ABSTRACT
Human hallmarks of sarcopenia include muscle weakness and a blunted response to exercise. Nicotinamide N-methyltransferase inhibitors (NNMTis) increase strength and promote the regenerative capacity of aged muscle, thus offering a promising treatment for sarcopenia. Since human hallmarks of sarcopenia are recapitulated in aged (24-month-old) mice, we treated mice from 22 to 24 months of age with NNMTi, intensive exercise, or a combination of both, and compared skeletal muscle adaptations, including grip strength, longitudinal running capacity, plantarflexor peak torque, fatigue, and muscle mass, fiber type, cross-sectional area, and intramyocellular lipid (IMCL) content. Exhaustive proteome and metabolome analyses were completed to identify the molecular mechanisms underlying the measured changes in skeletal muscle pathophysiology. Remarkably, NNMTi-treated aged sedentary mice showed ~ 40% greater grip strength than sedentary controls, while aged exercised mice only showed a 20% increase relative to controls. Importantly, the grip strength improvements resulting from NNMTi treatment and exercise were additive, with NNMTi-treated exercised mice developing a 60% increase in grip strength relative to sedentary controls. NNMTi treatment also promoted quantifiable improvements in IMCL content and, in combination with exercise, significantly increased gastrocnemius fiber CSA. Detailed skeletal muscle proteome and metabolome analyses revealed unique molecular mechanisms associated with NNMTi treatment and distinct molecular mechanisms and cellular processes arising from a combination of NNMTi and exercise relative to those given a single intervention. These studies suggest that NNMTi-based drugs, either alone or combined with exercise, will be beneficial in treating sarcopenia and a wide range of age-related myopathies.
Subject(s)
Aging , Muscle, Skeletal , Nicotinamide N-Methyltransferase , Physical Conditioning, Animal , Sarcopenia , Animals , Nicotinamide N-Methyltransferase/metabolism , Muscle, Skeletal/metabolism , Muscle, Skeletal/drug effects , Mice , Aging/physiology , Sarcopenia/metabolism , Sarcopenia/drug therapy , Male , Muscle Strength/drug effects , Mice, Inbred C57BL , Enzyme Inhibitors/pharmacologyABSTRACT
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.
ABSTRACT
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.
Subject(s)
Muscle, Skeletal , Tumor Suppressor Protein p53 , Animals , Mice , Cellular Senescence , Hypertrophy , Muscle Fibers, Skeletal/physiology , Muscle, Skeletal/metabolism , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/pharmacologyABSTRACT
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.
Subject(s)
Anterior Cruciate Ligament Injuries , Knee Injuries , Humans , Mice , Animals , Anterior Cruciate Ligament Injuries/complications , Anterior Cruciate Ligament Injuries/genetics , Anterior Cruciate Ligament Injuries/surgery , Antioxidants/metabolism , Reactive Oxygen Species/metabolism , Muscular Atrophy/genetics , Muscular Atrophy/prevention & control , Muscle Weakness/genetics , Muscle Weakness/complications , Knee Injuries/complications , Knee Injuries/surgery , Oxidative Stress/physiology , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolismABSTRACT
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.
Subject(s)
Anterior Cruciate Ligament Injuries , Osteoarthritis , Animals , Humans , Mice , Anterior Cruciate Ligament Injuries/complications , Anterior Cruciate Ligament Injuries/drug therapy , Anterior Cruciate Ligament Injuries/surgery , Disease Models, Animal , Muscle, Skeletal/pathology , Myostatin/genetics , Osteoarthritis/drug therapy , Osteoarthritis/etiology , Osteoarthritis/pathologyABSTRACT
Exercise promotes functional improvements in aged tissues, but the extent to which it simulates partial molecular reprogramming is unknown. Using transcriptome profiling from (1) a skeletal muscle-specific in vivo Oct3/4, Klf4, Sox2 and Myc (OKSM) reprogramming-factor expression murine model; (2) an in vivo inducible muscle-specific Myc induction murine model; (3) a translatable high-volume hypertrophic exercise training approach in aged mice; and (4) human exercise muscle biopsies, we collectively defined exercise-induced genes that are common to partial reprogramming. Late-life exercise training lowered murine DNA methylation age according to several contemporary muscle-specific clocks. A comparison of the murine soleus transcriptome after late-life exercise training to the soleus transcriptome after OKSM induction revealed an overlapping signature that included higher JunB and Sun1. Also, within this signature, downregulation of specific mitochondrial and muscle-enriched genes was conserved in skeletal muscle of long-term exercise-trained humans; among these was muscle-specific Abra/Stars. Myc is the OKSM factor most induced by exercise in muscle and was elevated following exercise training in aged mice. A pulse of MYC rewired the global soleus muscle methylome, and the transcriptome after a MYC pulse partially recapitulated OKSM induction. A common signature also emerged in the murine MYC-controlled and exercise adaptation transcriptomes, including lower muscle-specific Melusin and reactive oxygen species-associated Romo1. With Myc, OKSM and exercise training in mice, as well habitual exercise in humans, the complex I accessory subunit Ndufb11 was lower; low Ndufb11 is linked to longevity in rodents. Collectively, exercise shares similarities with genetic in vivo partial reprogramming. KEY POINTS: Advances in the last decade related to cellular epigenetic reprogramming (e.g. DNA methylome remodelling) toward a pluripotent state via the Yamanaka transcription factors Oct3/4, Klf4, Sox2 and Myc (OKSM) provide a window into potential mechanisms for combatting the deleterious effects of cellular ageing. Using global gene expression analysis, we compared the effects of in vivo OKSM-mediated partial reprogramming in skeletal muscle fibres of mice to the effects of late-life murine exercise training in muscle. Myc is the Yamanaka factor most induced by exercise in skeletal muscle, and so we compared the MYC-controlled transcriptome in muscle to Yamanaka factor-mediated and exercise adaptation mRNA landscapes in mice and humans. A single pulse of MYC is sufficient to remodel the muscle methylome. We identify partial reprogramming-associated genes that are innately altered by exercise training and conserved in humans, and propose that MYC contributes to some of these responses.
Subject(s)
Aging , Cellular Reprogramming , Exercise , Muscle, Skeletal , Animals , Humans , Mice , Cellular Reprogramming/genetics , Disease Models, Animal , DNA Methylation , Exercise/physiology , Gene Expression Profiling , Membrane Proteins/metabolism , Mitochondrial Proteins/metabolism , Muscle, Skeletal/metabolism , Aging/genetics , Aging/physiologyABSTRACT
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.
Subject(s)
Anterior Cruciate Ligament Injuries , Young Adult , Humans , Mice , Animals , Anterior Cruciate Ligament Injuries/pathology , Muscular Atrophy/etiology , Muscular Atrophy/pathology , Quadriceps Muscle/physiology , Muscle Fibers, Skeletal , Muscle ProteinsABSTRACT
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.
Subject(s)
Extracellular Matrix , Muscle Fibers, Skeletal , Collagen/metabolism , Extracellular Matrix/metabolism , Humans , Hypertrophy/metabolism , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/metabolismABSTRACT
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.
Subject(s)
Muscle Fibers, Skeletal , Physical Conditioning, Animal , Mice , Animals , Muscle Fibers, Skeletal/metabolism , Motor Activity , Muscle, Skeletal/blood supply , Physical Conditioning, Animal/physiology , Adaptation, Physiological/geneticsABSTRACT
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.
Subject(s)
Muscle, Skeletal , Senotherapeutics , Animals , Humans , Male , Mice , Hypertrophy/pathology , Mice, Inbred C57BL , Muscle, Skeletal/pathologyABSTRACT
There are functional benefits to exercise in muscle, even when performed late in life, but the contributions of epigenetic factors to late-life exercise adaptation are poorly defined. Using reduced representation bisulfite sequencing (RRBS), ribosomal DNA (rDNA) and mitochondrial-specific examination of methylation, targeted high-resolution methylation analysis, and DNAge™ epigenetic aging clock analysis with a translatable model of voluntary murine endurance/resistance exercise training (progressive weighted wheel running, PoWeR), we provide evidence that exercise may mitigate epigenetic aging in skeletal muscle. Late-life PoWeR from 22-24 months of age modestly but significantly attenuates an age-associated shift toward promoter hypermethylation. The epigenetic age of muscle from old mice that PoWeR-trained for eight weeks was approximately eight weeks younger than 24-month-old sedentary counterparts, which represents ~8% of the expected murine lifespan. These data provide a molecular basis for exercise as a therapy to attenuate skeletal muscle aging.
Subject(s)
Aging/genetics , Epigenomics/methods , Muscle, Skeletal/physiopathology , Physical Conditioning, Animal/physiology , Animals , Male , MiceABSTRACT
Systemic deletion of senescent cells leads to robust improvements in cognitive, cardiovascular, and whole-body metabolism, but their role in tissue reparative processes is incompletely understood. We hypothesized that senolytic drugs would enhance regeneration in aged skeletal muscle. Young (3 months) and old (20 months) male C57Bl/6J mice were administered the senolytics dasatinib (5 mg/kg) and quercetin (50 mg/kg) or vehicle bi-weekly for 4 months. Tibialis anterior (TA) was then injected with 1.2% BaCl2 or PBS 7- or 28 days prior to euthanization. Senescence-associated ß-Galactosidase positive (SA ß-Gal+) cell abundance was low in muscle from both young and old mice and increased similarly 7 days following injury in both age groups, with no effect of D+Q. Most SA ß-Gal+ cells were also CD11b+ in young and old mice 7- and 14 days following injury, suggesting they are infiltrating immune cells. By 14 days, SA ß-Gal+/CD11b+ cells from old mice expressed senescence genes, whereas those from young mice expressed higher levels of genes characteristic of anti-inflammatory macrophages. SA ß-Gal+ cells remained elevated in old compared to young mice 28 days following injury, which were reduced by D+Q only in the old mice. In D+Q-treated old mice, muscle regenerated following injury to a greater extent compared to vehicle-treated old mice, having larger fiber cross-sectional area after 28 days. Conversely, D+Q blunted regeneration in young mice. In vitro experiments suggested D+Q directly improve myogenic progenitor cell proliferation. Enhanced physical function and improved muscle regeneration demonstrate that senolytics have beneficial effects only in old mice.
Subject(s)
Muscle, Skeletal/drug effects , Regeneration/physiology , Satellite Cells, Skeletal Muscle/metabolism , Senotherapeutics/therapeutic use , Animals , Humans , Male , Mice , Senotherapeutics/pharmacologyABSTRACT
BACKGROUNDSkeletal muscle maladaptation accompanies chronic kidney disease (CKD) and negatively affects physical function. Emphasis in CKD has historically been placed on muscle fiber-intrinsic deficits, such as altered protein metabolism and atrophy. However, targeted treatment of fiber-intrinsic dysfunction has produced limited improvement, whereas alterations within the fiber-extrinsic environment have scarcely been examined.METHODSWe investigated alterations to the skeletal muscle interstitial environment with deep cellular phenotyping of biopsies from patients with CKD and age-matched controls and performed transcriptome profiling to define the molecular underpinnings of CKD-associated muscle impairments. We examined changes in muscle maladaptation following initiation of dialysis therapy for kidney failure.RESULTSPatients with CKD exhibited a progressive fibrotic muscle phenotype, which was associated with impaired regenerative capacity and lower vascular density. The severity of these deficits was strongly associated with the degree of kidney dysfunction. Consistent with these profound deficits, CKD was associated with broad alterations to the muscle transcriptome, including altered ECM organization, downregulated angiogenesis, and altered expression of pathways related to stem cell self-renewal. Remarkably, despite the seemingly advanced nature of this fibrotic transformation, dialysis treatment rescued these deficits, restoring a healthier muscle phenotype. Furthermore, after accounting for muscle atrophy, strength and endurance improved after dialysis initiation.CONCLUSIONThese data identify a dialysis-responsive muscle fibrotic phenotype in CKD and suggest the early dialysis window presents a unique opportunity of improved muscle regenerative capacity during which targeted interventions may achieve maximal impact.TRIAL REGISTRATIONNCT01452412FUNDINGNIH, NIH Clinical and Translational Science Awards (CTSA), and Einstein-Mount Sinai Diabetes Research Center.
Subject(s)
Fibrosis/etiology , Muscular Diseases/etiology , Renal Dialysis/methods , Renal Insufficiency, Chronic/complications , Case-Control Studies , Female , Fibrosis/pathology , Humans , Male , Middle Aged , Muscular Diseases/pathology , Renal Insufficiency, Chronic/pathology , Renal Insufficiency, Chronic/therapy , Risk FactorsABSTRACT
Dysregulation and fibrosis of the extracellular matrix (ECM) in skeletal muscle is a consequence of injury. Current ECM assessment necessitates muscle biopsies to evaluate alterations to the muscle ECM, which is often not practical in humans. The goal of this study was to evaluate the potential of a magnetic resonance imaging sequence that quantifies T1ρ relaxation time to predict ECM collagen composition and organization. T1ρ imaging was performed and muscle biopsies obtained from the involved and non-involved vastus lateralis muscle on 27 subjects who had an anterior cruciate ligament (ACL) tear. T1ρ times were quantified via monoexponential decay curve fitted to a series of T1ρ-weighted images. Several ECM indices, including collagen content and organization, were obtained using immunohistochemistry and histochemistry in addition to hydroxyproline. Model selection with multiple linear regression was used to evaluate the relationships between T1ρ times and ECM composition. Additionally, the ACL-deficient and healthy limb were compared to determine sensitivity of T1ρ to detect early adaptations in the muscle ECM following injury. We show that T1ρ relaxation time was strongly associated with collagen unfolding (t = 4.093, P = 0.0007) in the ACL-deficient limb, and collagen 1 abundance in the healthy limb (t = 2.75, P = 0.014). In addition, we show that T1ρ relaxation time is significantly longer in the injured limb, coinciding with significant differences in several indices of collagen content and remodelling in the ACL-deficient limb. These results support the use of T1ρ to evaluate ECM composition in skeletal muscle in a non-invasive manner. KEY POINTS: Dysregulation and fibrotic transformation of the skeletal muscle extracellular matrix (ECM) is a common pathology associated with injury and ageing. Studies of the muscle ECM in humans have necessitated the use of biopsies, which are impractical in many settings. Non-invasive MRI T1ρ relaxation time was validated to predict ECM collagen composition and organization with aligned T1ρ imaging and biopsies of the vastus lateralis in the healthy limb and anterior cruciate ligament (ACL)-deficient limb of 27 subjects. T1ρ relaxation time was strongly associated with collagen abundance and unfolding in the ACL-deficient limb, and T1ρ relaxation time was strongly associated with total collagen abundance in the healthy limb. T1ρ relaxation time was significantly longer in the ACL-deficient limb, coinciding with significant increases in several indices of muscle collagen content and remodelling supporting the use of T1ρ to non-invasively evaluate ECM composition and pathology in skeletal muscle.
Subject(s)
Anterior Cruciate Ligament Injuries , Anterior Cruciate Ligament Injuries/diagnostic imaging , Collagen , Humans , Magnetic Resonance Imaging , Muscle, Skeletal/diagnostic imaging , Quadriceps Muscle/diagnostic imagingABSTRACT
Satellite cells are required for postnatal development, skeletal muscle regeneration across the lifespan, and skeletal muscle hypertrophy prior to maturity. Our group has aimed to address whether satellite cells are required for hypertrophic growth in mature skeletal muscle. Here, we generated a comprehensive characterization and transcriptome-wide profiling of skeletal muscle during adaptation to exercise in the presence or absence of satellite cells in order to identify distinct phenotypes and gene networks influenced by satellite cell content. We administered vehicle or tamoxifen to adult Pax7-DTA mice and subjected them to progressive weighted wheel running (PoWeR). We then performed immunohistochemical analysis and whole-muscle RNA-seq of vehicle (SC+) and tamoxifen-treated (SC-) mice. Further, we performed single myonuclear RNA-seq to provide detailed information on how satellite cell fusion affects myonuclear transcription. We show that while skeletal muscle can mount a robust hypertrophic response to PoWeR in the absence of satellite cells, growth, and adaptation are ultimately blunted. Transcriptional profiling reveals several gene networks key to muscle adaptation are altered in the absence of satellite cells.
Subject(s)
Physical Conditioning, Animal , Satellite Cells, Skeletal Muscle , Mice , Animals , Motor Activity , Muscle, Skeletal , Hypertrophy , TamoxifenABSTRACT
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.
ABSTRACT
Using in vivo muscle stem cell (satellite cell)-specific extracellular vesicle (EV) tracking, satellite cell depletion, in vitro cell culture, and single-cell RNA sequencing, we show satellite cells communicate with other cells in skeletal muscle during mechanical overload. Early satellite cell EV communication primes the muscle milieu for proper long-term extracellular matrix (ECM) deposition and is sufficient to support sustained hypertrophy in adult mice, even in the absence of fusion to muscle fibers. Satellite cells modulate chemokine gene expression across cell types within the first few days of loading, and EV delivery of miR-206 to fibrogenic cells represses Wisp1 expression required for appropriate ECM remodeling. Late-stage communication from myogenic cells during loading is widespread but may be targeted toward endothelial cells. Satellite cells coordinate adaptation by influencing the phenotype of recipient cells, which extends our understanding of their role in muscle adaptation beyond regeneration and myonuclear donation.
ABSTRACT
Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.
Subject(s)
Knee Joint/physiology , Muscle, Skeletal/physiology , Animals , Area Under Curve , Calibration , Data Analysis , Electrodes , Humans , Isometric Contraction/physiology , Mice, Inbred C57BL , Muscle Strength/physiology , Software , TorqueABSTRACT
The inability of older adults to maintain independence is a consequence of sarcopenia and frailty. In order to identify the molecular mechanisms responsible for decreased physical function, it will be critical to utilize a small animal model. The main purpose of this study was to develop a composite Comprehensive Functional Assessment Battery (CFAB) of well-validated tests to determine physical function and exercise capacity in 3 age groups of male C57BL/6 mice (6 months old, n = 29; 24 months old, n = 24; 28+ months old, n = 28). To measure physical function in mice, we used rotarod (overall motor function), grip meter (forelimb strength), treadmill (endurance), inverted cling (strength/endurance), voluntary wheel running (volitional exercise and activity rate), and muscle performance with in vivo contractile physiology (dorsiflexor torque). We hypothesized that CFAB would be a valid means to assess the physical function of a given mouse across the life span. In addition, we proposed that CFAB could be used to determine relationships between different parameters associated with sarcopenia. We found that there was an overall age-related significant decline (p < .05) in all measurements, and the CFAB score demonstrated that some individual mice (the upper quartile) retained the functional capacity of average mice 1 cohort younger. We conclude that the CFAB is a powerful, repeatable, and noninvasive tool to assess and compare physical function and assess complex motor task ability in mice, which will enable researchers to easily track performance at the individual mouse level.
Subject(s)
Aging/physiology , Exercise Test , Exercise Tolerance/physiology , Animals , Frailty/physiopathology , Male , Mice, Inbred C57BL , Physical Conditioning, Animal , Sarcopenia/physiopathologyABSTRACT
Severe burn injury induces a myriad of deleterious effects to skeletal muscle, resulting in impaired function and delayed recovery. Following burn, catabolic signaling and myofiber atrophy are key fiber-intrinsic determinants of weakness; less well understood are alterations in the interstitial environment surrounding myofibers. Muscle quality, specifically alterations in the extracellular matrix (ECM), modulates force transmission and strength. We sought to determine the impact of severe thermal injury on adaptation to the muscle ECM and quantify muscle fibrotic burden. After a 30% total body surface area dorsal burn, spinotrapezius muscle was harvested from mice at 7 (7d, n = 5), 14 (14d, n = 4), and 21 days (21d, n = 4), and a sham control group was also examined (Sham, n = 4). Expression of transforming growth factor-ß (TGFß), myostatin, and downstream effectors and proteases involved in fibrosis and collagen remodeling were measured by immunoblotting, and immunohistochemical and biochemical analyses assessed fibrogenic cell abundance and collagen deposition. Myostatin signaling increased progressively through 21 days postburn alongside fibrogenic/adipogenic progenitor cell expansion, with abundance peaking at 14 days postburn. Postburn, elevated expression of tissue inhibitor of matrix metalloproteinase 1 supported collagen remodeling resulting in a net accumulation of muscle collagen content. Collagen accumulation peaked at 14 days postburn but remained elevated through 21 days postburn, demonstrating minimal resolution of burn-induced fibrosis. These findings highlight a progressive upregulation of fibrogenic processes following burn injury, eliciting a fibrotic muscle phenotype that hinders regenerative capacity and is not resolved with 21 days of recovery.