Does moderate intensity impact exercise and non-impact exercise induce acute changes in collagen biochemical markers related to osteoarthritis? - An exploratory randomized cross-over trial.

OBJECTIVE: To investigate acute changes in biochemical markers of cartilage turnover in response to moderate intensity exercise with and without joint impact in humans with knee osteoarthritis. DESIGN: We conducted a randomized, cross-over, exploratory clinical study. Twenty subjects with knee osteoarthritis (OA) were randomized, of which twenty completed 30 min of cycling and 15 completed 30 min of running on days 1 week apart. Fasting blood samples were taken before, immediately after and 1, 2, 3, and 24 h after activity was initiated. Midstream spot urine was sampled before and after activity. Serum samples were analyzed for concentrations of fragment of type II collagen degradation, C2M, fragment of type VI collagen degradation, C6M, cartilage oligomeric matrix protein, COMP, marker of type II collagen formation, PRO-C2, and urine for marker of crosslinked type II collagen degradation, CTX-II. To establish a reference, all subjects had similar samples taken during rest on a separate day. Data was analyzed in a restricted maximum likelihood based random effects linear mixed model. RESULTS: C2M trended to increase after cycling compared running (13.49%, 95%CI: -0.36-27.34%) and resting (12.88%, 95%CI: 0.2-25.6%) and the type II collagen formation/degradation ratio switched towards degradation after cycling, but not running. C6M trended to decrease after cycling (-8.1%, 95%CI: -14.8 to -1.4%) and running (-6.8%, 95%CI: -14.16-0.55%). CONCLUSION: In persons with knee OA moderate intensity exercise without joint impact may induce acute changes in circulating levels of biochemical markers reflecting type II and VI collagen degradation.

An anti-inflammatory phenotype in visceral adipose tissue of old lean mice, augmented by exercise.

Visceral adipose tissue is an immunogenic tissue, which turns detrimental during obesity by activation of proinflammatory macrophages. During aging, chronic inflammation increases proportional to visceral adipose tissue (VAT) mass and associates with escalating morbidity and mortality. Here, we utilize a mouse model to investigate the inflammatory status of visceral adipose tissue in lean aging mice and assess the effects of exercise training interventions. We randomized adult (11 months; n = 21) and old (23 months; n = 27) mice to resistance training (RT) or endurance training (ET), or to a sedentary control group (S). Strikingly, we observed an anti-inflammatory phenotype in the old mice, consisting of higher accumulation of M2 macrophages and IL-10 expression, compared to the adult mice. In concordance, old mice also had less VAT mass and smaller adipocytes compared to adult mice. In both age groups, exercise training enhanced the anti-inflammatory phenotype and increased PGC1-α mRNA expression. Intriguingly, the brown adipose tissue marker UCP1 was modestly higher in old mice, while remained unchanged by the intervention. In conclusion, in the absence of obesity, visceral adipose tissue possesses a pronounced anti-inflammatory phenotype during aging which is further enhanced by exercise.

The effect of resistance exercise upon age-related systemic and local skeletal muscle inflammation.

AIM: Chronic inflammation increases with age and is correlated positively to visceral fat mass, but inversely to muscle mass. We investigated the hypothesis that resistance training would increase muscle mass and strength together with a concomitant drop in local and systemic inflammation level independent of any changes in visceral fat tissue in elderly. METHODS: 25 subjects (mean 67, range 62-70 years) were randomized to 1 year of heavy resistance training (HRT) or control (CON), and tested at 0, 4 and 12 months for physical performance, body composition (DXA), vastus lateralis muscle area (MRI) local and systemic inflammation (blood and muscle). In addition, systemic and local muscle immunological responses to acute exercise was determined before and after the training period. RESULTS: Increases in muscle mass (≈2%, p < 0.05), vastus lateralis area (≈9%. P < 0.05), isometric (≈15%) and dynamic (≈15%) muscle strength (p < 0.05) were found in the HRT group after 12 months training. HRT did not alter overall or visceral fat mass (p > 0.05). Blood C-Reactive Protein declined over time in both groups (p < 0.05), whereas muscle inflammation markers were unchanged to 1 year of HRT. Acute exercise increased plasma IL-6 and FGF-19 (p < 0.05), decreased FGF-21 (p < 0.05) and CCL-20 (p < 0.05), and increased GDNF in muscle (p < 0.001) similarly before and after 1 year in both groups. CONCLUSION: Long term resistance training increased muscle strength and improved muscle mass, but did not alter visceral fat mass and did not show any specific effect upon resting or exercise induced markers of inflammation.

Remodeling of muscle fibers approaching the human myotendinous junction.

The myotendinous junction (MTJ) is at high risk of strain injuries, due to high amounts of energy that is transferred through this structure. The risk of strain injury is significantly reduced by heavy resistance training (HRT), indicating a remodeling capacity of MTJ. We investigated the degree of remodeling of muscle fibers near the human MTJ. In 8 individuals, samples were taken from the semitendinosus and gracilis MTJ and they were stained immunohistochemically for myonuclei (DAPI), fibroblasts (TCF7L2), and satellite cells (CD56). A high portion of the muscle fibers adjacent to the MTJ contained a centrally located myonucleus (47 ± 8%, mean ± SD) and half of the muscle fibers were CD56 positive. The number of satellite cells and fibroblasts were not higher than what has previously been reported from muscle bellies. The immunohistochemical findings suggest that the rate of remodeling of muscle fibers near the MTJ is very high. The finding that there was no increased number of satellite cells and fibroblasts could be explained as a dynamic phenomenon. The effect of HRT should be evaluated in a randomized setting.

Skeletal muscle morphology and regulatory signalling in endurance-trained and sedentary individuals: The influence of ageing.

Muscle mass in humans is inversely associated with circulating levels of inflammatory cytokines, but the interaction between ageing and training on muscle composition and the intra-muscular signalling behind inflammation and contractile protein synthesis and degradation is unknown. We studied 15 healthy life-long endurance runners, 12 age-matched untrained controls, 10 young trained and 12 young untrained individuals. Thigh muscle composition was investigated by magnetic resonance imaging (MRI), where non-contractile intramuscular tissue (NCIT) area (fat and connective tissue) was found to be greater in older but lower in trained individuals. Subcutaneous adipose tissue was also lower in trained individuals but was not affected by age. In vastus lateralis biopsies, no influence of age or training was found on levels of endomysial collagen, determined by Sirius Red and Collagen III staining, whereas perimysial organisation tended to be more complex in older individuals. No clear difference with training was seen on intramuscular inflammatory signalling, whereas lower protein levels of NFkB subunits p105, p50 and p65 were observed with ageing. Gene expression of IL6 and TNFα was not different between groups, while IL1-receptor and TNFα-receptor1 levels were lower with age. Myostatin mRNA was lower in older and trained groups, while expression of MuRF1 was lower in trained individuals and FoxO3 expression was greater in aged groups. The association of increased muscle NCIT with age-associated muscle loss in humans is not accompanied by any major alterations in intramuscular signalling for inflammation, but rather by direct regulatory factors for protein synthesis and proteolysis in skeletal muscle.

Composition and adaptation of human myotendinous junction and neighboring muscle fibers to heavy resistance training.

The myotendinous junction (MTJ) is a common site of strain injury and yet understanding of its composition and ability to adapt to loading is poor. The main aims of this study were to determine the profile of selected collagens and macrophage density in human MTJ and adjoining muscle fibers, and to investigate whether heavy exercise loading would alter this profile. Fifteen individuals scheduled for anterior cruciate ligament repair surgery were randomized into three groups: control, acute or 4 weeks heavy resistance training. MTJ samples were collected from the semitendinosus and gracilis muscles and were sectioned and stained immunohistochemically for collagen types I, III, VI, XII, XIV, XXII, Tenascin-C and CD68. Macrophage density and distribution was evaluated and the amount of each collagen type in muscle and MTJ was graded. Collagen XXII was observed solely at the MTJ, while all other collagens were abundant at the MTJ and in muscle perimysium or endomysium. The endomysial content of collagen XIV, macrophages and Tenascin-C increased following 4 weeks of training. These findings illustrate the heterogeneity of collagen type composition of human MTJ. The increase in collagen XIV following 4 weeks of training may reflect a training-induced protection against strain injuries in this region.

The human myotendinous junction: an ultrastructural and 3D analysis study.

The myotendinous junction (MTJ) is a specialized structure in the musculotendinous system, where force is transmitted from muscle to tendon. Animal models have shown that the MTJ takes form of tendon finger-like processes merging with muscle tissue. The human MTJ is largely unknown and has never been described in three dimensions (3D). The aim of this study was to describe the ultrastructure of the human MTJ and render 3D reconstructions. Fourteen subjects (age 25 ± 3 years) with isolated injury of the anterior cruciate ligament (ACL), scheduled for reconstruction with a semitendinosus/gracilis graft were included. Semitendinosus and gracilis tendons were stripped as grafts for the ACL reconstruction. The MTJ was isolated from the grafts and prepared for transmission electron microscopy (TEM) and focused ion beam/scanning electron microscopy. It was possible to isolate recognizable MTJ tissue from all 14 patients. TEM images displayed similarities to observations in animals: Sarcolemmal evaginations observed as finger-like processes from the tendon and endomysium surrounding the muscle fibers, with myofilaments extending from the final Z-line of the muscle fiber merging with the tendon tissue. The 3D reconstruction revealed that tendon made ridge-like protrusions, which interdigitiated with groove-like indentations in the muscle cell.

Differential satellite cell density of type I and II fibres with lifelong endurance running in old men.

AIM: To investigate the influence of lifelong endurance running on the satellite cell pool of type I and type II fibres in healthy human skeletal muscle. METHODS: Muscle biopsies were collected from 15 healthy old trained men (O-Tr) who had been running 43 ± 16 (mean ± SD) kilometres a week for 28 ± 9 years. Twelve age-matched untrained men (O-Un) and a group of young trained and young untrained men were recruited for comparison. Frozen sections were immunohistochemically stained for Pax7, type I myosin and laminin, from which fibre area, the number of satellite cells, and the relationship between these variables were determined. RESULTS: In O-Un and O-Tr, type II fibres were smaller and contained fewer satellite cells than type I fibres. However, when expressed relative to fibre area, the difference in satellite cell content between fibre types was eliminated in O-Tr, but not O-Un. A strong positive relationship between fibre size and satellite cell content was detected in trained individuals. In line with a history of myofibre repair, a greater number of fibres with centrally located myonuclei were detected in O-Tr. CONCLUSION: Lifelong endurance training (i) does not deplete the satellite cell pool and (ii) is associated with a similar density of satellite cells in type I and II fibres despite a failure to preserve the equal fibre type distribution of satellite cells observed in young individuals. Taken together, these data reveal a differential regulation of satellite cell content between fibre types, in young and old healthy men with dramatically different training histories.

Life-long endurance exercise in humans: circulating levels of inflammatory markers and leg muscle size.

Human aging is associated with a loss of skeletal muscle and an increase in circulating inflammatory markers. It is unknown whether endurance training (Tr) can prevent these changes. Therefore we studied 15 old trained (O-Tr) healthy males and, for comparison, 12 old untrained (O-Un), 10 Young-Tr (Y-Tr) and 12 Young-Un (Y-Un). Quadriceps size, VO2 peak, CRP, IL-6, TNF-α and its receptors, suPAR, lipid profile, leucocytes and glucose homeostasis were measured. Tr was associated with an improved insulin profile (p<0.05), and lower leucocyte (p<0.05) and triglyceride levels (p<0.05), independent of age. Aging was associated with poorer glucose control (p<0.05), independent of training. The age-related changes in waist circumference, VO2 peak, cholesterol, LDL, leg muscle size, CRP and IL-6 were counteracted by physical activity (p<0.05). A significant increase in suPAR with age was observed (p<0.05). Most importantly, life-long endurance exercise was associated with a lower level of the inflammatory markers CRP and IL-6 (p<0.05), and with a greater thigh muscle area (p<0.05), compared to age-matched untrained counterparts. These findings in a limited group of individuals suggest that regular physical endurance activity may play a role in reducing some markers of systemic inflammation, even within the normal range, and in maintaining muscle mass with aging.

Automated image segmentation of haematoxylin and eosin stained skeletal muscle cross-sections.

The ability to accurately and efficiently quantify muscle morphology is essential to determine the physiological relevance of a variety of muscle conditions including growth, atrophy and repair. There is agreement across the muscle biology community that important morphological characteristics of muscle fibres, such as cross-sectional area, are critical factors that determine the health and function (e.g. quality) of the muscle. However, at this time, quantification of muscle characteristics, especially from haematoxylin and eosin stained slides, is still a manual or semi-automatic process. This procedure is labour-intensive and time-consuming. In this paper, we have developed and validated an automatic image segmentation algorithm that is not only efficient but also accurate. Our proposed automatic segmentation algorithm for haematoxylin and eosin stained skeletal muscle cross-sections consists of two major steps: (1) A learning-based seed detection method to find the geometric centres of the muscle fibres, and (2) a colour gradient repulsive balloon snake deformable model that adopts colour gradient in Luv colour space. Automatic quantification of muscle fibre cross-sectional areas using the proposed method is accurate and efficient, providing a powerful automatic quantification tool that can increase sensitivity, objectivity and efficiency in measuring the morphometric features of the haematoxylin and eosin stained muscle cross-sections.

Ageing is associated with diminished muscle re-growth and myogenic precursor cell expansion early after immobility-induced atrophy in human skeletal muscle.

Recovery of skeletal muscle mass from immobilisation-induced atrophy is faster in young than older individuals, yet the cellular mechanisms remain unknown. We examined the cellular and molecular regulation of muscle recovery in young and older human subjects subsequent to 2 weeks of immobility-induced muscle atrophy. Retraining consisted of 4 weeks of supervised resistive exercise in 9 older (OM: mean age) 67.3, range 61-74 yrs) and 11 young (YM: mean age 24.4, range 21-30 yrs) males. Measures of myofibre area (MFA), Pax7-positive satellite cells (SCs) associated with type I and type II muscle fibres, as well as gene expression analysis of key growth and transcription factors associated with local skeletal muscle milieu, were performed after 2 weeks immobility (Imm) and following 3 days (+3d) and 4 weeks (+4wks) of retraining. OM demonstrated no detectable gains in MFA (vastus lateralis muscle) and no increases in number of Pax7-positive SCs following 4wks retraining, whereas YM increased their MFA (P < 0.05), number of Pax7-positive cells, and had more Pax7-positive cells per type II fibre than OM at +3d and +4wks (P < 0.05). No age-related differences were observed in mRNA expression of IGF-1Ea, MGF, MyoD1 and HGF with retraining, whereas myostatin expression levels were more down-regulated in YM compared to OM at +3d (P < 0.05). In conclusion, the diminished muscle re-growth after immobilisation in elderly humans was associated with a lesser response in satellite cell proliferation in combination with an age-specific regulation of myostatin. In contrast, expression of local growth factors did not seem to explain the age-related difference in muscle mass recovery.

The expression of heat shock protein in human skeletal muscle: effects of muscle fibre phenotype and training background.

AIM: Exercise-induced adaptations of skeletal muscle are related to training mode and can be muscle fibre type specific. This study aimed to investigate heat shock protein expression in type I and type II muscle fibres in resting skeletal muscle of subjects with different training backgrounds. METHODS: Three groups of subjects were included: healthy active not engaged in any training programme (ACT, n = 12), resistance trained (RES, n = 6) and endurance trained (END, n = 8). Biopsies were obtained from vastus lateralis, and immunohistochemistry was performed using monoclonal antibodies against myosin heavy chain I and IIA, αB-crystallin, HSP27, HSP60 and HSP70. RESULTS: In ACT and RES, but not in END, a fibre type-specific expression with higher staining intensity in type I than type II fibres was seen for αB-crystallin. The opposite (II > I) was found for HSP27 in subjects from ACT (6 of 12 subjects) and RES (3 of 6), whereas all subjects from END displayed uniform staining. HSP60 showed no fibre-specific expression. HSP70 displayed a fibre-specific expression pattern (I > II) in ACT (4 of 12), but not in END or RES. CONCLUSION: This study shows that the level of expression of the different HSPs in human skeletal muscle is influenced by muscle fibre phenotype. The fibre type-specific expression of HSP70 is influenced by resistance and endurance training, whereas those of αB-crystallin and HSP27 is influenced only by endurance training, suggesting the existence of a training-modality-specific action on the adaptive processes including heat shock proteins in human skeletal muscle.

GH/IGF-I axis and matrix adaptation of the musculotendinous tissue to exercise in humans.

Exercise is not only associated with adaptive responses within skeletal muscle fibers but also with induction of collagen synthesis both in muscle and adjacent connective tissue. Additionally, exercise and training leads to activation of the systemic growth hormone/insulin-like growth factor I axis (GH/IGF-I), as well as increased local IGF-I expression. Studies in humans with pathologically high levels of GH/IGF-I, and in healthy humans who receive either weeks of GH administration or acute injection of IGF-I into connective tissue, demonstrate increased expression and synthesis of collagen in muscle and tendon. These observations support a stimulatory effect of GH/IGF-I on the connective tissue in muscle and tendon, which appears far more potent than the effect on contractile proteins of skeletal muscle. However, GH/IGF-I may play an additional role in skeletal muscle by regulation of stem cells (satellite cells), as increased satellite cell numbers are found in human muscle with increased GH/IGF-I levels, despite no change in myofibrillar protein synthesis. Although advanced age is associated with both a reduction in the GH/IGF-I axis activity, and in skeletal muscle mass (sarcopenia) as well as in tendon connective tissue, there is no direct proof linking age-related changes in the musculotendinous tissue to an impaired GH/IGF-I axis.

Rehabilitation of muscle after injury - the role of anti-inflammatory drugs.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely consumed among athletes worldwide in relation to muscle injury and soreness. This review aims to provide an overview of studies investigating their effects on skeletal muscle, in particular the repair processes in injured muscle. Muscle injury occurs in diverse situations and the nature of muscle injuries varies significantly, complicating extrapolations between experimental models and "real life." Classical muscle strain injuries occur at the interphase between the muscle fibers and connective tissue, most often in the myotendinuous junction, whereas contusion or overload injury can damage both myofibers and intramuscular connective tissue. The role of NSAIDs in muscle repair is complicated by differences in injury models used, variables evaluated, and time point(s) selected for evaluations. While the temporal pattern of the influence of NSAIDs on muscle repair is difficult to settle on, it appears that a potential beneficial effect of NSAIDs in the early phase after injury is not maintained in the long term, or is even negated by a long-term repair deficit. At the cellular level, evidence exists for a negative influence of NSAIDs on the muscle stem cell population (satellite cells). At a structural level, it is known that muscle connective tissue undergoes significant remodeling during muscle regeneration, but the potential of NSAID exposure to alter this response in humans needs investigation.

Structural, biochemical, cellular, and functional changes in skeletal muscle extracellular matrix with aging.

The extracellular matrix (ECM) of skeletal muscle is critical for force transmission and for the passive elastic response of skeletal muscle. Structural, biochemical, cellular, and functional changes in skeletal muscle ECM contribute to the deterioration in muscle mechanical properties with aging. Structural changes include an increase in the collagen concentration, a change in the elastic fiber system, and an increase in fat infiltration of skeletal muscle. Biochemical changes include a decreased turnover of collagen with potential accumulation of enzymatically mediated collagen cross-links and a buildup of advanced glycation end-product cross-links. Altered mechanotransduction, poorer activation of satellite cells, poorer chemotactic and delayed inflammatory responses, and a change in modulators of the ECM are important cellular changes. It is possible that the structural and biochemical changes in skeletal muscle ECM contribute to the increased stiffness and impairment in force generated by the contracting muscle fibers seen with aging. The cellular interactions provide and potentially coordinate an adaptation to mechanical loading and ensure successful regeneration after muscle injury. Some of the changes in skeletal muscle ECM with aging may be preventable with resistance or weight training, but it is clear that more human studies are needed on the topic.

Myogenic response of human skeletal muscle to 12 weeks of resistance training at light loading intensity.

There is strong evidence for enhanced numbers of satellite cells with heavy resistance training. The satellite cell response to very light muscle loading is, however, unknown. We, therefore, designed a 12-week training protocol where volunteers trained one leg with a high load (H) and the other leg with a light load (L). Twelve young healthy men [mean age 25 ± 3 standard deviation (SD) years] volunteered for the study. Muscle biopsies were collected from the m. vastus lateralis of both legs before and after the training period and satellite cells were visualized by CD56 immunohistochemistry. A significant main effect of time was observed (P<0.001) for the number of CD56+ cells per fiber (L: from 0.11 ± 0.02 to 0.13 ± 0.03; H: from 0.12 ± 0.03 to 0.15 ± 0.05, mean ± SD). The finding that 12 weeks of training skeletal muscle even with very light loads can induce an increase in the number of satellite cells reveals a new aspect of myogenic precursor cell activation and suggests that satellite cells may play a role in skeletal muscle adaptation over a broad physiological range.

Local NSAID infusion inhibits satellite cell proliferation in human skeletal muscle after eccentric exercise.

Despite the widespread consumption of nonsteroidal anti-inflammatory drugs (NSAIDs), the influence of these drugs on muscle satellite cells is not fully understood. The aim of the present study was to investigate the effect of a local NSAID infusion on satellite cells after unaccustomed eccentric exercise in vivo in human skeletal muscle. Eight young healthy males performed 200 maximal eccentric contractions with each leg. An NSAID was infused via a microdialysis catheter into the vastus lateralis muscle of one leg (NSAID leg) before, during, and for 4.5 h after exercise, with the other leg working as a control (unblocked leg). Muscle biopsies were collected before and 8 days after exercise. Changes in satellite cells and inflammatory cell numbers were investigated by immunohistochemistry. Satellite cells were identified using antibodies against neural cell adhesion molecule and Pax7. The number of Pax7(+) cells per myofiber was increased by 96% on day 8 after exercise in the unblocked leg (0.14 +/- 0.04, mean +/- SE) compared with the prevalue (0.07 +/- 0.02, P < 0.05), whereas the number of Pax7(+) cells was unchanged in the leg muscles exposed to the NSAID (0.07 +/- 0.01). The number of inflammatory cells (CD68(+) or CD16(+) cells) was not significantly increased in either of the legs 8 days after exercise and was unaffected by the NSAID. The main finding in the present study was that the NSAID infusion for 7.5 h during the exercise day suppressed the exercise-induced increase in the number of satellite cells 8 days after exercise. These results suggest that NSAIDs negatively affect satellite cell activity after unaccustomed eccentric exercise.

Immunohistochemical changes in the expression of HSP27 in exercised human vastus lateralis muscle.

AIM: The role of HSP27 in the adaptive process of skeletal muscle to exercise, especially in humans, is not well understood. The objective of this study was to investigate immunohistochemical changes in HSP27 expression in human vastus lateralis muscle following resistance and endurance exercises. METHODS: Two different exercise protocols were used: (1) one-leg ergometer cycling (EC, n = 6) consisting of two 30-min bouts at 40% and 75% of peak oxygen uptake, respectively, and (2) leg extension resistance exercise (RE, n = 9) including 10 sets of eight repetitions at a load corresponding to 70% of one maximal repetition (1RM). Immunohistochemistry using specific monoclonal antibodies was used to determine the location of HSP27 protein in muscle biopsies from human vastus lateralis. RESULTS: Our results show that RE, but not EC, induced a significant appearance of scattered accumulations of HSP27 protein in muscle fibres from five of nine subjects. The number of fibres with accumulation of HSP27 in RE ranged from 0% to 32% with a mean of 6.3% of the total number of fibres. CONCLUSION: We conclude that this rapid HSP27 protein relocation after RE is an important player in the cellular remodelling of human muscle fibres in response to exercise involving high-force contractions, but not in response to endurance exercises.