Journal of Applied Physiology Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 102: 573-581, 2007. First published October 12, 2006; doi:10.1152/japplphysiol.00866.2006
8750-7587/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
102/2/573    most recent
00866.2006v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Heinemeier, K. M.
Right arrow Articles by Kjaer, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Heinemeier, K. M.
Right arrow Articles by Kjaer, M.

Short-term strength training and the expression of myostatin and IGF-I isoforms in rat muscle and tendon: differential effects of specific contraction types

K. M. Heinemeier,1,* J. L. Olesen,1,* P. Schjerling,3,4 F. Haddad,2 H. Langberg,1 K. M. Baldwin,2 and M. Kjaer1

1Institute of Sports Medicine, Bispebjerg Hospital, Copenhagen; 2Department of Physiology and Biophysics, University of California, Irvine, California; 3Department of Molecular Muscle Biology, Copenhagen Muscle Research Centre, Rigshospitalet, Copenhagen; and 4Department of Medical Biochemistry and Genetics, University of Copenhagen, Copenhagen Denmark

Submitted 7 August 2006 ; accepted in final form 5 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
In skeletal muscle, an increased expression of insulin like growth factor-I isoforms IGF-IEa and mechano-growth factor (MGF) combined with downregulation of myostatin is thought to be essential for training-induced hypertrophy. However, the specific effects of different contraction types on regulation of these factors in muscle are still unclear, and in tendon the functions of myostatin, IGF-IEa, and MGF in relation to training are unknown. 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 during general anesthesia. mRNA levels for myostatin, IGF-IEa, and MGF in muscle and Achilles' tendon were measured by real-time RT-PCR. Muscle myostatin mRNA decreased in response to all types of training (2- to 8-fold) (P < 0.05), but the effect of eccentric training was greater than concentric and isometric training (P < 0.05). In tendon, myostatin mRNA was detected, but no changes were seen after exercise. IGF-IEa and MGF increased in muscle (up to 15-fold) and tendon (up to 4-fold) in response to training (P < 0.01). In tendon no difference was seen between training types, but in muscle the effect of eccentric training was greater than concentric training for both IGF-IEa and MGF (P < 0.05), and for IGF-IEa isometric training had greater effect than concentric (P < 0.05). The results indicate a possible role for IGF-IEa and MGF in adaptation of tendon to training, and the combined changes in myostatin and IGF-IEa/MGF expression could explain the important effect of eccentric actions for muscle hypertrophy.

insulin-like growth factor-I; skeletal muscle; eccentric loading

MUSCLE GROWTH AND ADAPTATION of the supporting connective tissue in adults is dependent on a balance of negative and positive regulators of protein synthesis. Myostatin and insulin like growth factor-I (IGF-I) are regarded as two of the major players in regulation of muscle homeostasis (16, 33). They have contrasting effects on muscle growth, and regulation of their expression is suggested to be crucial in relation to exercise-induced muscle hypertrophy. Although evidence suggests adaptive changes in tendon in response to exercise (8, 31, 32, 39, 48), the role of myostatin and IGF-I in connective tissue is largely unknown.

Myostatin, a member of the transforming growth factor-beta (TGF-beta) superfamily, is a negative regulator of muscle growth, and the absence of functional myostatin is associated with hypermuscular phenotypes in mice and cattle (36, 37). In vitro studies indicate that myostatin inhibits myoblast proliferation during myogenesis (53) as well as satellite cell activation and protein synthesis in adult mouse muscle cells (35, 52). In addition, a number of studies show a decrease in myostatin expression in response to both long- and short-term resistance training, as well as acute resistance exercise, in humans, and in response to short-term resistance and endurance training in rats (17, 18, 26, 27, 34, 47, 49). These observations point to myostatin as an important regulator of muscle growth, and a reduction in myostatin expression could be essential for exercise-induced hypertrophy of skeletal muscle. However, the exact role of myostatin in relation to different types of training, e.g., concentric vs. eccentric contractions, is unknown, and likewise nothing is known regarding its role in regulation of the force-transmitting tendon tissue.

In contrast to myostatin, IGF-I is a positive regulator of muscle growth. It appears to act on several levels, including satellite cell activation (5), gene transcription, and protein translation (6), and seems essential in mediating the loading-induced hypertrophy of skeletal muscle (15). Different splice variants of IGF-I are expressed in muscle tissue (20, 44), and whereas IGF-IEa (liver-type IGF-I) is produced both in liver and muscle tissue, the splice variant IGF-IEb (mechano-growth factor; MGF) is thought to be expressed mostly as a local growth factor in skeletal muscle (15). The expression of both IGF-IEa and MGF is increased in response to several types of muscle loading (4, 7, 10, 11, 16, 17, 20, 24). This increased expression has been shown to persist during long-term resistance training in humans (19) but seems to be initiated already in the early phase of long-term functional loading in rat muscle (4, 11, 56) and has also been observed after a single resistance exercise bout in rats (17). Recent studies show a more rapid increase in MGF expression, compared with IGF-IEa, in rat muscle in response to stimulated isometric contractions (17) and stretch combined with stimulation (17, 24), suggesting that the two isoforms could influence different temporal stages of muscle hypertrophy. This is supported by in vitro observations on mouse myoblasts, which indicate that MGF induces myoblast proliferation, but not differentiation, while IGF-IEa seems important for differentiation of myoblasts into myotubes (58). The role of IGF-IEa and MGF in relation to muscle adaptation to loading has received a great deal of attention. However, it is still unclear whether different contraction types have different effects on the expression of the IGF-I isoforms, as the few studies, which have addressed this subject, show conflicting results (3, 7). In the present study, we have investigated the specific effects of different contraction types on IGF-IEa/MGF expression.

With regard to tendon tissue, the role of IGF-I in relation to exercise/training-induced adaptation has not been described. However, IGF-I is known to induce collagen synthesis in tendon cells (1, 2), and both IGF-I protein and mRNA have been localized in tendon tissue from humans (42) and several animal species (40). Thus IGF-I could mediate the increase in collagen synthesis observed in response to exercise/training (31, 32, 39). No distinction has been made between the IGF-I mRNA splice variants in tendon in earlier studies, but recently we have found both IGF-IEa and MGF mRNA to be present in rat tendon (43). With regard to myostatin, no evidence exists on its presence in tendon.

Our aim was to investigate whether myostatin is expressed in tendon tissue and to test if the expression of myostatin, IGF-IEa, and MGF in tendon is changed in response to short-term training. Additionally, we compared the effects of short-term concentric, eccentric, and isometric training on the expression of these substances in both muscle and tendon. No data exist to our knowledge on any differential effects of contraction types on tendon adaptation, whereas several studies indicate that long-term resistance training, which includes an eccentric component, has a superior effect on muscle hypertrophy in both men and women (14, 21, 23, 25). As myostatin, IGF-IEa, and MGF are known to be important for regulation of muscle growth, it is hypothesized that eccentric loading has a greater potential than concentric and isometric loading for changing the expression of these factors in skeletal muscle and potentially also in tendon tissue.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals.   Young adult female Sprague-Dawley rats, weighing 238 ± 2 (mean ± SE) g, were assigned randomly to three groups (minimum 7 in each group). Each group was subjected to involuntary concentric training (Con), eccentric training (Ecc), or isometric training (Iso). Rats were housed in groups in standard vivarium cages on a 12:12-h light-dark cycle and had ad libitum access to standard rat chow and water. The study was conducted according to the American Physiological Society's "Guiding Principles in the Care and Use of Animals," and the protocol was approved by the University of California Institutional Animal Care and Use Committee.

Training protocol.   The model used in the present study for training of rats was identical to the model employed previously by Adams et al. (3). However, it should be noted that the number of repetitions per set and the number of training sessions in the present study were not identical to those of the study of Adams et al.

Electrical muscle stimulation.   Before each training bout, the rats were lightly anesthetized with ketamine (30 mg/kg), xylazine (4 mg/kg), and acepromazine (1 mg/kg). Stainless steel wire electrodes, coated with Teflon, were used for stimulation. The electrodes were introduced subcutaneously in the region adjacent to the popliteal fossa, via 22-gauge hypodermic needles (which were withdrawn, leaving the electrode in place). Before insertion, a section of the Teflon was removed, leaving the wire exposed in the area lateral and medial of the sciatic nerve and allowing for field stimulation of the nerve. The electrodes were attached to the output poles of a Grass stimulus isolation unit interfaced with a Grass S8 stimulator. This allowed for delivery of current to the sciatic nerve and thus muscle activation.

When stimulation electrodes were in place, the rats were positioned in a specially designed training platform described previously (9). The right leg was positioned in a footplate, which was attached to the shaft of a Cambridge model H ergometer. The voltage and stimulation frequency (~50 Hz) were adjusted to produce maximal isometric tension.

Training.   During all training bouts the sciatic nerve of the right leg was stimulated for 2 s with 18 s between stimulations. Sets consisted of 10 stimulations and were separated by 5 min rest. Rats were trained for 4 consecutive days with two sets on day 1, three sets on day 2, and four sets on days 3 and 4. After each training session the electrodes were removed. The left leg served as control.

The training protocols were controlled by computer via a digital-to-analog board (model DDA-06, Keithley Instruments) used to control footplate movement and to trigger the stimulus. A separate analog-to-digital board (DAS-16) was used to acquire force measurements (100-Hz acquisition). Data acquisition, control of stimulus triggering, and footplate movements were programmed by using LabTech Note-book (Laboratory Technologies). Data analysis was conducted by using Acqknowledge software (Biopac Systems). Force output was monitored in real time on the computer screen during each contraction.

Rats of the Iso group had their right foot placed in the footplate with an angle of ~ 44° relative to the tibia. The footplate angle was fixed during muscle stimulation. For the Con group, the ergometer allowed the footplate to move from 44° to 64° after the development of maximal isometric tension in the beginning of muscle contraction. For the Ecc group, the footplate was moved from 44° to 24° after the development of maximal isometric tension. The movement rate was limited to 29°/s in the Con and Ecc groups to maintain force development.

The choice of the present short-term training protocol was based on previous observations by Adams et al. (3), who found that 10 days of training spread over a 20-day period resulted in muscle hypertrophy and increased IGF-I mRNA, combined with the findings of Haddad and Adams (17), which indicated that many of the signals for protein synthesis are turned on after only two training sessions. It should be noted that muscles trained under these conditions generate forces that are significantly different among the three training modes, i.e., eccentric-trained muscles generated the most force, while the concentric-trained muscles generated the least amount of force (3).

Tissue sampling and storage.   Twenty-four hours after the last training bout, the rats were killed via an injection of Pentosol euthanasia solution (Med-Pharmex) at a dose of 0.4 ml/kg. The medial gastrocnemius including the Achilles tendon, from both legs, was dissected free, and the midsection of the muscle belly and the Achilles tendon were cut out and stored at –80°C for later use.

RNA extraction.   Total RNA from all tissue types was extracted according to the method described by Chomczynski and Sacchi (9a). Muscle tissue was homogenized in TRI-reagent (MRC) with a Polytron homogenizer (Ultra-Turrax T8, Ika labortechnik, Staufen, Germany), while tendon tissue was homogenized in TRI-reagent using a bead-mixer (Retsch, MM300) with the aid of 3 mm steel beads. The tubes containing tendon tissue, TRI-reagent, and beads were shaken at 24 Hz for 60 s and then cooled on ice. This was repeated seven times.

Following homogenization, 1-bromo-3-chloropropane (BCP) (MRC) was added (100 µl per 1,000 µl TRI-reagent) to separate the samples into an aqueous and an organic phase. In tendon samples glycogen was added (120 µg per 1,000 µl TRI-reagent) to improve RNA precipitation. Following isolation of the aqueous phase, RNA was precipitated using isopropanol. The RNA pellet was then washed in ethanol and subsequently dissolved in RNase-free water. All samples were weighed before RNA extraction.

RNA concentrations were determined by spectroscopy at 260 nm. To account for absorbance of the glycogen added to the tendon samples, mock extractions were made in which no tissue was present, and the mean absorbance of these negative controls (n = 3) was subtracted from all test values. RNA purity was ensured by 260/240 and 260/280 nm ratio, and RNA quality was ensured by gel electrophoresis.

Real-time PCR.   Five hundred nanograms total RNA from muscle and 200 ng from tendon was converted into cDNA in 20 µl using the OmniScript reverse transcriptase (Qiagen) according to the manufacturer's protocol. For each target mRNA, 0.25 µl cDNA was amplified in a 25-µl SYBR Green PCR reaction containing 1x Quantitect SYBR Green Master Mix (Qiagen) and 100 nM of each primer (Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1. Primers for real-time RT-PCR

 
The amplification was monitored in real time using the MX3000P Real-time PCR machine (Stratagene). The cycle threshold (Ct) values were related to a standard curve made with the cloned PCR products, and specificity was confirmed by melting curve analysis after amplification. The general range of Ct values was 15–30. The large ribosomal protein P0 (RPLP0) had been chosen as internal control, assuming RPLPO mRNA to be constitutively expressed (12). To validate this assumption, another unrelated "constitutive" RNA, GAPDH mRNA, was measured, and RPLP0 was normalized to GAPDH. However, the RPLP0-to-GAPDH ratio was not stable, and we chose to normalize mRNA data to the weight of the tissue needed for extracting the amount of RNA used for cDNA synthesis (500 ng RNA for muscle and 200 ng RNA for tendon). The changes in RPLP0-to-GAPDH ratio are described in RESULTS. mRNA data are presented as fold changes relative to the mean of all control values for muscle or tendon.

Number of samples in each group.   For muscle, all samples were successfully analyzed, and all groups included seven to nine samples. For tendon, five, six, and eight samples for control Con, Ecc, and Iso groups, respectively, and six, seven, and nine samples for trained Con, Ecc, and Iso groups, respectively, were included.

Statistics.   All data [except RNA concentration (Fig. 1, I and J)] were log-transformed before statistical analyses and are presented as geometric means ± backtransformed SE.


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 1. Training-induced changes in reference genes and RNA concentration. A and B: large ribosomal protein P0 (RPLP0) mRNA normalized to GAPDH mRNA in muscle (A) and tendon (B). C and D: RPLP0 mRNA normalized to tissue weight in muscle (C) and tendon (D). E and F: GAPDH mRNA normalized to tissue weight in muscle (E) and tendon (F). G and H: RPLP0 normalized to total RNA in muscle (G) and tendon (H). Values are presented as fold changes relative to the mean of all control values in gastrocnemius muscle and Achilles tendon subjected to concentric (Con), eccentric (Ecc), or isometric (Iso) training (gray bars) vs. contralateral controls (open bars). Values are geometric means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, ~P = 0.07. I and J: RNA concentration (ng/mg) in muscle (I) and tendon (J) subjected to concentric, eccentric, or isometric training (gray bars) vs. contralateral controls (open bars). Values are means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001.

 
A two-way ANOVA on contraction x training was performed using SAS procedure mixed with autoregressive modeling. If the two-way ANOVA was significant, individual differences between trained and untrained leg within each contraction type, and between contraction types, were tested with a post hoc test (Bonferroni). Otherwise, training effect was tested against control on all contraction types combined. Differences were considered significant when P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Normalization of mRNA data.   To validate the use of RPLP0 as internal reference for mRNA data, we normalized it to GAPDH, which is also assumed to be constitutively expressed. However, the RPLP0-to-GAPDH ratio was found to increase in both muscle and tendon in response to loading (P < 0.05) (Fig. 1, A and B). When normalizing to tissue weight, we found that RPLP0 increased in both tissue types (P < 0.05) (Fig. 1, C and D) and that GAPDH decreased in muscle tissue (P < 0.05) (Fig. 1, E and F), which explains the marked increase in the RPLP0-to-GAPDH ratio in muscle (Fig. 1, A and B). The RNA concentration increased up to 2- and 1.6-fold, respectively, in tendon and muscle after training (P < 0.05) (Fig. 1, I and J). This could indicate a general increase in all RNA molecules as a result of a general increase in transcriptional activity and/or an increased cell population. However, if this were the case, we would likely have found corresponding increases in all the measured mRNA species relative to tissue weight. As this was not the case (see RESULTS), it is more likely that the increase in RNA concentration reflects a general increase in the expression of ribosome elements, including ribosomal RNA and ribosomal proteins, such as RPLP0. This is supported by the pronounced changes in RPLP0 relative to tissue weight (Fig. 1, C and D). Furthermore, the RPLP0 mRNA level increased even when normalized to total RNA (P < 0.05) (Fig. 1, G and H), and in combination with the changes observed in the RPLP0-to-GAPDH ratio this underlines the importance of investigating whether housekeeping genes are constantly expressed to validate their use as internal controls. In the present study we have to rule out the use of these housekeeping genes, and as an alternative, we have chosen to normalize all mRNA data to the weight of the tissue corresponding to the amount of RNA used for cDNA synthesis for individual samples.

Myostatin.   Myostatin mRNA was reduced in muscle tissue in response to all types of training (P < 0.05). Ecc training induced an ~8-fold decrease in myostatin mRNA (P < 0.001), while Con and Iso training led to ~2- and ~4 fold decreases, respectively (P < 0.05) (Fig. 2A); the effect of Ecc training was significantly greater than the effects of both Con and Iso training (P < 0.05) (Fig. 2A). In tendon tissue, myostatin mRNA was present but at levels far lower than in muscle (relative to total RNA) (data not shown) and with large variations. No significant changes were seen in myostatin mRNA in tendon in response to training (P > 0.05) (Fig. 2B).


Figure 2
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 2. Myostatin mRNA normalized to tissue weight, presented as fold changes relative to the mean of all control values, in gastrocnemius muscle (A) and Achilles tendon (B) subjected to concentric, eccentric, and isometric training (gray bars) vs. contralateral controls (open bars). Values are geometric means ± SE. A: myostatin mRNA decreased in muscle in response to all types of training (*P < 0.05, ***P < 0.001), and myostatin mRNA was lower in eccentric-trained muscle than in concentric and isometric trained (#P < 0.05). B: no significant changes in myostatin mRNA in tendon (P > 0.05).

 
IGF-IEa.   All types of training led to significant increases in IGF-IEa mRNA (P < 0.01) in muscle tissue, but the levels of IGF-IEa mRNA were higher after Ecc and Iso training than after Con training (P < 0.05) (Fig. 3A). In tendon the level of IGF-IEa mRNA (relative to total RNA) was similar to the level in muscle tissue (data not shown), and an increase was seen in response to all types of training (P < 0.05). No difference was seen between training types (P > 0.05) (Fig. 3B).


Figure 3
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 3. Insulin-like growth factor-I isoform Ea (IGF-IEa) mRNA normalized to tissue weight, presented as fold changes relative to the mean of all control values, in gastrocnemius muscle (A) and Achilles tendon (B) subjected to concentric, eccentric, and isometric training (gray bars) vs. contralateral controls (open bars). Values are geometric means ± SE. A: IGF-IEa mRNA increased in response to all types of training in muscle (**P < 0.01, ***P < 0.001), and IGF-IEa was higher in eccentric- and isometric-trained muscle than in concentric trained (#P < 0.05). B: IGF-IEa mRNA increased in tendon in response to all types of training (*P < 0.05, **P < 0.01).

 
MGF.   The regulation pattern for MGF mRNA was quite similar to that of IGF-IEa, although fold changes appeared to be higher for MGF than IGF-IEa in both muscle and tendon (Fig. 4). In muscle, MGF mRNA increased in response to all three types of training (P < 0.001) (Fig. 4A), but the effects of Ecc loading was significantly greater than the effect of Con loading (P < 0.01) (Fig. 4A). As with IGF-IEa, the level of MGF (relative to total RNA) in tendon was in a similar range as in muscle tissue (data not shown), and an increase in MGF was seen in response to all training types (P < 0.05). No difference was seen between training types (P > 0.05) (Fig. 4B).


Figure 4
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 4. Mechano-growth factor (MGF) mRNA normalized to tissue weight, presented as fold changes relative to the mean of all control values, in gastrocnemius muscle (A) and Achilles tendon (B) subjected to concentric, eccentric, and isometric training (gray bars) vs. contralateral controls (open bars). Values are geometric means ± SE. A: MGF mRNA increased in muscle in response to all types of training (***P < 0.001), and the level was higher in eccentric-trained muscle compared with concentric-trained muscle (##P < 0.01). B: MGF mRNA increased in tendon in response to all types of training (*P < 0.05, ***P < 0.001).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The data presented here support the view that myostatin downregulation is a significant part of loading-induced muscle growth and is in line with other studies that demonstrate a decrease in myostatin mRNA levels of skeletal muscle in response to resistance type muscle loading (26, 27, 50). Furthermore, in the present study it is shown that eccentric training has a greater potential than concentric and isometric training for reducing myostatin expression in female rat skeletal muscle in the early phase of resistance training. Considering the inhibiting effect of myostatin on muscle growth, this finding could help to explain the superior effect of eccentric training on muscle hypertrophy seen in earlier studies in both men and women (14, 21, 23, 25). The only previous study describing the specific effect of eccentric loading on myostatin regulation showed a transient increase in myostatin mRNA, peaking at 3–6 h after one acute bout of muscle-damaging eccentric exercise in male rats (45). This finding appears to contradict our results and the majority of studies on myostatin that show a downregulation in response to loading (33). However, in that study muscle was only subjected to acute exercise and not to repeated resistance training bouts, and it should be considered that the effect of one acute exercise bout differs significantly from the effect of a series of bouts with regard to expression of several other factors involved in regulation of muscle growth (17). In addition, the acute response to eccentric muscle loading could entail a transient increase in myostatin expression. Such an acute effect might also explain the increase in myostatin mRNA found in untrained young men in response to long-term heavy resistance training by Willoughby (54), as he obtained muscle samples immediately after the last training bout. Meanwhile, a very recent study shows a decrease in muscle levels of myostatin mRNA only 4 h after a moderate bout of resistance exercise in young and old women (47), and thus it must be acknowledged that regulation of myostatin in response to acute exercise, and in response to eccentric training, is still unclear. Our results support that a myostatin downregulation is important for loading-induced adaptations of skeletal muscle and indicate that the effect of short-term eccentric training on myostatin regulation is greater than the effects of concentric and isometric training. However, it should be considered that changes in mRNA are only indicative of changes in protein levels, and furthermore myostatin action appears to be dependent on enzymatic activation (55). Thus further experiments investigating protein levels of both active and inactive myostatin, as well as IGF-I protein, should be performed to confirm the findings of the present study.

In tendon tissue, we were able to detect myostatin mRNA, but the concentration was highly variable, and no changes were seen in response to training. Thus we found no indications that myostatin regulation is important for tendon adaptation. In contrast, our results indicate that both IGF-IEa and MGF are possible players in adaptation of tendon tissue to increased loading. As shown earlier (43), we found mRNA for both IGF-I splice variants to be present in tendon and furthermore that the concentrations of these mRNA species were similar in tendon and muscle tissue. Interestingly, we saw that their expression increased in response to all types of training (Figs. 3B and 4B). This corresponds well with our earlier observations of increased IGF-IEa and MGF expression in female rat plantaris tendon after functional loading (43). IGF-IEa is known to induce collagen synthesis in rabbit tendon tissue (1, 2), and the observed increase in IGF-IEa mRNA points to this growth factor as a mediator of the increased collagen synthesis seen in human (male and female) tendon in response to acute exercise (32, 38, 39). The effect of MGF on collagen regulation, and on tendon tissue turnover in general, has not been investigated. However, our observations indicate a likely involvement of this growth factor in tendon adaptation, and the function of MGF in tendon tissue remains an attractive area for future investigation.

The roles of IGF-IEa and MGF in skeletal muscle are extensively described, and both splice variants are known to be involved in loading-induced hypertrophy (16). In line with earlier studies that show increased IGF-I mRNA levels in the early phase of muscle loading (4, 7, 10, 11), we found increased muscle levels of IGF-IEa and MGF mRNA after 4 days of resistance training. Recent studies have shown a discrepancy in the timing of MGF and IGF-IEa expression in response to mechanical loading in rat muscle (17, 24). Haddad and Adams (17) found a more rapid increase in MGF expression compared with that of IGF-IEa in response to stimulated isometric contractions in female rat muscle, and a similar picture was shown in response to stretch combined with stimulation by Hill and Goldspink (24). These findings suggest that after muscle loading the IGF-I precursor mRNA is first spliced toward the MGF variant and then later toward the IGF-IEa variant (24). In the present study, the fold increases in MGF mRNA appeared higher than the increases in IGF-IEa mRNA in both muscle and tendon tissue (Figs. 3 and 4), indicating a relatively greater increase in MGF expression compared with IGF-IEa.

With regard to the specific effect of contraction type, we found eccentric training to have greater effect on muscle IGF-IEa and MGF mRNA expression than concentric training. In addition, the effect of isometric training on IGF-IEa mRNA was superior to concentric training. To our knowledge only two studies have been published that compare the effects of pure eccentric loading to other loading types on expression of IGF-I in muscle tissue. Bamman et al. (7) measured muscle IGF-I mRNA 48 h after a single bout of eccentric or concentric exercise in young men and women and found an increase only in response to eccentric exercise. In conflict with this, Adams et al. (3) observed no effect of long-term eccentric training on levels of IGF-IEa or MGF in female rat muscle, while increases were found in response to both concentric and isometric training. The different nature of the muscle stimuli applied in these studies complicates the comparison, and it should be noted that, conversely to the present study, no significant differences between contraction types were reported in either study (3, 7). Our results could be expected to concur with the findings of Adams et al. (3), as the set-up for training was similar. Meanwhile, we applied four consecutive days of training and a considerably higher workload per session, and considering the known effect of repeated eccentric contractions (46), the possibility of muscle damage induced by eccentric training was presumably greater in the present study compared with the work by Adams et al. (3). Muscle damage has been shown to increase local IGF-I protein levels in rats and humans (22, 51, 57), and if muscle damage was induced in response to eccentric loading with our training approach, and not with the training applied in the study of Adams et al., this might explain the discrepancy with regard to the expression of IGF-IEa and MGF. However, if the increase in IGF-IEa and MGF in response to eccentric training were based primarily on muscle damage, and a possible inflammatory response, eccentric loading would be expected to have a greater effect than isometric loading, and although the fold changes seen after eccentric loading appeared higher than after isometric loading, no significant difference was observed between these training types. Thus no conclusive explanations can be given for the diverging observations on the effect of eccentric exercise on IGF-I regulation.

When taking into account the changes we found in myostatin, IGF-IEa, and MGF, and considering the contrasting effects of myostatin and the IGF-I splice variants on muscle growth, eccentric training does appear to have greater potential for inducing muscle growth than other training types. With an identical set-up for training as the one employed in the present study, Adams et al. (3) showed a torque production during eccentric contraction, which was approximately 1.9- and 3-fold higher than during isometric and concentric contraction, although the stimulation parameters were constant. Thus, as discussed in several earlier studies (e.g., Refs. 21, 23), the obvious explanation for the superior effect of eccentric loading is the higher total workload imposed on the muscle. This could lead to a greater response, not only locally in the eccentrically loaded muscle tissue, but also in the central hormones (e.g., growth hormone) (29), which could in turn affect skeletal muscle expression of growth factors (19). In support of this, we found the extent of mRNA changes to be ranked in an order corresponding to the level of torque production (eccentric > isometric > concentric), although differences between eccentric and isometric training were not significant for IGF-IEa and MGF (Figs. 2A, 3A, and 4A). Meanwhile our results on tendon tissue add an interesting dimension to this discussion, as we saw no differences in the induction of IGF-IEa and MGF mRNA between contraction types in this tissue. This indicates that muscle tissue is either more sensitive than tendon to changes in workload or that it contains a sensing mechanism that enables it to detect differences between the specific contraction modes, perhaps involving detection of lateral forces or shear stress. In support of this, data reported by Moore et al. (41) indicate a higher myofibrillar protein synthesis rate in young men in response to acute lengthening contractions compared with shortening contractions, even though workloads were identical.

Limitations of the study.   In the present study we chose to investigate only pure contraction modes. Earlier work indicates that a combination of concentric and eccentric action may be the optimal training type in relation to hypertrophy, strength gain, and hormonal response (13, 21, 29), and such a combination of concentric and eccentric muscle action may elicit an even greater response than the one seen in response to eccentric loading. Thus, in future studies it would be relevant to include a training type, which combines concentric and eccentric muscle action. Furthermore, it should be kept in mind that the training employed in the present study was based on involuntary muscle contractions, and we cannot be certain that the effect of this type of training is identical to that of a voluntary type (28). Finally, it should be considered that female rats were investigated, and because of differences in hormonal response between genders (30), the observed responses may not have been identical in male rats.

In conclusion, we have demonstrated that short-term training increases tendon levels of both IGF-IEa and MGF mRNA, indicating a possible role for these growth factors in the adaptation of tendon to training. Furthermore, we found that eccentric training was more effective in downregulating myostatin expression than other loading types, and in combination with the effect of eccentric loading on IGF-IEa and MGF expression, this may well explain the strong contributions of eccentric actions in resistance training-induced muscle hypertrophy.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This study was supported by grants from the Danish Rheumatism Association, The Danish Ministry of Culture Committee for Sports Research, Danish Medical Research Counsel (22-04-0191), Lundbeck Foundation (128/03), Novo Nordic Foundation, and Medical Faculty, University of Copenhagen Grant NSBRI-NC9-58 to K. M. Heinemeier.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We address a special thanks to Ann-Christina, Christina Bøg Sørensen, Lisbeth Tranholm Andersen, and Satu Koskinen for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. M. Heinemeier, Institute of Sports Medicine, Bispebjerg Hospital-Bldg. 8, 1st Floor, 23 Bispebjerg Bakke, DK-2400 Copenhagen NV, Denmark (e-mail: katjaheinemeier{at}hotmail.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* K. M. Heinemeier and J. L. Olesen contributed equally to this study. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. Abrahamsson SO, Lohmander S. Differential effects of insulin-like growth factor-I on matrix and DNA synthesis in various regions and types of rabbit tendons. J Orthop Res 14: 370–376, 1996.[CrossRef][ISI][Medline]
  2. Abrahamsson SO, Lundborg G, Lohmander LS. Recombinant human insulin-like growth factor-I stimulates in vitro matrix synthesis and cell proliferation in rabbit flexor tendon. J Orthop Res 9: 495–502, 1991.[CrossRef][ISI][Medline]
  3. Adams GR, Cheng DC, Haddad F, Baldwin KM. Skeletal muscle hypertrophy in response to isometric, lengthening, and shortening training bouts of equivalent duration. J Appl Physiol 96: 1613–1618, 2004.[Abstract/Free Full Text]
  4. Adams GR, Haddad F. The relationships among IGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy. J Appl Physiol 81: 2509–2516, 1996.[Abstract/Free Full Text]
  5. Adams GR, McCue SA. Localized infusion of IGF-I results in skeletal muscle hypertrophy in rats. J Appl Physiol 84: 1716–1722, 1998.[Abstract/Free Full Text]
  6. Baldwin KM, Haddad F. Skeletal muscle plasticity: cellular and molecular responses to altered physical activity paradigms. Am J Phys Med Rehabil 81: S40–S51, 2002.[CrossRef][ISI][Medline]
  7. Bamman MM, Shipp JR, Jiang J, Gower BA, Hunter GR, Goodman A, McLafferty CLJ, Urban RJ. Mechanical load increases muscle IGF-I and androgen receptor mRNA concentrations in humans. Am J Physiol Endocrinol Metab 280: E383–E390, 2001.[Abstract/Free Full Text]
  8. Birch HL, McLaughlin L, Smith RK, Goodship AE. Treadmill exercise-induced tendon hypertrophy: assessment of tendons with different mechanical functions. Equine Vet J Suppl 30: 222–226, 1999.[Medline]
  9. Caiozzo VJ, Ma E, McCue SA, Smith E, Herrick RE, Baldwin KM. A new animal model for modulating myosin isoform expression by altered mechanical activity. J Appl Physiol 73: 1432–1440, 1992.[Abstract/Free Full Text]
  10. Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.[ISI][Medline]
  11. Czerwinski SM, Martin JM, Bechtel PJ. Modulation of IGF mRNA abundance during stretch-induced skeletal muscle hypertrophy and regression. J Appl Physiol 76: 2026–2030, 1994.[Abstract/Free Full Text]
  12. DeVol DL, Rotwein P, Sadow JL, Novakofski J, Bechtel PJ. Activation of insulin-like growth factor gene expression during work-induced skeletal muscle growth. Am J Physiol Endocrinol Metab 259: E89–E95, 1990.[Abstract/Free Full Text]
  13. Dheda K, Huggett JF, Bustin SA, Johnson MA, Rook G, Zumla A. Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques 37: 112–119, 2004.[ISI][Medline]
  14. Dudley GA, Tesch PA, Miller BJ, Buchanan P. Importance of eccentric actions in performance adaptations to resistance training. Aviat Space Environ Med 62: 543–550, 1991.[Medline]
  15. Farthing JP, Chilibeck PD. The effects of eccentric and concentric training at different velocities on muscle hypertrophy. Eur J Appl Physiol 89: 578–586, 2003.[CrossRef][ISI][Medline]
  16. Goldspink G. Gene expression in muscle in response to exercise. J Muscle Res Cell Motil 24: 121–126, 2003.[CrossRef][ISI][Medline]
  17. Goldspink G. Mechanical signals, IGF-I gene splicing, and muscle adaptation. Physiology 20: 232–238, 2005.[Abstract/Free Full Text]
  18. Haddad F, Adams GR. Selected contribution: acute cellular and molecular responses to resistance exercise. J Appl Physiol 93: 394–403, 2002.[Abstract/Free Full Text]
  19. Haddad F, Adams GR. Aging-sensitive cellular and molecular mechanisms associated with skeletal muscle hypertrophy. J Appl Physiol 100: 1188–1203, 2006.[Abstract/Free Full Text]
  20. Hameed M, Lange KH, Andersen JL, Schjerling P, Kjaer M, Harridge SD, Goldspink G. The effect of recombinant human growth hormone and resistance training on IGF-I mRNA expression in the muscles of elderly men. J Physiol 555: 231–240, 2004.[Abstract/Free Full Text]
  21. Hameed M, Orrell RW, Cobbold M, Goldspink G, Harridge SD. Expression of IGF-I splice variants in young and old human skeletal muscle after high resistance exercise. J Physiol 547: 247–254, 2003.[Abstract/Free Full Text]
  22. Hather BM, Tesch PA, Buchanan P, Dudley GA. Influence of eccentric actions on skeletal muscle adaptations to resistance training. Acta Physiol Scand 143: 177–185, 1991.[ISI][Medline]
  23. Hellsten Y, Hansson HA, Johnson L, Frandsen U, Sjodin B. Increased expression of xanthine oxidase and insulin-like growth factor I (IGF-I) immunoreactivity in skeletal muscle after strenuous exercise in humans. Acta Physiol Scand 157: 191–197, 1996.[CrossRef][ISI][Medline]
  24. Higbie EJ, Cureton KJ, Warren GL III, Prior BM. Effects of concentric and eccentric training on muscle strength, cross-sectional area, and neural activation. J Appl Physiol 81: 2173–2181, 1996.[Abstract/Free Full Text]
  25. Hill M, Goldspink G. Expression and splicing of the insulin-like growth factor gene in rodent muscle is associated with muscle satellite (stem) cell activation following local tissue damage. J Physiol 549: 409–418, 2003.[Abstract/Free Full Text]
  26. Hortobagyi T, Dempsey L, Fraser D, Zheng D, Hamilton G, Lambert J, Dohm L. Changes in muscle strength, muscle fibre size and myofibrillar gene expression after immobilization and retraining in humans. J Physiol 524: 293–304, 2000.[Abstract/Free Full Text]
  27. Jones SW, Hill RJ, Krasney PA, O'Conner B, Peirce N, Greenhaff PL. Disuse atrophy and exercise rehabilitation in humans profoundly affects the expression of genes associated with the regulation of skeletal muscle mass. FASEB J 18: 1025–1027, 2004.[Abstract/Free Full Text]
  28. Kim JS, Cross JM, Bamman MM. Impact of resistance loading on myostatin expression and cell cycle regulation in young and older men and women. Am J Physiol Endocrinol Metab 288: E1110–E1119, 2005.[Abstract/Free Full Text]
  29. Kjaer M, Secher NH, Bangsbo J, Perko G, Horn A, Mohr T, Galbo H. Hormonal and metabolic responses to electrically induced cycling during epidural anesthesia in humans. J Appl Physiol 80: 2156–2162, 1996.[Abstract/Free Full Text]
  30. Kraemer WJ, Dudley GA, Tesch PA, Gordon SE, Hather BM, Volek JS, Ratamess NA. The influence of muscle action on the acute growth hormone response to resistance exercise and short-term detraining. Growth Horm IGF Res 11: 75–83, 2001.[CrossRef][ISI][Medline]
  31. Kraemer WJ, Ratamess NA. Hormonal responses and adaptations to resistance exercise and training. Sports Med 35: 339–361, 2005.[CrossRef][ISI][Medline]
  32. Langberg H, Rosendal L, Kjaer M. Training-induced changes in peritendinous type I collagen turnover determined by microdialysis in humans. J Physiol 534: 297–302, 2001.[Abstract/Free Full Text]
  33. Langberg H, Skovgaard D, Petersen LJ, Bulow J, Kjaer M. Type I collagen synthesis and degradation in peritendinous tissue after exercise determined by microdialysis in humans. J Physiol 521: 299–306, 1999.[Abstract/Free Full Text]
  34. Matsakas A, Diel P. The growth factor myostatin, a key regulator in skeletal muscle growth and homeostasis. Int J Sports Med 26: 83–89, 2005.[CrossRef][ISI][Medline]
  35. Matsakas A, Friedel A, Hertrampf T, Diel P. Short-term endurance training results in a muscle-specific decrease of myostatin mRNA content in the rat. Acta Physiol Scand 183: 299–307, 2005.[CrossRef][ISI][Medline]
  36. McCroskery S, Thomas M, Maxwell L, Sharma M, Kambadur R. Myostatin negatively regulates satellite cell activation and self-renewal. J Cell Biol 162: 1135–1147, 2003.[Abstract/Free Full Text]
  37. McPherron AC, Lawler AM, Lee SJ. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387: 83–90, 1997.[CrossRef][Medline]
  38. McPherron AC, Lee SJ. Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci USA 94: 12457–12461, 1997.[Abstract/Free Full Text]
  39. Miller BF, Hansen M, Olesen JL, Flyvbjerg A, Schwarz P, Babraj JA, Smith K, Rennie MJ, Kjaer M. No effect of menstrual cycle on myofibrillar and connective tissue protein synthesis in contracting skeletal muscle. Am J Physiol Endocrinol Metab 290: E163–E168, 2006.[Abstract/Free Full Text]
  40. Miller BF, Olesen JL, Hansen M, Dossing S, Crameri RM, Welling RJ, Langberg H, Flyvbjerg A, Kjaer M, Babraj JA, Smith K, Rennie MJ. Coordinated collagen and muscle protein synthesis in human patella tendon and quadriceps muscle after exercise. J Physiol 567: 1021–1033, 2005.[Abstract/Free Full Text]
  41. Molloy T, Wang Y, Murrell G. The roles of growth factors in tendon and ligament healing. Sports Med 33: 381–394, 2003.[CrossRef][ISI][Medline]
  42. Moore DR, Phillips SM, Babraj JA, Smith K, Rennie MJ. Myofibrillar and collagen protein synthesis in human skeletal muscle in young men after maximal shortening and lengthening contractions. Am J Physiol Endocrinol Metab 288: E1153–E1159, 2005.[Abstract/Free Full Text]
  43. Olesen J, Heinemeier K, Langberg H, Magnusson P, Flyvbjerg A, Kjaer M. Expression, content and localization of IGF-I in human Achilles tendon. Connect Tissue Res 47: 200–206, 2006.[CrossRef][ISI][Medline]
  44. Olesen JL, Heinemeier KM, Haddad F, Langberg H, Flyvbjerg A, Kjaer M, Baldwin KM. Expression of insulin-like growth factor I, insulin-like growth factor binding proteins, and collagen mRNA in mechanically loaded plantaris tendon. J Appl Physiol 101: 183–188, 2006.[Abstract/Free Full Text]
  45. Owino V, Yang SY, Goldspink G. Age-related loss of skeletal muscle function and the inability to express the autocrine form of insulin-like growth factor-1 (MGF) in response to mechanical overload. FEBS Lett 505: 259–263, 2001.[CrossRef][ISI][Medline]
  46. Peters D, Barash IA, Burdi M, Yuan PS, Mathew L, Friden J, Lieber RL. Asynchronous functional, cellular and transcriptional changes after a bout of eccentric exercise in the rat. J Physiol 553: 947–957, 2003.[Abstract/Free Full Text]
  47. Proske U, Allen TJ. Damage to skeletal muscle from eccentric exercise. Exerc Sport Sci Rev 33: 98–104, 2005.[CrossRef][ISI][Medline]
  48. Raue U, Slivka D, Jemiolo B, Hollon C, Trappe SW. Myogenic gene expression at rest and following a bout of resistance exercise in young (18–30 yr) and old (80–89 yr) women. J Appl Physiol 101: 53–59, 2006.[Abstract/Free Full Text]
  49. Rosager S, Aagaard P, Dyhre-Poulsen P, Neergaard K, Kjaer M, Magnusson SP. Load-displacement properties of the human triceps surae aponeurosis and tendon in runners and non-runners. Scand J Med Sci Sports 12: 90–98, 2002.[CrossRef][ISI][Medline]
  50. Roth SM, Martel GF, Ferrell RE, Metter EJ, Hurley BF, Rogers MA. Myostatin gene expression is reduced in humans with heavy-resistance strength training: a brief communication. Exp Biol Med (Maywood) 228: 706–709, 2003.[Abstract/Free Full Text]
  51. Roth SM, Walsh S. Myostatin: a therapeutic target for skeletal muscle wasting. Curr Opin Clin Nutr Metab Care 7: 259–263, 2004.[ISI][Medline]
  52. Singh MA, Ding W, Manfredi TJ, Solares GS, O'Neill EF, Clements KM, Ryan ND, Kehayias JJ, Fielding RA, Evans WJ. Insulin-like growth factor I in skeletal muscle after weight-lifting exercise in frail elders. Am J Physiol Endocrinol Metab 277: E135–E143, 1999.[Abstract/Free Full Text]
  53. Taylor WE, Bhasin S, Artaza J, Byhower F, Azam M, Willard DH Jr, Kull FC Jr, Gonzalez-Cadavid N. Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells. Am J Physiol Endocrinol Metab 280: E221–E228, 2001.[Abstract/Free Full Text]
  54. Thomas M, Langley B, Berry C, Sharma M, Kirk S, Bass J, Kambadur R. Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation. J Biol Chem 275: 40235–40243, 2000.[Abstract/Free Full Text]
  55. Willoughby DS. Effects of heavy resistance training on myostatin mRNA and protein expression. Med Sci Sports Exerc 36: 574–582, 2004.
  56. Wolfman NM, McPherron AC, Pappano WN, Davies MV, Song K, Tomkinson KN, Wright JF, Zhao L, Sebald SM, Greenspan DS, Lee SJ. Activation of latent myostatin by the BMP-1/tolloid family of metalloproteinases. Proc Natl Acad Sci USA 100: 15842–15846, 2003.[Abstract/Free Full Text]
  57. Yamaguchi A, Fujikawa T, Shimada S, Kanbayashi I, Tateoka M, Soya H, Takeda H, Morita I, Matsubara K, Hirai T. Muscle IGF-I Ea, MGF, and myostatin mRNA expressions after compensatory overload in hypophysectomized rats. Pflügers Arch 453: 203–210, 2006.[CrossRef][ISI][Medline]
  58. Yan Z, Biggs RB, Booth FW. Insulin-like growth factor immunoreactivity increases in muscle after acute eccentric contractions. J Appl Physiol 74: 410–414, 1993.[Abstract/Free Full Text]
  59. Yang SY, Goldspink G. Different roles of the IGF-I Ec peptide (MGF) and mature IGF-I in myoblast proliferation and differentiation. FEBS Lett 522: 156–160, 2002.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
C. L. Mendias, K. I. Bakhurin, and J. A. Faulkner
Tendons of myostatin-deficient mice are small, brittle, and hypocellular
PNAS, January 8, 2008; 105(1): 388 - 393.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
K. M. Heinemeier, J. L. Olesen, F. Haddad, H. Langberg, M. Kjaer, K. M. Baldwin, and P. Schjerling
Expression of collagen and related growth factors in rat tendon and skeletal muscle in response to specific contraction types
J. Physiol., August 1, 2007; 582(3): 1303 - 1316.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
M. Kjaer
Matrix loaded and unloaded: can tendons grow when exercised?
J Appl Physiol, February 1, 2007; 102(2): 515 - 515.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
102/2/573    most recent
00866.2006v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Heinemeier, K. M.
Right arrow Articles by Kjaer, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Heinemeier, K. M.
Right arrow Articles by Kjaer, M.

<