Henning Langberg

Coordinated collagen and muscle protein synthesis in human patella tendon and quadriceps muscle after exercise

We hypothesized that an acute bout of strenuous, non‐damaging exercise would increase rates of protein synthesis of collagen in tendon and skeletal muscle but these would be less than those of muscle myofibrillar and sarcoplasmic proteins. Two groups (n= 8 and 6) of healthy young men were studied over 72 h after 1 h of one‐legged kicking exercise at 67% of maximum workload (Wmax). To label tissue proteins in muscle and tendon primed, constant infusions of [1‐13C]leucine or [1‐13C]valine and flooding doses of [15N] or [13C]proline were given intravenously, with estimation of labelling in target proteins by gas chromatography–mass spectrometry. Patellar tendon and quadriceps biopsies were taken in exercised and rested legs at 6, 24, 42 or 48 and 72 h after exercise. The fractional synthetic rates of all proteins were elevated at 6 h and rose rapidly to peak at 24 h post exercise (tendon collagen (0.077% h−1), muscle collagen (0.054% h−1), myofibrillar protein (0.121% h−1), and sarcoplasmic protein (0.134% h−1)). The rates decreased toward basal values by 72 h although rates of tendon collagen and myofibrillar protein synthesis remained elevated. There was no tissue damage of muscle visible on histological evaluation. Neither tissue microdialysate nor serum concentrations of IGF‐I and IGF binding proteins (IGFBP‐3 and IGFBP‐4) or procollagen type I N‐terminal propeptide changed from resting values. Thus, there is a rapid increase in collagen synthesis after strenuous exercise in human tendon and muscle. The similar time course of changes of protein synthetic rates in different cell types supports the idea of coordinated musculotendinous adaptation.

Monitoring tissue oxygen availability with near infrared spectroscopy (NIRS) in health and disease.

Near infrared spectroscopy (NIRS) is becoming a widely used research instrument to measure tissue oxygen (O2) status non‐invasively. Continuous‐wave spectrometers are the most commonly used devices, which provide semi‐quantitative changes in oxygenated and deoxygenated hemoglobin in small blood vessels (arterioles, capillaries and venules). Refinement of NIRS hardware and the algorithms used to deconvolute the light absorption signal have improved the resolution and validity of cytochrome oxidase measurements. NIRS has been applied to measure oxygenation in a variety of tissues including muscle, brain and connective tissue, and more recently it has been used in the clinical setting to assess circulatory and metabolic abnormalities. Quantitative measures of blood flow are also possible using NIRS and a light‐absorbing tracer, which can be applied to evaluate circulatory responses to exercise along with the assessment of tissue O2 saturation. The venular O2 saturation can be estimated with NIRS by applying venous occlusion and measuring changes in oxygenated vs. total hemoglobin. These various measurements provide the opportunity to evaluate several important metabolic and circulatory patterns in very localized regions of tissue and may be fruitful in the study of occupational syndromes and a variety of diseases.

Type I collagen synthesis and degradation in peritendinous tissue after exercise determined by microdialysis in humans

1 Physical activity is known to increase type I collagen synthesis measured as the concentration of biomarkers in plasma. By the use of microdialysis catheters with a very high molecular mass cut‐off value (3000 kDa) we aimed to determine local type I collagen synthesis and degradation in the peritendinous region by measuring interstitial concentrations of a collagen propeptide (PICP; 100 kDa) and a collagen degradation product (ICTP; 9 kDa) as well as an inflammatory mediator (PGE2). 2 Seven trained human runners were studied before and after (2 and 72 h) 3 h of running (36 km). Two microdialysis catheters were placed in the peritendinous space ventral to the Achilles’ tendon under ultrasound guidance and perfused with a Ringer‐acetate solution containing 3H‐labelled human type IV collagen and [15‐3H(N)]PGE2 for in vivo recovery determination. Relative recovery was 37–59% (range of the s.e.m. values) for both radioactively labelled substances. 3 PICP concentration decreased in both interstitial peritendinous tissue and arterial blood immediately after exercise, but rose 3‐fold from basal 72 h after exercise in the peritendinous tissue (55 ± 10 μg l−1, mean ± s.e.m. (rest) to 165 ± 40 μg l−1 (72 h), P < 0·05) and by 25% in circulating blood (160 ± 10 μg l−1 (rest) to 200 ± 12 μg l−1 (72 h), P < 0·05). ICTP concentration did not change in blood, but decreased transiently in tendon‐related tissue during early recovery after exercise only. PGE2 concentration increased in blood during running, and returned to baseline in the recovery period, whereas interstitial PGE2 concentration was elevated in the early recovery phase. 4 The findings of the present study indicate that acute exercise induces increased formation of type I collagen in peritendinous tissue as determined with microdialysis and using dialysate fibre with a very high molecular mass cut‐off. This suggests an adaptation to acute physical loading also in non‐bone‐related collagen in humans.

Myofibre damage in human skeletal muscle: effects of electrical stimulation versus voluntary contraction

Disruption to proteins within the myofibre after a single bout of unaccustomed eccentric exercise is hypothesized to induce delayed onset of muscle soreness and to be associated with an activation of satellite cells. This has been shown in animal models using electrical stimulation but not in humans using voluntary exercise. Untrained males (n= 8, range 22–27 years) performed 210 maximal eccentric contractions with each leg on an isokinetic dynamometer, voluntarily (VOL) with one leg and electrically induced (ES) with the other leg. Assessments from the skeletal muscle were obtained prior to exercise and at 5, 24, 96 and 192 h postexercise. Muscle tenderness rose in VOL and ES after 24 h, and did not differ between groups. Maximal isometric contraction strength, rate of force development and impulse declined in the VOL leg from 4 h after exercise, but not in ES (except at 24 h). In contrast, a significant disruption of cytoskeletal proteins (desmin) and a rise of myogenic growth factors (myogenin) occurred only in ES. Intracellular disruption and destroyed Z‐lines were markedly more pronounced in ES (40%) compared with VOL (10%). Likewise, the increase in satellite cell markers [neural cell adhesion molecule (N‐CAM) and paired‐box transcription factor (Pax‐7)] was more pronounced in ES versus VOL. Finally, staining of the intramuscular connective tissue (tenascin C) was increased equally in ES and VOL after exercise. The present study demonstrates that in human muscle, the delayed onset of muscle soreness was not significantly different between the two treatments despite marked differences in intramuscular histological markers, in particular myofibre proteins and satellite cell markers. An increase in tenascin C expression in the midbelly of the skeletal muscle in both legs provides further evidence of a potential role for the extracellular matrix in the phenomenon of delayed onset of muscle soreness.

Changes in satellite cells in human skeletal muscle after a single bout of high intensity exercise

No studies to date have reported activation of satellite cells in vivo in human muscle after a single bout of high intensity exercise. In this investigation, eight individuals performed a single bout of high intensity exercise with one leg, the contralateral leg being the control. A significant increase in mononuclear cells staining for the neural cell adhesion molecule (N‐CAM) and fetal antigen 1 (FA1) were observed within the exercised human vastus lateralis muscle on days 4 and 8 post exercise. In addition, a significant increase in the concentration of the FA1 protein was determined in intramuscular dialysate samples taken from the vastus lateralis muscle of the exercising leg (day 0: 1.89 ± 0.82 ng ml−1; day 2: 1.68 ± 0.37 ng ml−1; day 4: 3.26 ± 1.29 ng ml−1, P < 0.05 versus basal; day 8: 4.68 ± 2.06 ng ml−1, P < 0.05 versus basal and control). No change was noted in the control leg. Despite this increase in N‐CAM‐ and FA1‐positive mononuclear cells, an increased expression of myogenin and the neonatal isoform of the myosin heavy chain (MHCn) was not observed. Interestingly, myofibre lesions resulting from extensive damage to the proteins within the myofibre, particularly desmin or dystrophin, were not observed, and hence did not appear to induce the expression of either N‐CAM or FA1. We therefore propose that satellite cells can be induced to re‐enter the cell growth cycle after a single bout of unaccustomed high intensity exercise. However, a single bout of exercise is not sufficient for the satellite cell to undergo terminal differentiation.

Eccentric rehabilitation exercise increases peritendinous type I collagen synthesis in humans with Achilles tendinosis

It has been shown that 12 weeks of eccentric heavy resistance training can reduce pain in runners suffering from chronic Achilles tendinosis, but the mechanism behind the effectiveness of this treatment is unknown. The present study investigates the local effect of an eccentric training regime on elite soccer players suffering from chronic Achilles tendinosis on the turnover of the peritendinous connective tissue.

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.

Substantial elevation of interleukin‐6 concentration in peritendinous tissue, in contrast to muscle, following prolonged exercise in humans

Plasma interleukin‐6 (IL‐6) concentration has been shown to increase with exercise and various cell types and tissues have been suggested to be responsible for this increase. At present no studies have measured the interstitial concentration of IL‐6 in skeletal muscle and connective tissue. The present study represents the first attempt to simultaneously measure IL‐6 in plasma, skeletal muscle and peritendinous connective tissue in response to prolonged exercise. Six healthy well‐trained volunteers completed a 36 km run (flat, 12 km h−1). IL‐6 was measured before, 2 h post‐exercise and 24 h, 48 h, 72 h and 96 h post‐exercise in both the medial gastrocnemius muscle (not measured at rest due to risk of disabling the subsequent exercise, and 24 h and 72 h post‐exercise) and the peritendinous tissue around the Achilles tendon using microdialysis catheters with a high molecular mass cut‐off value (3000 kDa). The plasma concentration of IL‐6 was measured simultaneously, and in addition every hour during the exercise, by enzyme‐linked immunosorbent assay (ELISA). The plasma concentration of IL‐6 was found to increase throughout the exercise, reaching peak values immediately after completion of the run (50‐fold increase). Using the microdialysis technique, the interstitial concentration of IL‐6 was found to increase dramatically from 0 ± 0 pg ml−1 to 3618 ± 1239 pg ml−1 in the peritendinous tissue in the hours following the exercise. The pattern of changes was similar in plasma and peritendinous tissue, although approximately 100‐fold higher in the latter. For comparison the interstitial muscle concentration was found to be 465 ± 176 pg ml−1 when measured 2 h post‐exercise and 223 ± 113 pg ml−1 and 198 ± 96 pg ml−1 48 h and 96 h post‐exercise, respectively. The present study demonstrates that the connective tissue around the human Achilles tendon produces significant amounts of IL‐6 in response to prolonged physical activity, which might contribute to the exercise‐induced increase in IL‐6 found in plasma.

Metabolism and inflammatory mediators in the peritendinous space measured by microdialysis during intermittent isometric exercise in humans

1 The metabolic processes that occur around the tendon during mechanical loading and exercise are undescribed in man. These processes are important for understanding the development of overuse inflammation and injury. 2 A microdialysis technique was used to determine interstitial concentrations of glycerol, glucose, lactate, prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) as well as to calculate tissue substrate balance in the peritendinous region of the human Achilles tendon. Recovery of 48–62% (range) at rest and 70–77% during exercise were obtained for glycerol, glucose and PGE2. 3 Six young healthy humans were studied at rest, during 30 min of intermittent static plantar flexion of the ankle at a workload corresponding to individual body weight, and during 60 min of recovery. Microdialysis was performed in both legs with simultaneous determination of blood flow by 133Xe washout in the same area, and blood sampling from the radial artery. 4 With exercise, the net release of lactate as well as of glycerol from the peritendinous space of the Achilles tendon increased 2‐fold (P < 0.05). Furthermore a 100% increase in interstitial concentration of PGE2 and TXB2 was found, but it was only significant for TXB2(P < 0.05). As peritendinous blood flow increased 2‐ to 3‐fold during intermittent static contractions, this indicates also that the output of these substances from the tissue increased during exercise. 5 This study indicates that both lipid and carbohydrate metabolism as well as inflammatory activity is accelerated in the peritendinous region of the human Achilles tendon with dynamic loading.