The 24-Hour Time Course of Integrated Molecular Responses to Resistance Exercise in Human Skeletal Muscle Implicates MYC as a Hypertrophic Regulator That is Sufficient for Growth.

Molecular control of recovery after exercise in muscle is temporally dynamic. A time course of biopsies around resistance exercise (RE) combined with -omics is necessary to better comprehend the molecular contributions of skeletal muscle adaptation in humans. Vastus lateralis biopsies before and 30 minutes, 3-, 8-, and 24-hours after acute RE were collected. A time-point matched biopsy-only group was also included. RNA-sequencing defined the transcriptome while DNA methylomics and computational approaches complemented these data. The post-RE time course revealed: 1) DNA methylome responses at 30 minutes corresponded to upregulated genes at 3 hours, 2) a burst of translation- and transcription-initiation factor-coding transcripts occurred between 3 and 8 hours, 3) global gene expression peaked at 8 hours, 4) ribosome-related genes dominated the mRNA landscape between 8 and 24 hours, 5) methylation-regulated MYC was a highly influential transcription factor throughout the 24-hour recovery and played a primary role in ribosome-related mRNA levels between 8 and 24 hours. The influence of MYC in human muscle adaptation was strengthened by transcriptome information from acute MYC overexpression in mouse muscle. To test whether MYC was sufficient for hypertrophy, we generated a muscle fiber-specific doxycycline inducible model of pulsatile MYC induction. Periodic 48-hour pulses of MYC over 4 weeks resulted in higher muscle mass and fiber size in the soleus of adult female mice. Collectively, we present a temporally resolved resource for understanding molecular adaptations to RE in muscle and reveal MYC as a regulator of RE-induced mRNA levels and hypertrophy.

Skeletal muscle architecture and aging: A comparison of ultrasound techniques and an assessment of intrarater reliability.

OBJECTIVE: To assess intrarater reliability of ultrasound-determined measurements of skeletal muscle characteristics across different measurement outcomes, imaging techniques, and age groups. METHODS: 2D ultrasound images (B-mode) of the quadriceps were obtained from young (26 ± 4 year, n = 8 M, 8 F) and older (70 ± 7 year, n = 7 M, 5 F) adults on two occasions, separated by 6 ± 3 days. With participants in both standing and supine postures, images were collected from five anatomical sites along the anterior (two sites) and lateral (three sites) compartments of the thigh corresponding to 56%, 39%, and 22% (lateral only) of femur length. Images were analysed for muscle thickness, pennation angle, and echogenicity. Intraclass correlation coefficients (ICC) were used to assess reliability. RESULTS: Muscle thickness values were higher (p < 0.05) on images collected in the stand versus supine posture only for muscles of the anterior compartment, independent of age. Echogenicity values were higher (p < 0.05) in the vastus intermedius on images collected in the supine versus stand posture only in older adults. Pennation angle values were not impacted by imaging posture (p > 0.05). ICC values for thickness, echogenicity, and pennation angle were generally higher for analyses conducted on images collected in the supine versus stand posture. Imaging posture generated a greater difference in ICC values in the lateral versus anterior muscles and in older versus younger participants. CONCLUSION: Our findings suggest that participant posture during imaging impacts the absolute values and intrarater reliability of ultrasound-determined muscle characteristics in a muscle-specific fashion, and this effect is greater in older compared to younger individuals.

Sex Differences in Quadriceps Atrophy After Anterior Cruciate Ligament Tear.

BACKGROUND: Female athletes lag behind their male counterparts in recovery from anterior cruciate ligament (ACL) injury. Quadriceps muscle size and strength are crucial factors for regaining function after ACL injury, but little is known about how these metrics vary due to biological sex. HYPOTHESIS: Female patients have reduced vastus lateralis fiber cross-sectional area (CSA) and lower quadriceps strength after ACL injury than male patients. STUDY DESIGN: Cross-sectional study. LEVEL OF EVIDENCE: Level 4. METHODS: A total of 60 participants with recent ACL tear were evaluated for vastus lateralis muscle fiber CSA, isometric quadriceps peak torque, and quadriceps rate of torque development. Linear mixed models were fit to determine differences across sex and limb for each variable of interest. RESULTS: The female group averaged almost 20% atrophy between limbs (P < 0.01), while the male group averaged just under 4% (P = 0.05). Strength deficits between limbs were comparable between female and male groups. CONCLUSION: Immediately after ACL injury, female patients have greater between-limb differences in muscle fiber CSA but between-limb strength deficits comparable with those of male patients. CLINICAL RELEVANCE: These results indicate that the underpinnings of strength loss differ based on biological sex, and thus individual patients could benefit from a sex-specific treatment approach to ACL injury.

Inhibition of p53-MDM2 binding reduces senescent cell abundance and improves the adaptive responses of skeletal muscle from aged mice.

Skeletal muscle adaptation to external stimuli, such as regeneration following injury and hypertrophy in response to resistance exercise, are blunted with advanced age. The accumulation of senescent cells, along with defects in myogenic progenitor cell (MPC) proliferation, have been strongly linked as contributing factors to age-associated impairment in muscle adaptation. p53 plays an integral role in all these processes, as upregulation of p53 causes apoptosis in senescent cells and prevents mitotic catastrophe in MPCs from old mice. The goal of this study was to determine if a novel pharmaceutical agent (BI01), which functions by upregulating p53 through inhibition of binding to MDM2, the primary p53 regulatory protein, improves muscle regeneration and hypertrophy in old mice. BI01 effectively reduced the number of senescent cells in vitro but had no effect on MPC survival or proliferation at a comparable dose. Following repeated oral gavage with 2 mg/kg of BI01 (OS) or vehicle (OV), old mice (24 months) underwent unilateral BaCl2 injury in the tibialis anterior (TA) muscle, with PBS injections serving as controls. After 7 days, satellite cell number was higher in the TA of OS compared to OV mice, as was the expression of genes involved in ATP production. By 35 days, old mice treated with BI01 displayed reduced senescent cell burden, enhanced regeneration (higher muscle mass and fiber cross-sectional area) and restoration of muscle function relative to OV mice. To examine the impact of 2 mg/kg BI01 on muscle hypertrophy, the plantaris muscle was subjected to 28 days of mechanical overload (MOV) in OS and OV mice. In response to MOV, OS mice had larger plantaris muscles and muscle fibers than OV mice, particularly type 2b + x fibers, associated with reduced senescent cells. Together our data show that BI01 is an effective senolytic agent that may also augment muscle metabolism to enhance muscle regeneration and hypertrophy in old mice.

Overexpression of manganese superoxide dismutase mitigates ACL injury-induced muscle atrophy, weakness and oxidative damage.

Oxidative stress has been implicated in the etiology of skeletal muscle weakness following joint injury. We investigated longitudinal patient muscle samples following knee injury (anterior cruciate ligament tear). Following injury, transcriptomic analysis revealed downregulation of mitochondrial metabolism-related gene networks, which were supported by reduced mitochondrial respiratory flux rates. Additionally, enrichment of reactive oxygen species (ROS)-related pathways were upregulated in muscle following knee injury, and further investigation unveiled marked oxidative damage in a progressive manner following injury and surgical reconstruction. We then investigated whether antioxidant protection is effective in preventing muscle atrophy and weakness after knee injury in mice that overexpress Mn-superoxide dismutase (MnSOD+/-). MnSOD+/- mice showed attenuated oxidative damage, atrophy, and muscle weakness compared to wild type littermate controls following ACL transection surgery. Taken together, our results indicate that ROS-related damage is a causative mechanism of muscle dysfunction after knee injury, and that mitochondrial antioxidant protection may hold promise as a therapeutic target to prevent weakness and development of disability.

GDF8 inhibition enhances musculoskeletal recovery and mitigates posttraumatic osteoarthritis following joint injury.

Musculoskeletal disorders contribute substantially to worldwide disability. Anterior cruciate ligament (ACL) tears result in unresolved muscle weakness and posttraumatic osteoarthritis (PTOA). Growth differentiation factor 8 (GDF8) has been implicated in the pathogenesis of musculoskeletal degeneration following ACL injury. We investigated GDF8 levels in ACL-injured human skeletal muscle and serum and tested a humanized monoclonal GDF8 antibody against a placebo in a mouse model of PTOA (surgically induced ACL tear). In patients, muscle GDF8 was predictive of atrophy, weakness, and periarticular bone loss 6 months following surgical ACL reconstruction. In mice, GDF8 antibody administration substantially mitigated muscle atrophy, weakness, and fibrosis. GDF8 antibody treatment rescued the skeletal muscle and articular cartilage transcriptomic response to ACL injury and attenuated PTOA severity and deficits in periarticular bone microarchitecture. Furthermore, GDF8 genetic deletion neutralized musculoskeletal deficits in response to ACL injury. Our findings support an opportunity for rapid targeting of GDF8 to enhance functional musculoskeletal recovery and mitigate the severity of PTOA after injury.

Vitamin D status associates with skeletal muscle loss after anterior cruciate ligament reconstruction.

BACKGROUNDAlthough 25-hydroxyvitamin D [25(OH)D] concentrations of 30 ng/mL or higher are known to reduce injury risk and boost strength, the influence on anterior cruciate ligament reconstruction (ACLR) outcomes remains unexamined. This study aimed to define the vitamin D signaling response to ACLR, assess the relationship between vitamin D status and muscle fiber cross-sectional area (CSA) and bone density outcomes, and discover vitamin D receptor (VDR) targets after ACLR.METHODSTwenty-one young, healthy, physically active participants with recent ACL tears were enrolled (17.8 ± 3.2 years, BMI 26.0 ± 3.5 kg/m2). Data were collected through blood samples, vastus lateralis biopsies, dual energy x-ray bone density measurements, and isokinetic dynamometer measures at baseline, 1 week, 4 months, and 6 months after ACLR. The biopsies facilitated CSA, Western blotting, RNA-seq, and VDR ChIP-seq analyses.RESULTSACLR surgery led to decreased circulating bioactive vitamin D and increased VDR and activating enzyme expression in skeletal muscle 1 week after ACLR. Participants with less than 30 ng/mL 25(OH)D levels (n = 13) displayed more significant quadriceps fiber CSA loss 1 week and 4 months after ACLR than those with 30 ng/mL or higher (n = 8; P < 0.01 for post hoc comparisons; P = 0.041 for time × vitamin D status interaction). RNA-seq and ChIP-seq data integration revealed genes associated with energy metabolism and skeletal muscle recovery, potentially mediating the impact of vitamin D status on ACLR recovery. No difference in bone mineral density losses between groups was observed.CONCLUSIONCorrecting vitamin D status prior to ACLR may aid in preserving skeletal muscle during recovery.FUNDINGNIH grants R01AR072061, R01AR071398-04S1, and K99AR081367.

Depressed Protein Synthesis and Anabolic Signaling Potentiate ACL Tear-Resultant Quadriceps Atrophy.

BACKGROUND: Anterior cruciate ligament (ACL) tear (ACLT) leads to protracted quadriceps muscle atrophy. Protein turnover largely dictates muscle size and is highly responsive to injury and loading. Regulation of quadriceps molecular protein synthetic machinery after ACLT has largely been unexplored, limiting development of targeted therapies. PURPOSE: To define the effect of ACLT on (1) the activation of protein synthetic and catabolic signaling within quadriceps biopsy specimens from human participants and (2) the time course of alterations to protein synthesis and its molecular regulation in a mouse ACL injury model. STUDY DESIGN: Descriptive laboratory study. METHODS: Muscle biopsy specimens were obtained from the ACL-injured and noninjured vastus lateralis of young adult humans after an overnight fast (N = 21; mean ± SD, 19 ± 5 years). Mice had their limbs assigned to ACLT or control, and whole quadriceps were collected 6 hours or 1, 3, or 7 days after injury with puromycin injected before tissue collection for assessment of relative protein synthesis. Muscle fiber size and expression and phosphorylation of protein anabolic and catabolic signaling proteins were assessed at the protein and transcript levels (RNA sequencing). RESULTS: Human quadriceps showed reduced phosphorylation of ribosomal protein S6 (-41%) in the ACL-injured limb (P = .008), in addition to elevated phosphorylation of eukaryotic initiation factor 2α (+98%; P = .006), indicative of depressed protein anabolic signaling in the injured limb. No differences in E3 ubiquitin ligase expression were noted. Protein synthesis was lower at 1 day (P = .01 vs control limb) and 3 days (P = .002 vs control limb) after ACLT in mice. Pathway analyses revealed shared molecular alterations between human and mouse quadriceps after ACLT. CONCLUSION: (1) Global protein synthesis and anabolic signaling deficits occur in the quadriceps in response to ACL injury, without notable changes in measured markers of muscle protein catabolism. (2) Importantly, these deficits occur before the onset of significant atrophy, underscoring the need for early intervention. CLINICAL RELEVANCE: These findings suggest that blunted protein anabolism as opposed to increased catabolism likely mediates quadriceps atrophy after ACL injury. Thus, future interventions should aim to restore muscle protein anabolism rapidly after ACLT.

A glitch in the matrix: the pivotal role for extracellular matrix remodeling during muscle hypertrophy.

Multinuclear muscle fibers are the most voluminous cells in skeletal muscle and the primary drivers of growth in response to loading. Outside the muscle fiber, however, is a diversity of mononuclear cell types that reside in the extracellular matrix (ECM). These muscle-resident cells are exercise-responsive and produce the scaffolding for successful myofibrillar growth. Without proper remodeling and maintenance of this ECM scaffolding, the ability to mount an appropriate response to resistance training in adult muscles is severely hindered. Complex cellular choreography takes place in muscles following a loading stimulus. These interactions have been recently revealed by single-cell explorations into muscle adaptation with loading. The intricate ballet of ECM remodeling involves collagen production from fibrogenic cells and ECM modifying signals initiated by satellite cells, immune cells, and the muscle fibers themselves. The acellular collagen-rich ECM is also a mechanical signal-transducer and rich repository of growth factors that may directly influence muscle fiber hypertrophy once liberated. Collectively, high levels of collagen expression, deposition, and turnover characterize a well-trained muscle phenotype. The purpose of this review is to highlight the most recent evidence for how the ECM and its cellular components affect loading-induced muscle hypertrophy. We also address how the muscle fiber may directly take part in ECM remodeling, and whether ECM dynamics are rate limiting for muscle fiber growth.

Satellite cell depletion does not affect diaphragm adaptations to hypoxia.

The diaphragm is the main skeletal muscle responsible for inspiration and is susceptible to age-associated decline in function and morphology. Satellite cells in diaphragm fuse into unperturbed muscle fibers throughout life, yet their role in adaptations to hypoxia in diaphragm is unknown. Given their continual fusion, we hypothesize that satellite cell depletion will negatively impact adaptations to hypoxia in the diaphragm, particularly with aging. We used the Pax7CreER/CreER:R26RDTA/DTA genetic mouse model of inducible satellite cell depletion to investigate diaphragm responses to hypoxia in adult (6 mo) and aged (22 mo) male mice. The mice were subjected to normobaric hypoxia at 10% [Formula: see text] or normoxia for 4 wk. We showed that satellite cell depletion had no effect on diaphragm muscle fiber cross-sectional area, fiber-type distribution, myonuclear density, or regulation of extracellular matrix in either adult or aged mice. Furthermore, we showed lower muscle fiber cross-sectional area with hypoxia and age (main effects), while extracellular matrix content was higher and satellite cell abundance was lower with age (main effect) in diaphragm. Lastly, a greater number of Pax3-mRNA+ cells was observed in diaphragm muscle of satellite cell-depleted mice independent of hypoxia (main effect), potentially as a compensatory mechanism for the loss of satellite cells. We conclude that satellite cells are not required for diaphragm muscle adaptations to hypoxia in either adult or aged mice.NEW & NOTEWORTHY Satellite cells show consistent fusion into diaphragm muscle fibers throughout life, suggesting a critical role in maintaining homeostasis. Here, we report identical diaphragm adaptations to hypoxia with and without satellite cells in adult and aged mice. In addition, we propose that the higher number of Pax3-positive cells in satellite cell-depleted diaphragm muscle acts as a compensatory mechanism.

Muscle-Specific Cellular and Molecular Adaptations to Late-Life Voluntary Concurrent Exercise.

Murine exercise models can provide information on factors that influence muscle adaptability with aging, but few translatable solutions exist. Progressive weighted wheel running (PoWeR) is a simple, voluntary, low-cost, high-volume endurance/resistance exercise approach for training young mice. In the current investigation, aged mice (22-mo-old) underwent a modified version of PoWeR for 8 wk. Muscle functional, cellular, biochemical, transcriptional, and myonuclear DNA methylation analyses provide an encompassing picture of how muscle from aged mice responds to high-volume combined training. Mice run 6-8 km/d, and relative to sedentary mice, PoWeR increases plantarflexor muscle strength. The oxidative soleus of aged mice responds to PoWeR similarly to young mice in every parameter measured in previous work; this includes muscle mass, glycolytic-to-oxidative fiber type transitioning, fiber size, satellite cell frequency, and myonuclear number. The oxidative/glycolytic plantaris adapts according to fiber type, but with modest overall changes in muscle mass. Capillarity increases markedly with PoWeR in both muscles, which may be permissive for adaptability in advanced age. Comparison to published PoWeR RNA-sequencing data in young mice identified conserved regulators of adaptability across age and muscles; this includes Aldh1l1 which associates with muscle vasculature. Agrn and Samd1 gene expression is upregulated after PoWeR simultaneous with a hypomethylated promoter CpG in myonuclear DNA, which could have implications for innervation and capillarization. A promoter CpG in Rbm10 is hypomethylated by late-life exercise in myonuclei, consistent with findings in muscle tissue. PoWeR and the data herein are a resource for uncovering cellular and molecular regulators of muscle adaptation with aging.

Senolytic treatment rescues blunted muscle hypertrophy in old mice.

With aging, skeletal muscle plasticity is attenuated in response to exercise. Here, we report that senescent cells, identified using senescence-associated ß-galactosidase (SA ß-Gal) activity and p21 immunohistochemistry, are very infrequent in resting muscle, but emerge approximately 2 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 (MOV) of the plantaris muscle to model resistance training in adult (5-6-month) and old (23-24-month) male C57BL/6 J mice, we found increased senescent cells in both age groups during hypertrophy. Consistent with the human data, there were negligible senescent cells in plantaris muscle from adult and old sham controls, but old mice had significantly more senescent cells 7 and 14 days following MOV 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 days 7 and 10 during 14 days of MOV; 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 MOV, 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.

Hindlimb suspension in Wistar rats: Sex-based differences in muscle response.

Ground-based animal models have been used extensively to understand the effects of microgravity on various physiological systems. Among them, hindlimb suspension (HLS), developed in 1979 in rats, remains the gold-standard and allows researchers to study the consequences of total unloading of the hind limbs while inducing a cephalic fluid shift. While this model has already brought valuable insights to space biology, few studies have directly compared functional decrements in the muscles of males and females during HLS. We exposed 28 adult Wistar rats (14 males and 14 females) to 14 days of HLS or normal loading (NL) to better assess how sex impacts disuse-induced muscle deconditioning. Females better maintained muscle function during HLS than males, as shown by a more moderate reduction in grip strength at 7 days (males: -37.5 ± 3.1%, females: -22.4 ± 6.5%, compared to baseline), that remains stable during the second week of unloading (males: -53.3 ± 5.7%, females: -22.4 ± 5.5%, compared to day 0) while the males exhibit a steady decrease over time (effect of sex × loading p = 0.0002, effect of sex × time × loading p = 0.0099). This was further supported by analyzing the force production in response to a tetanic stimulus. Further functional analyses using force production were also shown to correspond to sex differences in relative loss of muscle mass and CSA. Moreover, our functional data were supported by histomorphometric analyzes, and we highlighted differences in relative muscle loss and CSA. Specifically, female rats seem to experience a lesser muscle deconditioning during disuse than males thus emphasizing the need for more studies that will assess male and female animals concomitantly to develop tailored, effective countermeasures for all astronauts.

Vitamin D Promotes Skeletal Muscle Regeneration and Mitochondrial Health.

Vitamin D is an essential nutrient for the maintenance of skeletal muscle and bone health. The vitamin D receptor (VDR) is present in muscle, as is CYP27B1, the enzyme that hydroxylates 25(OH)D to its active form, 1,25(OH)D. Furthermore, mounting evidence suggests that vitamin D may play an important role during muscle damage and regeneration. Muscle damage is characterized by compromised muscle fiber architecture, disruption of contractile protein integrity, and mitochondrial dysfunction. Muscle regeneration is a complex process that involves restoration of mitochondrial function and activation of satellite cells (SC), the resident skeletal muscle stem cells. VDR expression is strongly upregulated following injury, particularly in central nuclei and SCs in animal models of muscle injury. Mechanistic studies provide some insight into the possible role of vitamin D activity in injured muscle. In vitro and in vivo rodent studies show that vitamin D mitigates reactive oxygen species (ROS) production, augments antioxidant capacity, and prevents oxidative stress, a common antagonist in muscle damage. Additionally, VDR knockdown results in decreased mitochondrial oxidative capacity and ATP production, suggesting that vitamin D is crucial for mitochondrial oxidative phosphorylation capacity; an important driver of muscle regeneration. Vitamin D regulation of mitochondrial health may also have implications for SC activity and self-renewal capacity, which could further affect muscle regeneration. However, the optimal timing, form and dose of vitamin D, as well as the mechanism by which vitamin D contributes to maintenance and restoration of muscle strength following injury, have not been determined. More research is needed to determine mechanistic action of 1,25(OH)D on mitochondria and SCs, as well as how this action manifests following muscle injury in vivo. Moreover, standardization in vitamin D sufficiency cut-points, time-course study of the efficacy of vitamin D administration, and comparison of multiple analogs of vitamin D are necessary to elucidate the potential of vitamin D as a significant contributor to muscle regeneration following injury. Here we will review the contribution of vitamin D to skeletal muscle regeneration following injury.