Biology subjects

Mobley, C. B.

Publications and source records attributed to Mobley, C. B..

18 recordsLinked to original sources

bioRxiv2026

Exogenous L6 myotube mitochondrial transplantation attenuates hypertrophy and elicits a unique proteomic signature in phenylephrine-treated H9C2 cardiomyocytes

Mitochondrial transplantation has recently emerged as an alternative treatment for cardiovascular disease (CVD), aimed at increasing mitochondrial number and improving mitochondrial function. Though mitochondrial dysfunction is a key factor in the development of right ventricular hypertrophy/failure, the effects of mitochondrial transplantation on disease mitigation have been largely unexplored. Therefore, this in vitro study aimed to determine whether the transplantation of exogenous L6 myotube mitochondria mitigates negative outcomes in H9C2 cardiomyocytes that were stimulated to hypertrophy with phenylephrine. Control (CTL), control with mitochondrial transplantation (MitoTx), Phenylephrine only (Phe-only), and phenylephrine with mitochondrial transplantation (Phe+MitoTx) treatments were evaluated. Pilot experiments indicated mitochondrial transplantation into healthy cardiomyocytes acutely increased Complex I-linked oxidative phosphorylation (OXPHOS) capacity (p=0.005) and maximal respiratory capacity (p=0.022) within 24 hours, and these data alongside microscopic evidence of fluorescently labeled L6 mitochondria in H9C2 cardiomyocytes suggested successful transplantation. Regarding treatment comparisons, Phe-only showed a significant increase in cell area (p<0.05), while Phe+MitoTx blunted the hypertrophic cardiomyocyte response. Consistent with these results, proteomic analysis of 4,806 proteins showed that transplantation enriched the mitochondrial proteome, impacting pathways including OXPHOS, respiration, fatty acid and amino acid metabolism, while suppressing extracellular matrix remodeling and de-differentiation signatures. This response was observed in healthy and phenylephrine-stressed cardiomyocytes. In conclusion, our in vitro data indicates that transplantation of L6 skeletal muscle mitochondria mitigates negative effects induced by phenylephrine in H9C2 cardiomyocytes. However, more rigorous in vivo studies are needed to determine if this is a suitable approach for disease mitigation.

cell biology↗

bioRxiv2026

Acute molecular and chronic vastus lateralis adaptations to lengthened partial versus full range of motion resistance training in previously trained males

This study examined how lower-body lengthened partial (LP) versus full range of motion (FULL) resistance training affects acute post-exercise signaling, chronic hypertrophy, and cellular adaptations of the vastus lateralis (VL) muscle in resistance-trained men. Eight males (22{+/-}1 years old, 5.6{+/-}1.4 years training) completed a crossover study whereby VL biopsies were collected pre-exercise and 0, 3, and 24 hours following LP and FULL leg extension bouts for transcriptomic and anabolic signaling analyses (Experiment 1). Another 16 males (26{+/-}5 years old; 8.0{+/-}4.9 years training) completed an 8-week, twice-weekly lower-body intervention using a within-subject design (Experiment 2). One leg was assigned to FULL and the contralateral leg to LP training across three exercises (leg press, leg extension, and lying leg curl). Pre- and post-intervention outcomes included VL muscle cross-sectional area (mCSA) summed across five equidistant MRI-derived transverse slices and mid-thigh VL biopsy outcomes. As a secondary outcome, other hip and thigh muscles from Experiment 2 MRI scans were assessed. Condition x Time interactions for all outcomes were assessed using linear mixed-effects models. In Experiment 1, both conditions produced similar time-dependent changes in the VL transcriptome and anabolic (mTORC1 and Hippo) signaling, but minimal between-protocol interactions. In Experiment 2, VL summed mCSA significantly increased over time (mean change: 9.3 cm{superscript 2}, 95% CI [6.8, 11.8], P<0.001), but there was no clear evidence of differential change between protocols (LP-FULL change: -1.4 cm{superscript 2}, 95% CI [-6.1, 3.8], P=0.640). Additionally, no significant interactions existed for type I fiber CSA (P=0.476), type II fiber CSA (P=0.350), type I fiber myonuclei (P=0.813), type II fiber myonuclei (P=0.589), type I and II satellite cell number (P=0.102 and P=0.797, respectively), or total RNA content (P=0.537). Despite these null VL-centric findings, secondary Experiment 2 analyses provided some evidence that whole hamstring hypertrophy was greater following LP versus FULL (LP-FULL change: 3.9 cm{superscript 2}, 95% CI [-0.2, 7.9], P=0.058). In conclusion, 8 weeks of LP and FULL resistance training broadly elicit similar acute and chronic VL responses in previously trained men, though secondary hamstring findings suggest that differential responses may depend on exercises included in the resistance training program.

physiology↗

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

Proteomic profiling of skeletal muscle ribosomes from higher versus lower responders to 10 weeks of resistance training

Ribosome biogenesis is a key driver of resistance training (RT)-induced skeletal muscle hypertrophy in humans. However, high resolution insight into RT-induced compositional alterations in ribosomes remain unexplored. Therefore, the purpose of this study was twofold: 1) develop a protocol sufficient to enrich and examine the ribosomal proteome from a small amount of human muscle tissue, and 2) determine if ribosomal protein composition differs between higher and lower responders to RT. Fourteen participants completed 10 weeks of RT (23 sessions) and were stratified into higher (n=7) and lower (n=7) responders based on changes in vastus lateralis muscle cross-sectional area (VL mCSA) and mean myofiber cross-sectional area (fCSA) from baseline (PRE) to after the 23rd RT session (POST). Participants then performed a twenty fourth RT session and biopsies were collected 24 hours post-RT (POST-24h) to examine acute bout (POST to POST-24h) ribosomal proteome alterations to RT. Ribosome enrichment and analysis included ultracentrifugation (100,000 g, 3h, 2{degrees}C) through 20% sucrose gradients followed by shotgun proteomics to quantify ribosomal protein composition. Our protocol produced exceptional ribosome enrichment, with 74 distinct ribosomal proteins being detected (92% coverage of the putative 80 cytosolic ribosomal proteins) as well as [~]164-fold and [~]71-fold increases in large (RPL) and small (RPS) protein subunit abundances, respectively, compared to conventional tissue homogenization. Despite robust phenotypic differences in hypertrophy between responder groups to the 10-week RT protocol (mCSA: +31% versus +3%; fCSA: +29% versus -2%, P<0.05 for both outcomes), ribosomal protein composition was not significantly different between groups (higher vs. lower responders), nor was there a significant time (POST to POST-24h) or a group*time interaction (P>0.028 for all comparisons). In conclusion, we present a highly effective ribosome enrichment protocol requiring minimal human muscle tissue. Though preliminary analyses indicate that ribosomal protein composition was similar between hypertrophic phenotypes in response to an acute RT session after a 10-week RT intervention, future research leveraging our techniques with more sampling time points are needed to provide more definitive conclusions.

molecular biology↗

bioRxiv2025

Resistance training-induced appendicular lean tissue mass changes are largely unrelated to pre-training bone characteristics in a larger cohort of untrained adults

We sought to determine if pre-intervention bone characteristics measured by dual-energy x-ray absorptiometry (DXA) were associated with changes in bone-free lean tissue mass following a period of resistance training in a large cohort of untrained adults (n=119, 62M/57F, 26.0{+/-}4.7 kg/m2, age range = 18-70 years old). Participants completed 10-12 weeks of supervised whole-body resistance training twice weekly, and DXA scans were obtained approximately the same time of day prior to the intervention and 48-72 hours following the final training bout. Associations between baseline skeletal measures (e.g., appendicular bone characteristics, shoulder and hip widths) and training induced changes in appendicular lean mass were examined by estimating correlations between participant-level random slopes (reflecting change over time) and baseline skeletal measures. The same approach was used to evaluate associations between other participant attributes (e.g., age, training volume-load, self-reported energy intake) and appendicular lean tissue mass changes. Modeling was also used to explore whether baseline skeletal characteristics (e.g., shoulder and hip widths) moderated the change in appendicular lean tissue mass from training. All analyses used a Bayesian framework, and interpretation focused on estimated effect sizes and their associated credible intervals rather than formal null hypothesis testing. Strong positive associations were observed between pre-intervention characteristics including dual-arm lean tissue mass and dual-arm bone mineral content (r=0.90), dual-leg lean tissue mass and dual-leg bone mineral content (r=0.86), dual-leg lean tissue mass and pelvic mineral content (r=0.73), and dual-arm lean tissue mass and shoulder width (r=0.76). In contrast, weak associations were observed between training-induced changes in appendicular lean tissue mass versus bone characteristics, training volume-load, self-reported energy intake, self-reported protein intake, BMI, and age (-0.08[≤]r[≤] 0.24). After adjusting for sex, multivariable analyses indicated minimal evidence that skeletal characteristics moderated the hypertrophic response to training. These findings do not support a meaningful role of pre-training bone characteristics in influencing the lean tissue mass adaptations to shorter-term resistance training.

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

Microbial-Derived Exerkines Prevent Skeletal Muscle Atrophy

Regular exercise yields a multitude of systemic benefits, many of which may be mediated through the gut microbiome. Here, we report that cecal microbial transplants (CMTs) from exercise-trained vs. sedentary mice have modest benefits in reducing skeletal muscle atrophy using a mouse model of unilaterally hindlimb-immobilization. Direct administration of top microbial-derived exerkines from an exercise-trained gut microbiome preserved muscle function and prevented skeletal muscle atrophy.

physiology↗

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

The effects of resistance training on denervated myofibers, senescent cells, and associated protein markers in middle-aged adults

Denervated myofibers and senescent cells are hallmarks of skeletal muscle aging. However, sparse research has examined how resistance training affects these outcomes. We investigated the effects of unilateral leg extensor resistance training on denervated myofibers, senescent cells, and associated protein markers in middle-aged participants (MA, 55{+/-}8 years old, 17 females, 9 males). We obtained vastus lateralis (VL) muscle cross-sectional area (mCSA), VL biopsies, and strength assessments before and after training. Fiber cross-sectional area (fCSA), satellite cells (Pax7+), denervated myofibers (NCAM+), senescent cells (p16+ or p21+), senescence-related proteins, and senescence-associated secretory phenotype (SASP) proteins were analyzed from biopsied muscle. Leg extensor peak torque increased after training (p<0.001), while VL mCSA trended upward (p=0.082). No significant changes were observed for fCSA, NCAM+ myofibers, or senescent (p16+ or p21+) cells, albeit satellite cells increased after training (p=0.037). While >90% satellite cells were not p16+ or p21+, most p16+ and p21+ cells were Pax7+ (>90% on average). Training altered 13/46 proteins related to muscle-nerve communication (all upregulated, p<0.05) and 10/19 proteins related to cellular senescence (9 upregulated, p<0.05). Only 1/17 SASP proteins increased with training (IGFBP-3, p=0.031). In conclusion, resistance training upregulates proteins associated with muscle-nerve communication in MA participants but does not alter NCAM+ myofibers. Moreover, while training increases senescence-related proteins in skeletal muscle, this coincided with an increase in satellite cells but not alterations in senescent cell content or SASP proteins. Hence, we interpret these collective findings as resistance training being an unlikely inducer of cellular senescence in humans.

physiology↗

bioRxiv2023

A novel deep proteomic approach in human skeletal muscle unveils distinct molecular signatures affected by aging and resistance training

We examined the myofibrillar (MyoF) and non-myofibrillar (non-MyoF) proteomic profiles of the vastus lateralis (VL) muscle of younger (Y, 22{+/-}2 years old; n=5) and middle-aged participants (MA, 56{+/-}8 years old; n=6), and MA following eight weeks of knee extensor resistance training (RT, 2d/week). Shotgun/bottom-up proteomics in skeletal muscle typically yields wide protein abundance ranges that mask lowly expressed proteins. Thus, we adopted a novel approach whereby the MyoF and non-MyoF fractions were separately subjected to protein corona nanoparticle complex formation prior to digestion and Liquid Chromatography Mass Spectrometry (LC-MS) analysis. A total of 10,866 proteins (4,421 MyoF and 6,445 non-MyoF) were identified. Across all participants, the number of non-MyoF proteins detected averaged to be 5,645{+/-}266 (range: 4,888-5,987) and the number of MyoF proteins detected averaged to be 2,611{+/-}326 (range: 1,944-3,101). Differences in the non-MyoF (8.4%) and MyoF (2.5%) proteome were evident between age cohorts. Further, most of these age-related non-MyoF proteins (447/543) were more enriched in MA versus Y. Several biological processes in the non-MyoF fraction were predicted to be operative in MA versus Y including (but not limited to) increased cellular stress, mRNA splicing, translation elongation, and ubiquitin-mediated proteolysis. Non-MyoF proteins associated with splicing and proteostasis were further interrogated, and in agreement with bioinformatics, alternative protein variants, spliceosome-associated proteins (snRNPs), and proteolysis-related targets were more abundant in MA versus Y. RT in MA non-significantly increased VL muscle cross-sectional area (+6.5%, p=0.066) and significantly increased knee extensor strength (+8.7%, p=0.048). However, RT modestly altered the MyoF ([~]0.3%, 11 upregulated and two downregulated proteins) and non-MyoF proteomes ([~]1.0%, 56 upregulated and eight downregulated proteins, p<0.01). Further, RT did not affect predicted biological processes in either fraction. Although participant numbers were limited, these preliminary results using a novel deep proteomic approach in skeletal muscle suggest that aging and RT predominantly affects protein abundances in the non-contractile protein pool. However, the marginal proteome adaptations occurring with RT suggest either: a) this may be an aging-associated phenomenon, b) more rigorous RT may stimulate more robust effects, or c) RT, regardless of age, subtly affects skeletal muscle protein abundances in the basal state.

physiology↗

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↗

bioRxiv2022

Skeletal muscle nuclei in mice are not post-mitotic

The skeletal muscle research field generally accepts that nuclei in skeletal muscle fibers (i.e., myonuclei) are post-mitotic and unable to proliferate. Because our deuterium oxide (D2O) labeling studies showed DNA synthesis in skeletal muscle tissue, we hypothesized that resident myonuclei can replicate in vivo. To test this hypothesis, we used a mouse model that temporally labeled myonuclei with GFP followed by D2O labeling during normal cage activity, functional overload, and with satellite cell ablation. During normal cage activity, we observed deuterium enrichment into myonuclear DNA in 7 out of 7 plantaris (PLA), 6 out of 6 tibialis anterior (TA), 5 out of 7 gastrocnemius (GAST) and 7 out of 7 quadriceps (QUAD). The average fractional synthesis rates (FSR) of DNA in myonuclei were: 0.0202 {+/-} 0.0093 in PLA, 0.0239 {+/-} 0.0040 in TA, 0.0076 {+/-} 0. 0058 in GAST, and 0.0138 {+/-} 0.0039 in QUAD, while there was no replication in myonuclei from EDL. These FSR values were largely reproduced in the overload and satellite cell ablation conditions although there were higher synthesis rates in the overloaded PLA muscle. We further provided evidence that myonuclear replication is through endoreplication that results in polyploidy. These novel findings contradict the dogma that skeletal muscle nuclei are post-mitotic and open potential avenues to harness the intrinsic replicative ability of myonuclei for muscle maintenance and growth. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/513426v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1e4d210org.highwire.dtl.DTLVardef@19b52daorg.highwire.dtl.DTLVardef@5c9a53org.highwire.dtl.DTLVardef@197dc0d_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2020

Genetic and Epigenetic Regulation of Skeletal Muscle Ribosome Biogenesis with Exercise

Ribosomes are the macromolecular engines of protein synthesis. Skeletal muscle ribosome biogenesis is stimulated by exercise, but the contribution of ribosomal DNA (rDNA) copy number and methylation to exercise-induced rDNA transcription is unclear. To investigate the genetic and epigenetic regulation of ribosome biogenesis with exercise, a time course of skeletal muscle biopsies was obtained from 30 participants (18 men and 12 women; 31 {+/-}8 yrs, 25 {+/-}4 kg/m2) at rest and 30 min, 3h, 8h, and 24h after acute endurance (n=10, 45 min cycling, 70% VO2max) or resistance exercise (n=10, 4 x 7 x 2 exercises); 10 control participants underwent biopsies without exercise. rDNA transcription and dosage were assessed using qPCR and whole genome sequencing. rDNA promoter methylation was investigated using massARRAY EpiTYPER, and global rDNA CpG methylation was assessed using reduced-representation bisulfite sequencing. Ribosome biogenesis and MYC transcription were associated with resistance but not endurance exercise, indicating preferential up-regulation during hypertrophic processes. With resistance exercise, ribosome biogenesis was associated with rDNA gene dosage as well as epigenetic changes in enhancer and non-canonical MYC-associated areas in rDNA, but not the promoter. A mouse model of in vivo metabolic RNA labeling and genetic myonuclear fluorescent labeling validated the effects of an acute hypertrophic stimulus on ribosome biogenesis and Myc transcription, and corroborated rDNA enhancer and Myc-associated methylation alterations specifically in myonuclei. This study provides the first information on skeletal muscle genetic and rDNA gene-wide epigenetic regulation of ribosome biogenesis in response to exercise, revealing novel roles for rDNA dosage and CpG methylation. GRAPHICAL ABSTRACT O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

cell biology↗