Katja M. Heinemeier

Expression of collagen and related growth factors in rat tendon and skeletal muscle in response to specific contraction types

Acute exercise induces collagen synthesis in both tendon and muscle, indicating an adaptive response in the connective tissue of the muscle–tendon unit. However, the mechanisms of this adaptation, potentially involving collagen‐inducing growth factors (such as transforming growth factor‐β‐1 (TGF‐β‐1)), as well as enzymes related to collagen processing, are not clear. Furthermore, possible differential effects of specific contraction types on collagen regulation have not been investigated. Female Sprague–Dawley rats were subjected to 4 days of concentric, eccentric or isometric training (n= 7–9 per group) of the medial gastrocnemius, by stimulation of the sciatic nerve. RNA was extracted from medial gastrocnemius and Achilles tendon tissue 24 h after the last training bout, and mRNA levels for collagens I and III, TGF‐β‐1, connective tissue growth factor (CTGF), lysyl oxidase (LOX), metalloproteinases (MMP‐2 and ‐9) and their inhibitors (TIMP‐1 and 2) were measured by Northern blotting and/or real‐time PCR. In tendon, expression of TGF‐β‐1 and collagens I and III (but not CTGF) increased in response to all types of training. Similarly, enzymes/factors involved in collagen processing were induced in tendon, especially LOX (up to 37‐fold), which could indicate a loading‐induced increase in cross‐linking of tendon collagen. In skeletal muscle, a similar regulation of gene expression was observed, but in contrast to the tendon response, the effect of eccentric training was significantly greater than the effect of concentric training on the expression of several transcripts. In conclusion, the study supports an involvement of TGF‐β‐1 in loading‐induced collagen synthesis in the muscle–tendon unit and importantly, it indicates that muscle tissue is more sensitive than tendon to the specific mechanical stimulus.

From mechanical loading to collagen synthesis, structural changes and function in human tendon

The adaptive response of connective tissue to loading requires increased synthesis and turnover of matrix proteins, with special emphasis on collagen. Collagen formation and degradation in the tendon increases with both acute and chronic loading, and data suggest that a gender difference exists, in that females respond less than males with regard to an increase in collagen formation after exercise. It is suggested that estrogen may contribute toward a diminished collagen synthesis response in females. Conversely, the stimulation of collagen synthesis by other growth factors can be shown in both animal and human models where insulin‐like growth factor 1 (IGF‐I) and transforming growth factor‐β‐1 (TGF‐β‐1) expression increases to accompany or precede an increase in procollagen expression and collagen synthesis. In humans, it can be demonstrated that an increase in the interstitial concentration of TGF‐β, PGE2, IGF‐I plus its binding proteins and interleukin‐6 takes place after exercise. The increase in IGF‐I expression in tendon includes the isoform that has so far been thought only to exist in skeletal muscle (mechano growth factor). The increase in IGF‐I and procollagen expression showed a similar response whether the tendon was stimulated by concentric, isometric or eccentric muscle contraction, suggesting that strain rather that stress/torque determines the collagen‐synthesis stimulating response seen with exercise. The adaptation time to chronic loading is longer in tendon tissue compared with contractile elements of skeletal muscle or the heart, and only with very prolonged loading are significant changes in gross dimensions of the tendon observed, suggesting that habitual loading is associated with a robust change in the size and mechanical properties of human tendons. An intimate interplay between mechanical signalling and biochemical changes in the matrix is needed in tendon, such that chemical changes can be converted into adaptations in the morphology, structure and material properties.

Extracellular matrix adaptation of tendon and skeletal muscle to exercise.

The extracellular matrix (ECM) of connective tissues enables linking to other tissues, and plays a key role in force transmission and tissue structure maintenance in tendons, ligaments, bone and muscle. ECM turnover is influenced by physical activity, and both collagen synthesis and metalloprotease activity increase with mechanical loading. This can be shown by determining propeptide and proteinase activity by microdialysis, as well as by verifying the incorporation of infused stable isotope amino acids in biopsies. Local tissue expression and release of growth factors for ECM such as IGF‐1, TGF‐beta and IL‐6 is enhanced following exercise. For tendons, metabolic activity (e.g. detected by positron emission tomography scanning), circulatory responses (e.g. as measured by near‐infrared spectroscopy and dye dilution) and collagen turnover are markedly increased after exercise. Tendon blood flow is regulated by cyclooxygenase‐2 (COX‐2)‐mediated pathways, and glucose uptake is regulated by specific pathways in tendons that differ from those in skeletal muscle. Chronic loading in the form of physical training leads both to increased collagen turnover as well as to some degree of net collagen synthesis. These changes modify the mechanical properties and the viscoelastic characteristics of the tissue, decrease its stress‐susceptibility and probably make it more load‐resistant. The mechanical properties of tendon fascicles vary within a given human tendon, and even show gender differences. The latter is supported by findings of gender‐related differences in the activation of collagen synthesis with exercise. These findings may provide the basis for understanding tissue overloading and injury in both tendons and skeletal muscle.

Growth hormone stimulates the collagen synthesis in human tendon and skeletal muscle without affecting myofibrillar protein synthesis

In skeletal muscle and tendon the extracellular matrix confers important tensile properties and is crucially important for tissue regeneration after injury. Musculoskeletal tissue adaptation is influenced by mechanical loading, which modulates the availability of growth factors, including growth hormone (GH) and insulin‐like growth factor‐I (IGF‐I), which may be of key importance. To test the hypothesis that GH promotes matrix collagen synthesis in musculotendinous tissue, we investigated the effects of 14 day administration of 33–50 μg kg−1 day−1 recombinant human GH (rhGH) in healthy young individuals. rhGH administration caused an increase in serum GH, serum IGF‐I, and IGF‐I mRNA expression in tendon and muscle. Tendon collagen I mRNA expression and tendon collagen protein synthesis increased by 3.9‐fold and 1.3‐fold, respectively (P < 0.01 and P= 0.02), and muscle collagen I mRNA expression and muscle collagen protein synthesis increased by 2.3‐fold and 5.8‐fold, respectively (P < 0.01 and P= 0.06). Myofibrillar protein synthesis was unaffected by elevation of GH and IGF‐I. Moderate exercise did not enhance the effects of GH manipulation. Thus, increased GH availability stimulates matrix collagen synthesis in skeletal muscle and tendon, but without any effect upon myofibrillar protein synthesis. The results suggest that GH is more important in strengthening the matrix tissue than for muscle cell hypertrophy in adult human musculotendinous tissue.

Lack of tissue renewal in human adult Achilles tendon is revealed by nuclear bomb 14C

Tendons are often injured and heal poorly. Whether this is caused by a slow tissue turnover is unknown, since existing data provide diverging estimates of tendon protein half‐life that range from 2 mo to 200 yr. With the purpose of determining life‐long turnover of human tendon tissue, we used the 14C bomb‐pulse method. This method takes advantage of the dramatic increase in atmospheric levels of 14C, produced by nuclear bomb tests in 1955–1963, which is reflected in all living organisms. Levels of 14C were measured in 28 forensic samples of Achilles tendon core and 4 skeletal muscle samples (donor birth years 1945–1983) with accelerator mass spectrometry (AMS) and compared to known atmospheric levels to estimate tissue turnover. We found that Achilles tendon tissue retained levels of 14C corresponding to atmospheric levels several decades before tissue sampling, demonstrating a very limited tissue turnover. The tendon concentrations of 14C approximately reflected the atmospheric levels present during the first 17 yr of life, indicating that the tendon core is formed during height growth and is essentially not renewed thereafter. In contrast, 14C levels in muscle indicated continuous turnover. Our observation provides a fundamental premise for understanding tendon function and pathology, and likely explains the poor regenerative capacity of tendon tissue.—Heinemeier, K. M., Schjerling, P., Heinemeier, J., Magnusson, S. P., Kjaer, M. Lack of tissue renewal in human adult Achilles tendon is revealed by nuclear bomb 14C. FASEB J. 27, 2074–2079 (2013). www.fasebj.org

Effect of unloading followed by reloading on expression of collagen and related growth factors in rat tendon and muscle

Tendon tissue and the extracellular matrix of skeletal muscle respond to mechanical loading by increased collagen expression and synthesis. This response is likely a secondary effect of a mechanically induced expression of growth factors, including transforming growth factor-beta1 (TGF-beta1) and insulin-like growth factor-I (IGF-I). It is not known whether unloading of tendon tissue can reduce the expression of collagen and collagen-inducing growth factors. Furthermore, the coordinated response of tendon and muscle tissue to disuse, followed by reloading, is unclear. Female Sprague-Dawley rats were subjected to hindlimb suspension (HS) for 7 or 14 days, followed by 2, 4, 8, or 16 days of reload (RL) (n = 8 in each group). Age-matched controls were included for day 0, day 14 HS, and day 16 RL (n = 8). mRNA expression levels for collagen I (COL1A1), collagen III (COL3A1), TGF-beta1, connective tissue growth factor (CTGF), myostatin, and IGF-I isoforms were measured by real-time RT-PCR in Achilles tendon and soleus muscle. The tendon mass was unchanged, while the muscle mass was reduced by 50% after HS (P < 0.05) and returned to control levels during RL. Collagen I and III, TGF-beta1, and CTGF mRNA levels were unaltered by HS, although collagen III tended to decrease in muscle at day 7 HS. IGF-I isoforms were significantly induced in tendon after 7 days of HS (P < 0.001), and mechanogrowth factor increased in muscle at day 14 HS (P < 0.05). Reload increased muscle collagen I and III mRNA (>10-fold) (P < 0.001) and growth factor expression (P < 0.05), while the tendon response was limited to a moderate induction of collagen expression (2-fold) (P < 0.05). Unloading of tendon and muscle tissue did not reduce expression of collagen and collagen-inducing growth factors, indicating that the response to unloading is not opposite that of loading. Furthermore, the tendon response was clearly different and less pronounced than the muscle tissue response.

Aging affects the transcriptional regulation of human skeletal muscle disuse atrophy.

Important insights concerning the molecular basis of skeletal muscle disuse-atrophy and aging related muscle loss have been obtained in cell culture and animal models, but these regulatory signaling pathways have not previously been studied in aging human muscle. In the present study, muscle atrophy was induced by immobilization in healthy old and young individuals to study the time-course and transcriptional factors underlying human skeletal muscle atrophy. The results reveal that irrespectively of age, mRNA expression levels of MuRF-1 and Atrogin-1 increased in the very initial phase (2–4 days) of human disuse-muscle atrophy along with a marked reduction in PGC-1α and PGC-1β (1–4 days) and a ∼10% decrease in myofiber size (4 days). Further, an age-specific decrease in Akt and S6 phosphorylation was observed in young muscle within the first days (1–4 days) of immobilization. In contrast, Akt phosphorylation was unchanged in old muscle after 2 days and increased after 4 days of immobilization. Further, an age-specific down-regulation of MuRF-1 and Atrogin-1 expression levels was observed following 2 weeks of immobilization, along with a slowing atrophy response in aged skeletal muscle. Neither the immediate loss of muscle mass, nor the subsequent age-differentiated signaling responses could be explained by changes in inflammatory mediators, apoptosis markers or autophagy indicators. Collectively, these findings indicate that the time-course and regulation of human skeletal muscle atrophy is age dependent, leading to an attenuated loss in aging skeletal muscle when exposed to longer periods of immobility-induced disuse.

Expression of extracellular matrix components and related growth factors in human tendon and muscle after acute exercise.

Acute kicking exercise induces collagen synthesis in both tendon and muscle in humans, but it is not known if this relates to increased collagen transcription and if other matrix genes are regulated. Young men performed 1 h of one‐leg kicking at 67% of max workload. Biopsies were taken from the patellar tendon and vastus lateralis muscle of each leg at 2 (n = 10), 6 (n = 11), or 26 h (n = 10) after exercise. Levels of messenger ribonucleic acid mRNA for collagens, noncollagenous matrix proteins, and growth factors were measured with real‐time reverse transcription polymerase chain reaction. In tendon, gene expression was unchanged except for a decrease in insulin‐like growth factor‐IEa (IGF‐IEa; P < 0.05). In muscle, collagen expression was not significantly altered, while levels of connective tissue growth factor (CTGF), IGF‐IEa, transforming growth factor‐β1, ‐2 (TGF‐β), and the TGF‐β receptor II mRNA were increased (P < 0.05). Matrix components tenascin‐C, fibronectin, and decorin were also induced in loaded muscle (P < 0.05), while fibromodulin was unaffected. In conclusion, the relatively robust changes in matrix components and related growth factors in muscle indicate a stimulation of extracellular matrix even with moderate exercise. However, in tendon tissue, this exercise model does not appear to induce any anabolic response on the transcriptional level.

Dynamic Adaptation of Tendon and Muscle Connective Tissue to Mechanical Loading

The connective tissue of tendon and skeletal muscle is a crucial structure for force transmission. A dynamic adaptive capacity of these tissues in healthy individuals is evident from reports of altered gene expression and protein levels of the fibrillar and network-forming collagens, when subjected to mechanical loading. While it appears that the fibroblast is a key player in sensing and responding to loading, the issue of how these signals are converted into changed gene expression is not fully understood. It is clear, however, that the loading-induced response involves a variety of growth factors, in particular TGF-β-1, and matrix remodelling enzymes such as MMP-2. Furthermore, it is under hormonal influence. In skeletal muscle, the extracellular matrix demonstrates its potential for cross-talk by regulating the activity of cells with which it is in contact. Taken together, the studies highlighted in this article provide strong evidence for the highly adaptable nature of connective tissue in muscle and tendon.

Local NSAID infusion does not affect protein synthesis and gene expression in human muscle after eccentric exercise

Unaccustomed exercise leads to satellite cell proliferation and increased skeletal muscle protein turnover. Several growth factors and cytokines may be involved in the adaptive responses. Non‐steroidal anti‐inflammatory drugs (NSAIDs) negatively affect muscle regeneration and adaptation in animal models, and inhibit the exercise‐induced satellite cell proliferation and protein synthesis in humans. However, the cellular mechanisms eliciting these responses remain unknown. Eight healthy male volunteers performed 200 maximal eccentric contractions with each leg. To block prostaglandin synthesis locally in the skeletal muscle, indomethacin (NSAID) was infused for 7.5 h via microdialysis catheters into m. vastus lateralis of one leg. Protein synthesis was determined by the incorporation of 1,2‐13C2 leucine into muscle protein from 24 to 28 h post‐exercise. Furthermore, mRNA expression of selected genes was measured in muscle biopsies (5 h and 8 days post‐exercise) by real‐time reverse transcriptase PCR. Myofibrillar and collagen protein synthesis were unaffected by the local NSAID infusion. Five hours post‐exercise, the mRNA expression of cyclooxygenase‐2 (COX2) was sixfold higher in the NSAID leg (P=0.016) compared with the unblocked leg. The expression of growth factors and matrix‐related genes were unaffected by NSAID. Although NSAIDs inhibit the exercise‐induced satellite cell proliferation, we observed only limited effects on gene expression, and on post‐exercise protein synthesis.