BACKGROUND:The extent of inter-individual variability in response to heavy resistance exercise training (HReT), and the possible existence of non-responders, remains unclear. This study aimed to determine the degree of variability in response to prolonged HReT in healthy older men. METHODS:We conducted a secondary analysis of an 8- and 16-week intervention involving thrice-weekly HReT (EX) or continuation of a sedentary lifestyle (SED). Fifty-eight healthy men (age 72 ± 5) were randomized to EX (n = 38) or SED (n = 20). Assessments were conducted at baseline, 8-weeks, and 16-weeks for five outcomes: maximal voluntary contraction strength (MVC), rate of force development (RFD), quadriceps cross-sectional area (qCSA), and type I and II myofibre cross-sectional area (fCSA). Inter-individual variability was assessed using the standard deviation of individual responses (SDIR). Individual changes relative to a Typical Error were used to classify responders as Poor, Trivial, Robust, or Excellent. RESULTS:16 weeks of EX led to group-level increases in MVC (19 ± 14%), RFD (58 ± 80%), qCSA (3 ± 4%), and type II fCSA (14 ± 25%), with no changes in SED. Substantial inter-individual variability was observed. After 16 weeks, 82% of EX participants were classified as Robust or Excellent responders; only 5% were Poor responders. Training compliance and 1RM progression did not explain this variability. Lower baseline levels were linked to greater improvements but did not fully account for response differences. CONCLUSIONS:This study provides strong evidence of inter-individual variability in response to HReT among healthy older men. Given the rarity of true non-responders, our data support HReT as the universally recommended first-line strategy for enhancing muscle mass and strength.

Mechanical loading drives structural and functional improvements in muscle and tendon, protecting against injury at their interface, at the myotendinous junction (MTJ), and within the tendon matrix. However, the early cellular and molecular events that initiate these adaptations in humans remain poorly understood. To investigate this, we applied single-nucleus RNA sequencing and in situ hybridization to map the acute transcriptional response of the human muscle-tendon unit to a single bout of eccentric resistance exercise, with a focus on extracellular matrix (ECM) regulation. We identified four transcriptionally distinct fibroblast subtypes expressing key ECM components, including COL1A1 and DCN. Three of these subtypes were localized to the tendon and responded to exercise: two were spatially restricted to the collagen fascicles or the MTJ, while the third, enriched in the interfascicular matrix (IFM), exhibited the strongest response. This IFM population, marked by PDGFRA, upregulated PRG4 and VCAN, ECM genes linked to tissue lubrication and resilience. In parallel, exercise induced dynamic ECM regulation in myonuclei, particularly in a distinct subset of type II myonuclei at the MTJ, that expanded in number and robustly upregulated COL22A1, a collagen essential for MTJ integrity. Together, these findings uncover a spatially organized, cell type-specific program of ECM remodeling in response to mechanical load, offering new insight into the early molecular events of human muscle-tendon adaptation.NEW & NOTEWORTHY We provide the first high-resolution, in vivo single-nucleus map of the acute human muscle-tendon response to mechanical loading. Within 4 hours of resistance exercise, spatially distinct tendon fibroblasts and myonuclei activate coordinated extracellular matrix programs. Interfascicular fibroblasts upregulate PRG4 and VCAN in the tendon, while myonuclei at the myotendinous junction induce COL22A1 and LAMA2. These findings reveal a new principle of human mechanobiology, where exercise rapidly engages niche-specific programs to initiate extracellular matrix adaptation.

BACKGROUND:Anabolic-androgenic steroids (AASs) are synthetic derivatives of testosterone that are abused by athletes to enhance their physical appearance and performance. AAS abusers have an increased risk of tendon ruptures compared with nonusers, and it has been proposed that AASs damage tendon tissue. Only a few human studies have investigated the effect of AASs on tendon tissue, and to our knowledge, there are no data on female sex. PURPOSE/HYPOTHESIS:The purpose was to investigate the effect of current and former abuse of AASs on the patellar tendon (PT). It was hypothesized that AASs would not affect tendon tissue. STUDY DESIGN:Cross-sectional study; Level of evidence, 3. METHODS:This study included recreational athletes with current (female: n = 4; male: n = 18; total: n = 22) or former (female: n = 5; male: n = 7; total: n = 12) AAS abuse and nonusers (female: n = 5; male: n = 9; total: n = 14). The authors investigated the proportion of tendon injuries, PT cross-sectional area by magnetic resonance imaging, mechanical properties by ultrasound, gene expression levels of connective tissue proteins, and cell density by histological staining from tendon biopsy samples. RESULTS:The combined AAS group (both current and former abusers) reported a higher proportion of upper body tendon injuries compared with nonusers (79.4% vs 28.6%, respectively; P = .002). There was no difference in PT cross-sectional area (P = .918) or cell density (fascicular matrix: P = .413; interfascicular matrix: P = .982) between current AAS abusers, former AAS abusers, and nonusers. There was a greater expression of IGF-1 mRNA in current AAS abusers compared with nonusers (P = .043), but there were no group differences in other mRNA targets. Former AAS abusers had significantly higher tendon deformation (P = .030) and strain (P = .026) at common force compared with nonusers. There were no significant differences between male and female participants in the effect of AASs on tendon tissue. CONCLUSION:These data show that the PT itself was not severely affected by AAS abuse.

Short-term disuse leads to rapid declines in muscle mass and strength. These declines are driven by changes at all levels of the neuromuscular system; the brain, spinal cord and skeletal muscle. In addition to neural input from the central and peripheral nervous systems to the muscle, molecular factors originating in the muscle can be transported to the central nervous system. These interactions highlight the interconnected nature of the neuromuscular system during exercise and disuse, and form the basis for this review. Although it is well known that physical activity confers a myriad of health benefits, a recent interest in targeted exercise before periods of disuse or immobility, termed prehabilitation, has emerged. Clinical studies within multiple medical specialities suggest positive effects of prehabilitative exercise on preserving muscle function, reducing adverse outcomes and shortening the length of hospital stay. Yet, the studies available are few and heterogeneous, and the underlying protective mechanisms of prehabilitative exercise remain elusive. In this review, we examine the ramifications of disuse across all levels of the neuromuscular system and explore how prehabilitation may counteract these effects. We summarize these mechanisms into three primary categories: (1) enhancing pre-disuse capacity; (2) establishing neural and muscle memory; and (3) fostering structural adaptations in both muscle and brain.

The myotendinous junction (MTJ) is a weak link in the musculoskeletal system. Here, we isolated the tips of single myofibres from healthy (non-injured) human hamstring muscles for confocal microscopy (n=6) and undertook RNAscope in situ hybridisation (n=6) to gain insight into the profiles of cells and myonuclei in this region, in a fibre type manner. A marked presence of mononuclear cells was observed coating the myofibre tips (confirmed by serial block face scanning electron microscopy and cryosection immunofluorescence), with higher numbers for type I (median 29; range 16-63) than type II (16; 9-23) myofibres (P<0.05). The number of these cells expressing COL22A1 was comparable between fibre types. Myonuclear number and density gradually increased from the myofibre proper towards the tip for both fibre types (P<0.05). COL22A1 was expressed by similar proportions of myonuclei in type I (median 26%; range 13-56) and type II (19%; 3-67) myofibre tips. 70% of the COL22A1-positive nuclei in the MTJ region were myonuclei, and the remaining 30% were MTJ cells. This insight refines our fundamental understanding of the human MTJ at the cell and structural levels.

A critical link in the chain of force transmission from muscle fiber cross-bridge to bone is the interface between muscle and tendon-the myotendinous junction (MTJ). To meet the challenge of connecting these two tissues, the MTJ is specialized molecularly and morphologically. Distinct transcriptional profiles are evident for the myonuclei at the myofiber tips and a population of mononuclear tendon cells at the MTJ, demonstrating support from both sides in MTJ maintenance. Paradoxically, despite this high degree of specialization, the MTJ remains susceptible to strain (rupture) injury and is often associated with failed tissue healing. Incomplete understanding of the nature of the MTJ and the elements contributing to its plasticity hinder tackling this unsolved clinical challenge. The goal of this review is to summarize key structural and molecular features of the MTJ, discuss MTJ adaptation in response to mechanical (un)loading, aging, and injury, and highlight the major unanswered questions surrounding the MTJ.

BACKGROUND:Sarcopenia represents a major clinical and societal challenge facing rapidly aging populations. Accessible and specific biomarkers represent valuable tools, both in diagnosis and assessing the efficacy of therapeutic interventions. C-terminal agrin fragment (CAF) is the most commonly used blood-based biomarker of neuromuscular junction degradation in aging, inactivity and disease, but large unexplained interindividual variation exists, limiting its diagnostic and prognostic value. Exercise and medication may explain some of this variation. The aim of this study was to investigate the influence of a single bout (1EX) or 48 bouts (48EX) of heavy resistance exercise (EX), with or without angiotensin II type I receptor blocker (losartan (LOS)) supplementation, on serum CAF levels in healthy older men. METHODS:Eighty-three healthy, normotensive older men were enrolled in one of two randomized placebo (PLA) controlled trials. 1EX: 25 participants (EX ± LOS), with a mean age of 70 ± 7 years, had blood drawn before and after (4.5 h, Days 1, 4 and 7) a single bout of unilateral heavy resistance exercise of the quadriceps muscles. 48EX: at baseline, and after 8 and 16 weeks of a progressive heavy resistance exercise program, 58 participants (LOS-EX, n = 20; LOS-SED, n = 20; PLA-EX, n = 18), with a mean age of 72 ± 5 years, had blood drawn, and specific force (strength per unit mass) was measured by dynamometer and magnetic resonance imaging of the quadriceps muscles. Serum CAF was measured by ELISA. RESULTS:At baseline, CAF showed weak correlations with age and leg lean mass (both R2 = 0.07, p < 0.05). With 48EX, specific force increased in both EX groups (LOS-EX + PLA-EX) by 13%-14% at 8 weeks and 14%-17% at 16 weeks (p < 0.0001), with no change in LOS-SED (p > 0.05), confirming the efficacy of the 48EX program. Serum CAF increased in LOS-EX and LOS-SED by 4%-7% at 8 weeks and 7%-9% at 16 weeks (p < 0.005) respectively, with no change in PLA-EX (p > 0.05). 1EX reduced CAF by 8% 1 day postexercise (p < 0.05), with no correlation to circulating creatine kinase levels (p > 0.05), a marker of muscle damage. CONCLUSIONS:Serum CAF was unaffected by 16 weeks of EX but increased by LOS supplementation. 1EX, performed with one leg, acutely lowered serum CAF, albeit with substantial interindividual variability. These findings collectively identify novel stimuli of serum CAF turnover-drug interaction and time from last exercise bout to blood sampling. These findings add value to CAF as a neuromuscular biomarker and highlight important experimental design aspects for future clinical studies.

BACKGROUND:We examined cardiorespiratory fitness and physical performance in long-term survivors of paediatric haematopoietic stem cell transplantation (HSCT) and explored how these are associated with the presence of metabolic syndrome (MetS). PROCEDURE:We included 90 survivors of paediatric HSCT (median age, 30.3 years; range, 19.6-53.0; median follow-up time, 20.2 years) and 32 healthy controls. Cardiorespiratory fitness was evaluated by cardiopulmonary exercise tests, and physical performance was assessed through sit-to-stand, handgrip strength, timed-up-and-go, walking pace and six-minute walk tests. We assessed for components of MetS (blood pressure, waist circumference, plasma lipids, and glucose). For comparison of physical capacity between survivors and controls and survivors with or without the presence of MetS, a multiple linear regression analysis corrected for age and sex was applied. RESULTS:Survivors demonstrated lower cardiorespiratory fitness compared with controls (mean ± SD VO2 peak 29.3 ± 7.0 mL/kg/min vs. 44.3 ± 6.8 mL/kg/min, p < 0.0001) and impairment in all physical performance outcomes, where the most prominent differences compared with controls were seen in the sit-to-stand test (33% reduction). Twenty-eight percent of survivors fulfilled the criteria for MetS. The presence of MetS associated with lower VO2 peak (p = 0.03), poorer outcomes in the six-minute walk test (p = 0.02), walking pace (p = 0.03) and the timed-up-and-go test (p = 0.003). CONCLUSIONS:Young adult survivors of paediatric HSCT are at risk of markedly reduced physical capacity compared with age- and sex-matched controls, and the high incidence of MetS observed among survivors was associated with this impairment. Overall, these data underline the importance of monitoring physical capacity in survivors of paediatric HSCT.

Certain skeletal muscles are specialized for their functional roles, yet direct comparisons of cellular morphology of distinct muscles beyond fibre type distribution are limited. This study investigated myofibre morphology in predominantly slow, fast and mixed fibre muscles in humans and mice, with the aim of establishing reference values for muscle‐specific myofibre size and shape. Nine healthy young men (Age: 26 ± 1 years, BMI: 23 ± 1 kg/m 2 ) had muscle biopsies taken from soleus, triceps brachii and vastus lateralis muscles. Additionally, the soleus and gastrocnemius muscles were harvested from 7 male C57BL/6 mice. Muscle samples were analysed by ATPase (human) or immunofluorescence (mouse) stainings of fibre type specific cross‐sectional area, perimeter and Shape Factor Index (SFI; fibre perimeter 2 /4 × π × fibre cross‐sectional area). In humans, type I fibres had 30%–40% larger CSA and 4%–7% higher SFI in soleus (1.54 ± 0.06) compared to triceps brachii (1.47 ± 0.05) and vastus lateralis (1.43 ± 0.04). Type IIa fibres SFI were 10%–11% higher in soleus (1.61 ± 0.08) compared to triceps brachii (1.45 ± 0.04) and vastus lateralis (1.45 ± 0.08). Soleus type I fibres were more heterogeneous in terms of size and shape compared to other muscles. Analyses of mouse muscle showed a similar pattern, in that CSA and SFI were higher in type I and IIa fibres of the soleus compared to the gastrocnemius. These findings suggest a consistent morphological characteristic of soleus fibres across species, with potentially important implications for future biomedical research.

Abstract: Skeletal muscle regeneration is a cardinal feature of muscle pathologies and is crucial for post-exercise recovery and traumatic sports injuries. Regeneration of damaged muscle in humans is a prolonged process and is accompanied by pain and physical dysfunction, highlighting the unmet need for effective interventions to accelerate the regenerative process. Through cellular and preclinical models, we have previously identified nicotinamide (NAM) and pyridoxine (PN) as potent stimulators of Muscle Stem Cells (MuSCs). Herein we investigated if a combination of NAM and PN could enhance MuSC activity and improve muscle regeneration in healthy volunteers during recovery from eccentric contractions. Methods: This randomized, double-blind, placebo-controlled trial enrolled male participants aged 18-50 years supplemented daily with 714mg NAM and 19mg PN (NAM/PN) or placebo for 8 days following unilateral eccentric muscle contractions using Neuromuscular Electrical Stimulation (NMES). MuSC was quantified by immunohistofluorescence on vastus lateralis muscle biopsies. Results: 39 out of 43 enrolled participants completed the study. Supplementation of NAM/PN was well tolerated and increased blood concentrations of NAM and PN vitamers. The NMES protocol caused myofiber necrosis and triggered a strong MuSC response. After 8 days, the number of Pax7, MyoD, and myogenin positive cells per damaged fiber was significantly higher in NAM/PN vs placebo groups (+29-67%). NAM/PN also increased the proportion of regenerating fibers re-expressing embryonic myosin (+37%). Conclusion: Daily oral NAM/PN supplementation following eccentric muscle damaging contractions enhances MuSC activity and accelerates muscle regeneration. These findings provide new possibilities for targeted therapeutic interventions in muscle repair. Trial registration: [NCT04874662][1] ### Competing Interest Statement J.M., E.M., S.K., J.N.F. & P.S. are employees of Societe des Produits Nestle SA; E.P.M., and O.E.J. are employees of Nestle Health Science SA. L.G.K. was an employee of Nestle Health Science SA and is on the scientific advisory boards of Vital Proteins and NUUN, has participated on advisory boards of Liquid I.V and has received personal fees from RNWY and Nestle Health Science; is a board member of Siftlink. G.H., A.D., K.K., B.W.H., W.H., M.K. & A.M. declare that they have no competing interests. ### Clinical Trial NCT04874662 ### Funding Statement Nestle, with support from: Lundbeck Foundation grant R344.2020.254 (A.L.M); BRIDGE Translational Excellence Programme (bridge.ku.dk) at the Faculty of Health and Medical Sciences, University of Copenhagen, funded by the Novo Nordisk Foundation (NNF20SA0064340) (G.H) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This randomized, double-blind, placebo-controlled study was approved by the Regional Scientific Ethical Committees of Copenhagen (protocol number H-20081080). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT04874662&atom=%2Fmedrxiv%2Fearly%2F2025%2F03%2F24%2F2025.03.24.25323226.atom

ABSTRACT 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.

Exercise preserves neuromuscular function in aging through unknown mechanisms. Skeletal muscle fibroblasts (FIB) and stem cells (MuSC) are abundant in skeletal muscle and reside close to neuromuscular junctions, but their relative roles in motor neuron maintenance remain undescribed. Using direct cocultures of embryonic rat motor neurons with either human MuSC or FIB, RNA sequencing revealed profound differential regulation of the motor neuron transcriptome, with FIB generally favoring neuron growth and cell migration and MuSC favoring production of ribosomes and translational machinery. Conditioned medium from FIB was superior to MuSC in preserving motor neurons and increasing their maturity. Lastly, we established the importance of donor age and exercise status and found an age-related distortion of motor neuron and muscle cell interaction that was fully mitigated by lifelong physical activity. In conclusion, we show that human muscle FIB and MuSC synergistically stimulate the growth and viability of motor neurons, which is further amplified by regular exercise.

BACKGROUND:Plantar fasciitis is a painful tendinous condition (tendinopathy) with a high prevalence in athletes. While a healthy tendon has limited blood flow, ultrasound has indicated elevated blood flow in tendinopathy, but it is unknown if this is related to a de facto increase in the tendon vasculature. Likewise, an accumulation of glycosaminoglycans (GAGs) is observed in tendinopathy, but its relationship to clinical pain is unknown.PURPOSE:To explore to what extent vascularization, inflammation, and fat infiltration were present in patients with plantar fasciitis and if they were related to clinical symptoms.STUDY DESIGN:Descriptive laboratory study.METHODS:Biopsy specimens from tendinopathic plantar fascia tissue were obtained per-operatively from both the primary site of tendon pain and tissue swelling ("proximal") and a region that appeared macroscopically healthy at 1 to 2 cm away from the primary site ("distal") in 22 patients. Biopsy specimens were examined with immunofluorescence for markers of blood vessels, tissue cell density, fat infiltration, and macrophage level. In addition, pain during the first step in the morning (registered during an earlier study) was correlated with the content of collagen and GAGs in tissue.RESULTS:High vascularization (and cellularity) was present in both the proximal (0.89%) and the distal (0.96%) plantar fascia samples, whereas inconsistent but not significantly different fat infiltration and macrophage levels were observed. The collagen content was similar in the 2 plantar fascia regions, whereas the GAG content was higher in the proximal region (3.2% in proximal and 2.8% in distal; P = .027). The GAG content in the proximal region was positively correlated with the subjective morning pain score in the patients with tendinopathy (n = 17).CONCLUSION:In patients with plantar fasciitis, marked tissue vascularization was present in both the painful focal region and a neighboring nonsymptomatic area. In contrast, the accumulation of hydrophilic GAGs was greater in the symptomatic region and was positively correlated with increased clinical pain levels in daily life.CLINICAL RELEVANCE:The accumulation of GAGs in tissue rather than the extent of vascularization appears to be linked with the clinical degree of pain symptoms of the disease.

A major challenge in sports medicine is to facilitate the fastest possible recovery from injury without increasing the risk of subsequent reruptures, and thus effective rehabilitation programs should balance between these two factors. The present review focuses on examining the role of different resistance training interventions in rehabilitation of acute muscle strain in the time frame from injury until return to sport (RTS), the rate of reinjuries, and tissue changes after injury. Randomized, controlled trials dealing with a component of resistance training in their rehabilitation protocols, as well as observational studies on tissue morphology and tissue changes as a result to muscle strain injuries, were included. The mean time for RTS varied from 15 to 86 days between studies (n = 8), and the mean rate of reinjury spanned from 0 to 70%. Eccentric resistance training at long muscle length and rapid introduction to rehabilitation postinjury led to significant improvement regarding RTS, and core-stabilizing exercises as well as implementing an individualized algorithm for rehabilitation seem to reduce the risk of reinjury in studies with a high rerupture rate. Independent of the rehabilitation program, structural changes appear to persist for a long time, if not permanently, after a strain injury.

Abstract Prematurity has physical consequences, such as lower birth weight, decreased muscle mass and increased risk of adult‐onset metabolic disease. Insulin‐like growth factor 1 (IGF‐1) has therapeutic potential to improve the growth and quality of muscle and tendon in premature births, and thus attenuate some of these sequalae. We investigated the effect of IGF‐1 on extensor carpi radialis muscle and biceps brachii tendon of preterm piglets. The preterm group consisted of 19‐day‐old preterm (10 days early) piglets, treated with either IGF‐1 or vehicle. Term controls consisted of groups of 9‐day‐old piglets (D9) and 19‐day‐old piglets (D19). Muscle samples were analysed by immunofluorescence to determine the cross‐sectional area (CSA) of muscle fibres, fibre type composition, satellite cell content and central nuclei‐containing fibres in the muscle. Tendon samples were analysed for CSA, collagen content and maturation, and vascularization. Gene expression of the tendon was measured by RT‐qPCR. Across all endpoints, we found no significant effect of IGF‐1 treatment on preterm piglets. Preterm piglets had smaller muscle fibre CSA compared to D9 and D19 control group. Satellite cell content was similar across all groups. For tendon, we found an effect of age on tendon CSA, and mRNA levels of COL1A1, tenomodulin and scleraxis. Immunoreactivity for elastin and CD31, and several markers of tendon maturation, were increased in D9 compared to the preterm piglets. Collagen content was similar across groups. IGF‐1 treatment of preterm‐born piglets does not influence the growth and maturation of skeletal muscle and tendon.

Morphological studies of skeletal muscle tissue provide insights into the architecture of muscle fibers, the surrounding cells, and the extracellular matrix (ECM). However, a spatial proteomics analysis of the skeletal muscle including the muscle-tendon transition zone is lacking. Here, we prepare cryotome muscle sections of the mouse soleus muscle and measure each slice using short liquid chromatography-mass spectrometry (LC-MS) gradients. We generate 3,000 high-resolution protein profiles that serve as the basis for a network analysis to reveal the complex architecture of the muscle-tendon junction. Among the protein profiles that increase from muscle to tendon, we find proteins related to neuronal activity, fatty acid biosynthesis, and the renin-angiotensin system (RAS). Blocking the RAS in cultured mouse tenocytes using losartan reduces the ECM synthesis. Overall, our analysis of thin cryotome sections provides a spatial proteome of skeletal muscle and reveals that the RAS acts as an additional regulator of the matrix within muscle-tendon junctions.

BACKGROUND:Early-onset osteoporosis is a frequent late effect after pediatric hematopoietic stem cell transplantation (HSCT). It remains unknown if physical training can improve bone formation in these patients, as the transplantation procedure may cause sustained dysregulation of the bone-forming osteoblast progenitor cells. OBJECTIVE:We aimed to explore the effect of resistance training on bone remodeling in long-term survivors of pediatric HSCT. PROCEDURE:In this prospective, controlled intervention study, we included seven HSCT survivors and 15 age- and sex-matched healthy controls. The participants completed a 12-week heavy load, lower extremity resistance training intervention with three weekly sessions. We measured fasting serum levels of the bone formation marker "N-terminal propeptide of type I procollagen" (P1NP), and the bone resorption marker "C-terminal telopeptide of type I collagen" (CTX). The hypothesis was planned before data collection began. The trial was registered at Clinicaltrials.gov before including the first participant, with trial registration no. NCT04922970. RESULTS:Resistance training led to significantly increased levels of fasting P1NP in both patients (from 57.62 to 114.99 ng/mL, p = .03) and controls (from 66.02 to 104.62 ng/mL, p < .001). No significant changes in fasting CTX levels were observed. CONCLUSIONS:Despite previous high-dose cytotoxic therapy, long-term survivors of pediatric HSCT respond to resistance training with improvement of bone formation, comparable to that of healthy controls. This suggests that resistance training might be a promising non-pharmacological approach to prevent the early decline in bone mass, and should be considered as part of a follow-up program to counteract long-term sequela after pediatric HSCT.

BACKGROUND:Age-related loss of strength is disproportionally greater than the loss of mass, suggesting maladaptations in the neuro-myo-tendinous system. Myofibers are often misshaped in aged and diseased muscle, but systematic analyses of large sample sets are lacking. Our aim was to investigate myofiber shape in relation to age, exercise, myofiber type, species and sex. METHODS:Vastus lateralis muscle biopsies (n = 265) from 197 males and females, covering an age span of 20-97 years, were examined. The gastrocnemius and soleus muscles of 11 + 22-month-old male C57BL/6 mice were also examined. Immunofluorescence and ATPase stainings of muscle cross-sections were used to measure myofiber cross-sectional area (CSA) and perimeter. From these, a shape factor index (SFI) was calculated in a fibre-type-specific manner (type I/II in humans; type I/IIa/IIx/IIb in mice), with higher values indicating increased deformity. Heavy resistance training (RT) was performed three times per week for 3-4 months by a subgroup (n = 59). Correlation analyses were performed comparing SFI and CSA with age, muscle mass, maximal voluntary contraction (MVC), rate of force development and specific force (MVC/muscle mass). RESULTS:In human muscle, SFI was positively correlated with age for both type I (R2 = 0.20) and II (R2 = 0.38) myofibers. When subjects were separated into age cohorts, SFI was lower for type I (4%, P < 0.001) and II (6%, P < 0.001) myofibers in young (20-36) compared with old (60-80) and higher for type I (5%, P < 0.05) and II (14%, P < 0.001) myofibers in the oldest old (>80) compared with old. The increased SFI in old muscle was observed in myofibers of all sizes. Within all three age cohorts, type II myofiber SFI was higher than that for type I myofiber (4-13%, P < 0.001), which was also the case in mice muscles (8-9%, P < 0.001). Across age cohorts, there was no difference between males and females in SFI for either type I (P = 0.496/0.734) or II (P = 0.176/0.585) myofibers. Multiple linear regression revealed that SFI, after adjusting for age and myofiber CSA, has independent explanatory power for 8/10 indices of muscle mass and function. RT reduced SFI of type II myofibers in both young and old (3-4%, P < 0.001). CONCLUSIONS:Here, we identify type I and II myofiber shape in humans as a hallmark of muscle ageing that independently predicts volumetric and functional assessments of muscle health. RT reverts the shape of type II myofibers, suggesting that a lack of myofiber recruitment might lead to myofiber deformity.

Myonuclei transcriptionally regulate muscle fibers during homeostasis and adaptation to exercise. Their subcellular location and quantity are important when characterizing phenotypes of myopathies, the effect of treatments, and understanding the roles of satellite cells in muscle adaptation and muscle "memory." Difficulties arise in identifying myonuclei due to their proximity to the sarcolemma and closely residing interstitial cell neighbors. We aimed to determine to what extent (pericentriolar material-1) PCM1 is a specific marker of myonuclei in vitro and in vivo. Single isolated myofibers and cross sections from mice and humans were studied from several models including wild-type and Lamin A/C mutant mice after functional overload and damage and recovery in humans following forced eccentric contractions. Fibers were immunolabeled for PCM1, Pax7, and DNA. C2C12 myoblasts were also studied to investigate changes in PCM1 localization during myogenesis. PCM1 was detected at not only the nuclear enve-lope of myonuclei in mature myofibers and in newly formed myotubes but also centrosomes in proliferating myogenic precur-sors, which may or may not fuse to join the myofiber syncytium. PCM1 was also detected in nonmyogenic nuclei near the sarcolemma, especially in regenerating areas of the Lmna+/DK32 mouse and damaged human muscle. Although PCM1 is not completely specific to myonuclei, the impact that PCM1 + macrophages and interstitial cells have on myonuclei counts would be small in healthy muscle. PCM1 may prove useful as a marker of satellite cell dynamics due to the distinct change in localization during differentiation, revealing satellite cells in their quiescent (PCM1-), proliferating (PCM1 + centrosome), and prefusion states (PCM1 + nuclear envelope).

Immunohistochemistry, fluorescent microscopy, and image analysis are key tools for visualizing and analyzing specific proteins in a variety of tissues. Together these techniques are readily available in most laboratories and in an infinite number of variations. Herein, a working example is used relating to the aging human skeletal muscle being characterized by denervated muscle fibers that undergo atrophy and eventual death. As such, proteins normally restricted to embryonic development, muscle regeneration, and the myotendinous and neuromuscular junctions of the muscle fiber are observed in denervated muscle fibers. We describe a workflow from muscle biopsy sampling to lab bench and to image analysis. Our aim is—through a concrete example—to provide the reader with knowledge and insight into how to design an effective analysis workflow, which can then be utilized in specific research contexts.