Widespread Vascularization and Correlation of Glycosaminoglycan Accumulation to Tendon Pain in Human Plantar Fascia Tendinopathy.

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.

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

Denervated myofibers and senescent cells are hallmarks of skeletal muscle aging. However, sparse research has examined how resistance training affects these outcomes. We investigated the effects of unilateral leg extensor resistance training (2 days/week for 8 weeks) on denervated myofibers, senescent cells, and associated protein markers in apparently healthy middle-aged participants (MA, 55 ± 8 years old, 17 females, 9 males). We obtained dual-leg 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+), proteins associated with denervation and senescence, and senescence-associated secretory phenotype (SASP) proteins were analyzed from biopsy specimens. Leg extensor peak torque increased after training (p < .001), while VL mCSA trended upward (interaction p = .082). No significant changes were observed for Type I/II fCSAs, NCAM+ myofibers, or senescent (p16+ or p21+) cells, albeit satellite cells increased after training (p = .037). While >90% satellite cells were not p16+ or p21+, most p16+ and p21+ cells were Pax7+ (>90% on average). Training altered 13 out of 46 proteins related to muscle-nerve communication (all upregulated, p < .05) and 10 out of 19 proteins related to cellular senescence (9 upregulated, p < .05). Only 1 out of 17 SASP protein increased with training (IGFBP-3, p = .031). In conclusion, resistance training upregulates proteins associated with muscle-nerve communication in MA participants but does not alter NCAM+ myofibers. Moreover, while training increased senescence-related proteins, this coincided with an increase in satellite cells but not alterations in senescent cell content or SASP proteins. These latter findings suggest shorter term resistance training is an unlikely inducer of cellular senescence in apparently healthy middle-aged participants. However, similar study designs are needed in older and diseased populations before definitive conclusions can be drawn.

Marked irregular myofiber shape is a hallmark of human skeletal muscle ageing and is reversed by heavy resistance training.

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.

Fusion of myofibre branches is a physiological feature of healthy human skeletal muscle regeneration.

BACKGROUND: The occurrence of hyperplasia, through myofibre splitting, remains a widely debated phenomenon. Structural alterations and fibre typing of skeletal muscle fibres, as seen during regeneration and in certain muscle diseases, can be challenging to interpret. Neuromuscular electrical stimulation can induce myofibre necrosis followed by changes in spatial and temporal cellular processes. Thirty days following electrical stimulation, remnants of regeneration can be seen in the myofibre and its basement membrane as the presence of small myofibres and encroachment of sarcolemma and basement membrane (suggestive of myofibre branching/splitting). The purpose of this study was to investigate myofibre branching and fibre type in a systematic manner in human skeletal muscle undergoing adult regenerative myogenesis. METHODS: Electrical stimulation was used to induce myofibre necrosis to the vastus lateralis muscle of one leg in 5 young healthy males. Muscle tissue samples were collected from the stimulated leg 30 days later and from the control leg for comparison. Biopsies were sectioned and stained for dystrophin and laminin to label the sarcolemma and basement membrane, respectively, as well as ATPase, and antibodies against types I and II myosin, and embryonic and neonatal myosin. Myofibre branches were followed through 22 serial Sects. (264 µm). Single fibres and tissue blocks were examined by confocal and electron microscopy, respectively. RESULTS: Regular branching of small myofibre segments was observed (median length 144 µm), most of which were observed to fuse further along the parent fibre. Central nuclei were frequently observed at the point of branching/fusion. The branch commonly presented with a more immature profile (nestin + , neonatal myosin + , disorganised myofilaments) than the parent myofibre, together suggesting fusion of the branch, rather than splitting. Of the 210 regenerating muscle fibres evaluated, 99.5% were type II fibres, indicating preferential damage to type II fibres with our protocol. Furthermore, these fibres demonstrated 7 different stages of "fibre-type" profiles. CONCLUSIONS: By studying the regenerating tissue 30 days later with a range of microscopy techniques, we find that so-called myofibre branching or splitting is more likely to be fusion of myotubes and is therefore explained by incomplete regeneration after a necrosis-inducing event.

Aging is associated with an altered macrophage response during human skeletal muscle regeneration.

Skeletal muscle injury in aged rodents is characterized by an asynchronous infiltration of pro- and anti-inflammatory macrophage waves, leading to improper and incomplete regeneration. It is unclear whether this aberration also occurs in aged human muscle. In this study, we quantified the macrophage responses in a human model of muscle damage and regeneration induced by electrical stimulation in 7 young and 21 older adults. At baseline, total resident macrophage (CD68+/DAPI+) content was not different between young and old subjects, but pro-inflammatory (CD206-/CD68+/DAPI+) macrophage content was lower in the old. Following damage, muscle Infiltration of CD206-/CD68+/DAPI+ macrophages was lower in old relative to young subjects. Further, only the increase in CD206-/CD68+ macrophages correlated with the change in muscle satellite cell content. Our data show that older individuals have a compromised macrophage response during muscle regeneration, pointing to an altered inflammatory response as a potential mechanism for reduced muscle regenerative efficacy in aged humans.

Cardiorespiratory fitness and physical performance after childhood hematopoietic stem cell transplantation: a systematic review and meta-analysis.

The effects of childhood hematopoietic stem cell transplantation (HSCT) on key organs can impair cardiorespiratory fitness, muscle strength, and physical performance. We aimed to provide an overview of childhood HSCT survivors' status on these parameters compared with healthy controls and discuss current insights into clinical risk factors. We performed a systematic search in six scientific databases, including studies published before April 2019 and performed a meta-analysis on cardiorespiratory fitness. Muscle strength and physical performance status were presented narratively. We included ten studies embodying 517 childhood HSCT survivors (mean 17.8 years at follow-up). The meta-analysis (n = 4 studies) showed that childhood HSCT survivors have lower cardiorespiratory fitness compared with healthy controls (Standard mean difference (SMD) -1.32 [95% CI -1-58 to -1.07]; I2 2%, p < 0.00001). Collectively, the studies indicated that childhood HSCT survivors have lower muscle strength (n = 4 studies) and physical performance (n = 3 studies) compared with healthy controls. Childhood HSCT survivors have impaired cardiorespiratory fitness years after ended treatment. Muscle strength and physical performance seem to be impaired, although these measures are insufficiently investigated. Associations between HSCT-specific clinical risk factors and cardiorespiratory fitness, muscle strength, and physical performance are required.

Collagens in primary frozen shoulder: expression of collagen mRNA isoforms in the different phases of the disease.

OBJECTIVES: Primary frozen shoulder (pFS) has three phases that differ in clinical presentation. It is characterized by contracture of the joint capsule. We hypothesized that there is a general upregulation of collagens in pFS, and that this is highest in the first phase of the disease. The aims of this study were to investigate the expression of various collagens and degradation of collagens in patients with primary pFS and relate this to the three phases of the condition. METHODS: From twenty-six patients with pFS and eight control patients with subacromial impingement, biopsies were obtained during shoulder arthroscopy from the middle glenohumeral ligament and the anterior capsule, and mRNA levels for collagens, MMP-2 and -14 and TGF-ß1, - ß2 and -ß3 in the tissue were analysed using real-time PCR. RESULTS: Genes for collagens type I, III, IV, V, VI and XIV, were activated in pFS, and the total mRNA for all collagens was increased (P < 0.05). This upregulation was independent of disease phases in pFS. In addition, MMP-2, MMP-14, TGF-ß1 and TGF-ß3 were upregulated in all phases of the disease. CONCLUSION: There is a general upregulation and an increased degradation of collagens in pFS in all three phases of the disease. This indicates a constantly increased turnover of the fibrotic tissue in the capsule from pFS. The difference in clinical presentation of pFS observed in the three phases of the disease is not primarily a result of variations in collagen production.

Proteomics identifies differences in fibrotic potential of extracellular vesicles from human tendon and muscle fibroblasts.

BACKGROUND: Fibroblasts are the powerhouses responsible for the production and assembly of extracellular matrix (ECM). Their activity needs to be tightly controlled especially within the musculoskeletal system, where changes to ECM composition affect force transmission and mechanical loading that are required for effective movement of the body. Extracellular vesicles (EVs) are a mode of cell-cell communication within and between tissues, which has been largely characterised in cancer. However, it is unclear what the role of healthy fibroblast-derived EVs is during tissue homeostasis. METHODS: Here, we performed proteomic analysis of small EVs derived from primary human muscle and tendon cells to identify the potential functions of healthy fibroblast-derived EVs. RESULTS: Mass spectrometry-based proteomics revealed comprehensive profiles for small EVs released from healthy human fibroblasts from different tissues. We found that fibroblast-derived EVs were more similar than EVs from differentiating myoblasts, but there were significant differences between tendon fibroblast and muscle fibroblast EVs. Small EVs from tendon fibroblasts contained higher levels of proteins that support ECM synthesis, including TGFß1, and muscle fibroblast EVs contained proteins that support myofiber function and components of the skeletal muscle matrix. CONCLUSIONS: Our data demonstrates a marked heterogeneity among healthy fibroblast-derived EVs, indicating shared tasks between EVs of skeletal muscle myoblasts and fibroblasts, whereas tendon fibroblast EVs could play a fibrotic role in human tendon tissue. These findings suggest an important role for EVs in tissue homeostasis of both tendon and skeletal muscle in humans. Video abstract.

Key Components of Human Myofibre Denervation and Neuromuscular Junction Stability are Modulated by Age and Exercise.

The decline in muscle mass and function with age is partly caused by a loss of muscle fibres through denervation. The purpose of this study was to investigate the potential of exercise to influence molecular targets involved in neuromuscular junction (NMJ) stability in healthy elderly individuals. Participants from two studies (one group of 12 young and 12 elderly females and another group of 25 elderly males) performed a unilateral bout of resistance exercise. Muscle biopsies were collected at 4.5 h and up to 7 days post exercise for tissue analysis and cell culture. Molecular targets related to denervation and NMJ stability were analysed by immunohistochemistry and real-time reverse transcription polymerase chain reaction. In addition to a greater presence of denervated fibres, the muscle samples and cultured myotubes from the elderly individuals displayed altered gene expression levels of acetylcholine receptor (AChR) subunits. A single bout of exercise induced general changes in AChR subunit gene expression within the biopsy sampling timeframe, suggesting a sustained plasticity of the NMJ in elderly individuals. These data support the role of exercise in maintaining NMJ stability, even in elderly inactive individuals. Furthermore, the cell culture findings suggest that the transcriptional capacity of satellite cells for AChR subunit genes is negatively affected by ageing.

The influence of direct and indirect fibroblast cell contact on human myogenic cell behavior and gene expression in vitro.

Underpinning skeletal muscle plasticity is the interplay between many cell types, of which fibroblasts are emerging as potent players, both negatively in the development of fibrosis but also positively in stimulating muscle repair through enhancing myogenesis. The mechanisms behind this interaction however remain unknown. To investigate this, waste hamstring muscle tissue was obtained from eight healthy young men undergoing reconstructive anterior cruciate ligament surgery and primary myoblasts and fibroblasts were isolated. Myoblasts were cultured alone or with fibroblasts, either in direct or indirect contact (separated by an insert with a permeable membrane). The myogenesis parameters proliferation, differentiation, and fusion were determined from immunostained cells, while, in replicate samples, gene expression levels of GAPDH, Ki67, Pax7, MyoD, myogenin, myomaker, MHC-Iß, TCF7L2, COL1A1, and p16 were determined by RT-PCR. We found only trends for an influence of skeletal muscle fibroblasts on myogenic cell proliferation and differentiation. While greater mRNA levels of GAPDH, Pax7, MyoD, myogenin, and MHC-Iß were observed in myogenic cells in indirect contact with fibroblasts (insert) when compared with cells cultured alone, a similar effect of an empty insert was also observed. In conclusion we find very little influence of skeletal muscle fibroblasts on myoblasts derived from the same tissue, although it cannot be excluded that a different outcome would be seen under less optimal myogenic growth conditions.NEW & NOTEWORTHY Using passage one primary myoblasts and fibroblasts isolated from human skeletal muscle, we found only a trend for an effect of skeletal muscle fibroblasts on myogenic cell proliferation and differentiation. This is contrary to previous reports and raises the possibility that fibroblasts of different tissue origins exert distinct roles.

Lack of muscle fibre hypertrophy, myonuclear addition, and satellite cell pool expansion with resistance training in 83-94-year-old men and women.

AIMS: To examine satellite cell and myonuclear content in very old (≥83 years) individuals, and the response to heavy resistance training. METHODS: A group of very old men and women (Old, 83-94 years, n = 29) was randomized to 12 weeks of heavy resistance training or untrained controls. A group of young men who did not resistance train (Young, 19-27 years, n = 9) were included for comparison. RESULTS: Compared to young men, prior to training the old men had smaller type II fibres (-38%, P < 0.001), lower satellite cell content (-52%, P < 0.001), smaller myonuclear domain (-30%, P < 0.001), and a trend for lower myonuclear content (-13%, P = 0.09). Old women were significantly different from old men for these parameters, except for satellite cell content. Resistance training had no effect on these parameters in these old men and women. Fibre-size specific analysis showed strong correlations between fibre size and myonuclei per fibre and between fibre size and myonuclear domain for both fibre types (r = 0.94-0.99, P < 0.0001). In contrast, muscle fibre perimeter per myonucleus seemed to be constant across the range in fibre size, particularly in type I fibres (r = -0.31, P = 0.17). CONCLUSIONS: The present data demonstrate that type II fibre size, satellite cell content and myonuclear domain is significantly smaller in very old men compared to young men, while myonuclear content is less affected. These parameters were not improved with heavy resistance training at the most advanced stage of ageing.

Integrated method for quantitative morphometry and oxygen transport modeling in striated muscle.

Identifying structural limitations in O2 transport is primarily restricted by current methods employed to characterize the nature of physiological remodeling. Inadequate resolution or breadth of available data has impaired development of routine diagnostic protocols and effective therapeutic strategies. Understanding O2 transport within striated muscle faces major challenges, most notably in quantifying how well individual fibers are supplied by the microcirculation, which has necessitated exploring tissue O2 supply using theoretical modeling of diffusive exchange. With capillary domains identified as a suitable model for the description of local O2 supply and requiring less computation than numerically calculating the trapping regions that are supplied by each capillary via biophysical transport models, we sought to design a high-throughput method for histological analysis. We present an integrated package that identifies optimal protocols for identification of important input elements, processing of digitized images with semiautomated routines, and incorporation of these data into a mathematical modeling framework with computed output visualized as the tissue partial pressure of O2 (Po2) distribution across a biopsy sample. Worked examples are provided using muscle samples from experiments involving rats and humans. NEW & NOTEWORTHY Progress in quantitative morphometry and analytical modeling has tended to develop independently. Real diagnostic power lies in harnessing both disciplines within one user-friendly package. We present a semiautomated, high-throughput tool for determining muscle phenotype from biopsy material, which also provides anatomically relevant input to quantify tissue oxygenation, in a coherent package not previously available to nonspecialist investigators.

Losartan has no additive effect on the response to heavy-resistance exercise in human elderly skeletal muscle.

Our purpose here was to investigate the potential of blocking the angiotensin II type I receptor (AT1R) on the hypertrophy response of elderly human skeletal muscle to 4 mo of heavy-resistance exercise training. Fifty-eight healthy elderly men (+65 yr) were randomized into three groups, consuming either AT1R blocker (losartan, 100 mg/day) or placebo for 4 mo. Two groups performed resistance training (RT) and were treated with either losartan or placebo, and one group did not train but was treated with losartan. Quadriceps muscle biopsies, MR scans, and strength tests were performed at baseline and after 8 and 16 wk. Biopsies were sectioned for immunohistochemistry to determine the number of satellite cells, capillaries, fiber type distribution, and fiber area. Gene expression levels of myostatin, connective tissue, and myogenic signaling pathways were determined by real-time RT-PCR. Four months of heavy-resistance training led in both training groups to expected improvements in quadriceps (∼3-4%) and vastus lateralis (∼5-6%), cross-sectional area, and type II fiber area (∼10-18%), as well as dynamic (∼13%) and isometric (∼19%) quadriceps peak force, but with absolutely no effect of losartan on these outcomes. Furthermore, no changes were seen in satellite cell number with training, and most gene targets failed to show any changes induced by training or losartan treatment. We conclude that there does not appear to be any effect of AT1R blocking in elderly men during 4 mo of resistance training. Therefore, we do not find any support for using AT1R blockers for promoting muscle adaptation to training in humans. NEW & NOTEWORTHY Animal studies have suggested that blocking angiotensin II type I receptor (AT1R) enhances muscle regeneration and prevents disuse atrophy, but studies in humans are limited. Focusing on hypertrophy, satellite cells, and gene expression, we found that AT1R blocking did not result in any greater responses with 4 mo of resistance training. These results do not support previous findings and question the value of blocking AT1R in the context of preserving aging human muscle.

Skeletal muscle morphology, protein synthesis, and gene expression in Ehlers-Danlos syndrome.

Patients with Ehlers-Danlos syndrome (EDS) are known to have genetically impaired connective tissue and skeletal muscle symptoms in form of pain, fatigue, and cramps; however earlier studies have not been able to link these symptoms to morphological muscle changes. We obtained skeletal muscle biopsies in patients with classic EDS [cEDS; n = 5 (Denmark)+ 8 (The Netherlands)] and vascular EDS (vEDS; n = 3) and analyzed muscle fiber morphology and content (Western blotting and muscle fiber type/area distributions) and muscle mRNA expression and protein synthesis rate (RT-PCR and stable isotope technique). The cEDS patients did not differ from healthy controls (n = 7-11) with regard to muscle fiber type/area, myosin/α-actin ratio, muscle protein synthesis rate, or mRNA expression. In contrast, the vEDS patients demonstrated higher expression of matrix proteins compared with cEDS patients (fibronectin and MMP-2). The cEDS patients had surprisingly normal muscle morphology and protein synthesis, whereas vEDS patients demonstrated higher mRNA expression for extracellular matrix remodeling in skeletal musculature compared with cEDS patients.NEW & NOTEWORTHY This study is the first of its kind to systematically investigate muscle biopsies from Ehlers-Danlos patients, focusing on muscle structure and function. These patients suffer from severe muscle symptoms, but in our study they show surprisingly normal muscle findings, which points toward indirect muscle symptoms originating from the surrounding connective tissue. These findings have basal physiological importance and implications for future physiotherapeutic treatment options for these patients.

Human skeletal muscle fibroblasts stimulate in vitro myogenesis and in vivo muscle regeneration.

KEY POINTS: Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. The extent of cross-talk between fibroblasts, as the source of matrix protein, and satellite cells in humans is unknown. We studied this in human muscle biopsies and cell-culture studies. We observed a strong stimulation of myogenesis by human fibroblasts in cell culture. In biopsies collected 30 days after a muscle injury protocol, fibroblast number increased to four times control levels, where fibroblasts were found to be preferentially located immediately surrounding regenerating muscle fibres. These novel findings indicate an important role for fibroblasts in supporting the regeneration of muscle fibres, potentially through direct stimulation of satellite cell differentiation and fusion, and contribute to understanding of cell-cell cross-talk during physiological and pathological muscle remodelling. ABSTRACT: Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. In addition to the indispensable role satellite cells play in muscle regeneration, there is emerging evidence in rodents for a regulatory influence on fibroblast activity. However, the influence of fibroblasts on satellite cells and muscle regeneration in humans is unknown. The purpose of this study was to investigate this in vitro and during in vivo regeneration in humans. Following a muscle injury protocol in young healthy men (n = 7), the number of fibroblasts (TCF7L2+), satellite cells (Pax7+), differentiating myogenic cells (myogenin+) and regenerating fibres (neonatal/embryonic myosin+) was determined from biopsy cross-sections. Fibroblasts and myogenic precursor cells (MPCs) were also isolated from human skeletal muscle (n = 4) and co-cultured using different cell ratios, with the two cell populations either in direct contact with each other or separated by a permeable membrane. MPC proliferation, differentiation and fusion were assessed from cells stained for BrdU, desmin and myogenin. On biopsy cross-sections, fibroblast number was seen to increase, along with myogenic cell number, by d7 and increase further by d30, where fibroblasts were observed to be preferentially located immediately surrounding regenerating muscle fibres. In vitro, the presence of fibroblasts in direct contact with MPCs was found to moderately stimulate MPC proliferation and strongly stimulate both MPC differentiation and MPC fusion. It thus appears, in humans, that fibroblasts exert a strong positive regulatory influence on MPC activity, in line with observations during in vivo skeletal muscle regeneration.

Activation of satellite cells and the regeneration of human skeletal muscle are expedited by ingestion of nonsteroidal anti-inflammatory medication.

With this study we investigated the role of nonsteroidal anti-inflammatory drugs (NSAIDs) in human skeletal muscle regeneration. Young men ingested NSAID [1200 mg/d ibuprofen (IBU)] or placebo (PLA) daily for 2 wk before and 4 wk after an electrical stimulation-induced injury to the leg extensor muscles of one leg. Muscle biopsies were collected from the vastus lateralis muscles before and after stimulation (2.5 h and 2, 7, and 30 d) and were assessed for satellite cells and regeneration by immunohistochemistry and real-time RT-PCR, and we also measured telomere length. After injury, and compared with PLA, IBU was found to augment the proportion of ActiveNotch1(+) satellite cells at 2 d [IBU, 29 ± 3% vs. PLA, 19 ± 2% (means ± sem)], satellite cell content at 7 d [IBU, 0.16 ± 0.01 vs. PLA, 0.12 ± 0.01 (Pax7(+) cells/fiber)], and to expedite muscle repair at 30 d. The PLA group displayed a greater proportion of embryonic myosin(+) fibers and a residual ∼2-fold increase in mRNA levels of matrix proteins (all P < 0.05). Endomysial collagen was also elevated with PLA at 30 d. Minimum telomere length shortening was not observed. In conclusion, ingestion of NSAID has a potentiating effect on Notch activation of satellite cells and muscle remodeling during large-scale regeneration of injured human skeletal muscle.-Mackey, A. L., Rasmussen, L. K., Kadi, F., Schjerling, P., Helmark, I. C., Ponsot, E., Aagaard, P., Durigan, J. L. Q., Kjaer, M. Activation of satellite cells and the regeneration of human skeletal muscle are expedited by ingestion of nonsteroidal anti-inflammatory medication.

Matters of fiber size and myonuclear domain: Does size matter more than age?

INTRODUCTION: The relationship between fiber size and myonuclear content is poorly understood. METHODS: Biopsy cross-sections from young and old trained and untrained healthy individuals were analyzed for fiber area and myonuclei, and 2 fiber-size-dependent cluster analyses were performed. RESULTS: When comparing fibers of similar size, no effect of training or age was found for myonuclear domain. There was a linear relationship between fiber area and myonuclei per fiber (r = 0.99; P < 0.001) and a non-linear relationship between fiber area and domain (r = 0.97-0.99; P < 0.0001), with a markedly smaller domain in fibers <3,000 µm(2). A higher proportion of type II fibers <3,000 µm(2) was observed in the old subjects. CONCLUSIONS: These findings suggest that age-related reductions in myonuclear domain size could be explained by the greater proportion of small fibers. The data also highlight the usefulness of determining fiber-size-based clusters for gaining mechanistic insight into the relationship between skeletal muscle fiber size and myonuclear content.

Morphological adaptation of muscle collagen and receptor of advanced glycation end product (RAGE) in osteoarthritis patients with 12 weeks of resistance training: influence of anti-inflammatory or glucosamine treatment.

The aim of this study was to investigate the effect of 12-week resistance training on morphological presence of collagen and RAGE (receptor for advanced glycation end products) in skeletal muscle of patients with knee osteoarthritis (OA). Little is known about the influence of exercise on the skeletal muscle matrix that supports joints affected by OA mainly when it is associated with medication taken by OA patients (non-steroid anti-inflammatory drugs (NSAID) and glucosamine). A biopsy was collected from the vastus lateralis muscle in all patients before and after 12-week period of training. The patients (age 55-69 years) were divided into three groups, treated with NSAID, glucosamine or placebo. In addition, the muscle samples were analysed by immunohistochemistry for collagen types, RAGE and capillaries ratio. An increment in immunoreactivity for type IV collagen after the training period was observed in 72 % of all biopsies when compared with their respective baseline samples. Reduced immunoreactivity of collagen type I was observed in all patients treated with glucosamine. A significant increase with training in the amount of RAGE was detected in the placebo group only (p < 0.05). Comparison of post-treatment states indicated significant differences between the placebo and glucosamine group data, demonstrating increased levels in the placebo group (p < 0.05). These findings suggest a basement membrane remodelling in favour of a strengthened extracellular matrix surrounding individual muscle fibres after 12 weeks of resistance training. Glucosamine with training appeared to attenuate RAGE accumulation more than was seen with NSAID or placebo in skeletal muscle of OA patients.

Differentially activated macrophages orchestrate myogenic precursor cell fate during human skeletal muscle regeneration.

Macrophages (MPs) exert either beneficial or deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for adult skeletal muscle repair, notably by acting on myogenic precursor cells. However, these interactions have not been fully characterized. Here, we explored both in vitro and in vivo, in human, the interactions of differentially activated MPs with myogenic precursor cells (MPCs) during adult myogenesis and skeletal muscle regeneration. We showed in vitro that through the differential secretion of cytokines and growth factors, proinflammatory MPs inhibited MPC fusion while anti-inflammatory MPs strongly promoted MPC differentiation by increasing their commitment into differentiated myocytes and the formation of mature myotubes. Furthermore, the in vivo time course of expression of myogenic and MP markers was studied in regenerating human healthy muscle after damage. We observed that regenerating areas containing proliferating MPCs were preferentially associated with MPs expressing proinflammatory markers. In the same muscle, regenerating areas containing differentiating myogenin-positive MPCs were preferentially coupled to MPs harboring anti-inflammatory markers. These data demonstrate for the first time in human that MPs sequentially orchestrate adult myogenesis during regeneration of damaged skeletal muscle. These results support the emerging concept that inflammation, through MP activation, controls stem cell fate and coordinates tissue repair.