DNA (mtDNA) methylome. Herein, ten older untrained males (65{+/-}7 years old) performed six weeks of full-body RT (twice weekly). Body composition and knee extensor torque were assessed prior to and 72 hours following the last RT session. Vastus lateralis (VL) biopsies were also obtained. VL DNA was subjected to reduced representation bisulfite sequencing providing excellent coverage across the ~16-kilobase mtDNA methylome (254 CpG sites). Various biochemical assays were also performed, and older male data were compared to younger trained males (22{+/-}2 years old, n=7). RT increased whole-body lean tissue mass (p=0.017), VL thickness (p=0.012), and knee extensor torque (p=0.029) in older males. RT also profoundly affected the mtDNA methylome in older males, as 63% (159/254) of the CpG sites demonstrated reduced methylation (p<0.05). Notably, several mtDNA sites presented a more "youthful" signature after RT in older males when comparisons were made to younger males. The 1.12 kilobase D-loop/control region on mtDNA, which regulates mtDNA replication and transcription, possessed enriched hypomethylation in older males following RT. Enhanced expression of mitochondrial H- and L-strand genes and increases in mitochondrial complex III and IV protein levels were also observed (p<0.05). This is the first study to show RT alters the mtDNA methylome in skeletal muscle. Observed methylome alterations may enhance mitochondrial transcription, and RT remarkably evokes mitochondrial methylome profiles to mimic a more youthful signature in older males.

molecular biology↗