DISCUSSION
The present study is the first to demonstrate that creatine supplementation in association with strength training amplifies the training-induced increase in the number of satellite cells (SC) and myonuclei in human skeletal muscle fibres. In response to strength training, both creatine (STR-CRE) and protein (STR-PRO) supplementation as well as unsupplemented training (STR-CON) were found to increase SC per fibre and relative number of satellite cells (Fig. 1). However, greater increases occurred with creatine supplementation at week 4 (compared to STR-CON) and at week 8 (compared to STR-PRO and STR-CON). Furthermore, creatine supplementation resulted in an increased number of myonuclei per fibre (Fig. 2) and in an amplified hypertrophy response to training as indicated by increased in MFA at week 4, 8 and 16 (Fig. 3), while protein supplementation caused MFA to increase at week 16 and unsupplemented training increased MFA at week 4 only.
Only few data exist on the change in SC number and activity in response to training in humans. In the present study, the unsupplemented strength training group (STR-CON) was comparable to the training groups used in previous studies (i.e. Kadi et al. 2004b), and from values around 5% at baseline (for all groups) the relative number of SC increased 27% and 44% in STR-CON at week 4 and 8, respectively. Similar increases were reported by Kadi et al. (2004b) where SC content increased by 1931% in response to unsupplemented strength training. Further, in the present study substantially larger gains in the relative number of SC were observed when strength training was combined with protein supplementation (+61% at week 4) and even more so with creatine supplementation (+84% and +99% at week 4 and 8, respectively), which yielded a peak SC content of 10% at week 8 (Fig. 1). Evidence exists to suggest that creatine supplementation can stimulate satellite cell proliferation in vitro (Vierck et al. 2003) and increase satellite cell mitotic activity (Dangott et al. 2000) (cellular effects are discussed in detail below), which could explain the present finding that relative SC content increased most markedly when strength training was combined with creatine supplementation. This effect may have been mediated, at least in part, via creatine-induced facilitation of myogenic regulatory factor (MRF) pathways (effects on MRFs are discussed in detail below). In previous training studies the relative proportion of SC increased 46% (from 3.7% to 5.4%) in the trapezius muscle following 10 weeks of resistance training in women (Kadi & Thornell, 2000). Likewise, Roth et al. (2001) found an increases of 18% (from 2.8% to 3.3%) in young men in response to 9 weeks of resistance training of the knee extensors. More recently, a 29% increased SC content (from 2.4% to 3.1%) was reported in elderly men in response to endurance training for 14 weeks (Charifi et al. 2003). These study differences in the relative proportion of SC both at baseline and in response to training may be explained by several factors. Firstly, age and training status of the subjects can affect baseline values of SC. It has been shown that SC proportion decrease with age, and possibly is further diminished by reduced physical activity with ageing (Kadi et al. 2004a). Secondly, differences in the magnitude and type of training may affect the magnitude of adaptive change in SC proportion. Based on its documented effect on muscle protein accretion, it is likely that strength training increases SC proportion more than endurance training. In the present study, both the intensity and the number of training sets were reduced in the initial phase of the strength training programme, while being progressively increased in the later weeks of training. Since the largest increases in SC content were observed already at week 4 in the present study it seems therefore that the duration and intensity of training are not the governing factors responsible for the increase in the relative proportion of SC, at least when training is combined with timed creatine or protein intake. Thirdly, SC content may differ between various muscles due to differences in fibre type composition and/or functional demand. And finally, different methods have been utilized, with immuno-histochemistry and light microscopy allowing analysis of a much larger number of fibres compared to electron microscopy.
As demonstrated for the first time, creatine supplementation induced superior gains in the number of SC and myonuclei with strength training in the present study (Fig. 1). This suggests an increased contribution of SC-derived myonuclei to the muscle fibres, which is expected to increase the capacity for mRNA transcription and thereby lead to elevated rates of myofibrillar protein synthesis (Kadi, 2000). In turn, this is likely to have contributed to the accelerated hypertrophy response presently observed in the creatine supplemented training group. In the present study, strength training without creatine or protein supplementation (i.e. STR-CON) did not lead to increases in number of myonuclei, in accordance with previous reports (Kadi et al. 2004b). Nevertheless, transient muscle fibre hypertrophy was observed in STR-CON despite the absence of elevated myonucleus number, as also reported previously (Kadi et al. 2004b). In contrast, creatine supplementation combined with training led to an elevated myonucleus number (+1417% at week 416), which was likely to be responsible for the accelerated time course and more marked muscle fibre hypertrophy observed in this training group (cf. Fig. 3). These amplified training responses were accompanied by a corresponding change in mechanical muscle function, since post-training maximum isometric muscle strength (MVC) was found to be greater when strength training was combined with creatine supplementation.
Previous studies have reported amplified muscle accretion and elevated fibre size gains in response to long-term strength training with creatine or protein supplementation. Thus, following 6 weeks of strength training lean tissue mass increased to a greater extent with combined creatineprotein compared to protein or carbohydrate supplementation, respectively, and for protein compared to carbohydrate supplementation (Burke et al. 2001). Similarly, amplified gains in muscle fibre size have been reported both in young (Andersen et al. 2005) and old individuals (Esmarck et al. 2001) when strength training was combined with timed intake of protein. In support of these findings, ingestion of amino acids results in a more positive net protein balance compared to carbohydrates when ingested acutely after exercise (Borsheim et al. 2004). Creatine supplementation in conjunction with strength training also appears to lead to greater gains in lean body mass (Vandenberghe et al. 1997; Kreider et al. 1998; Steenge, 1999), cross-sectional muscle area (Hespel et al. 2001) and single muscle fibre area (Volek et al. 1999; Becque et al. 2000) compared to carbohydrate supplementation alone.
In the present study a positive correlation between the training-induced increases in MFA and myonucleus number from baseline to week 16 was demonstrated with creatine supplementation (r = 0.67, P < 0.05). This finding supports that the ratio between myonucleus number and fibre cross-sectional area (myonuclear domain) remained constant during the process of myofibre hypertrophy when training was supplemented by creatine. The finding that SC content was no longer elevated at week 16 in STR-CRE (Fig. 1) suggests that creatine supplementation accelerated the incorporation of SC-derived myonuclei to the growing muscle fibres, establishing the appropriate myonuclear domain earlier than the other training groups. Interestingly, strength training with carbohydrate supplementation alone (STR-CON) transiently increased MFA at week 4 despite the lack of increased myonucleus number. As mentioned above, MFA has previously been found to increase along with no change in myonucleus number in response to unsupplemented training (Kadi et al. 2004b). Collectively therefore the findings of the present study indicate that while an increase in myonucleus number is not a permissive factor to achieve muscle fibre hypertrophy, it does seem to set the limit for fibre hypertrophy likely by regulating the nuclear domain of the muscle cell. Notably, the substantial increase in myonucleus number in the STR-CRE group appeared to be the result of training-mediated creatine action on myonucleus number that occurred independently of the change in fibre area.
Cellular effects of creatine supplementation recently have been documented, in which creatine was found to affect satellite cell proliferation and differentiation in cell cultures (Vierck et al. 2003). Furthermore it has been shown in rats that creatine supplementation during increased functional loading and compensatory hypertrophy (synergist ablation) induced increased satellite cell mitotic activity (Dangott et al. 2000). The increase in SC number, myonuclei and MFA in the present study supports a role for creatine in activating myogenic satellite cells, thereby adding nuclei and augmenting the training-induced accretion of muscle mass, especially in the early part of the time course of training. As an osmotically active substance creatine can cause water retention in the muscle fibres (Ziegenfuss et al. 1998), and increased osmotic pressure and resultant cell swelling due to increased creatine concentration and muscle glycogen content (Op't Eijnde et al. 2001a,b) may represent an anabolic stimulus on cellular protein synthesis (Haussinger, 1993), and further it may stimulate satellite cells to proliferate and fuse with the enlarging myofibres (Dangott et al. 2000). In the present study, muscle creatine concentration increased significantly in STR-CRE and was higher compared to STR-PRO and STR-CON at week 8 (data not shown). The myogenic effect of elevated muscle creatine concentration probably is linked to the activity of training, since creatine supplementation without training does not seem to lead to increases in satellite cell mitotic activity (Dangott et al. 2000) or muscle fibre area (Steenge, 1999). Recently, it was reported that creatine per se did not increase myofibrillar and sarcoplasmatic protein synthesis at rest or after an acute bout of exercise at a fixed absolute intensity (Louis et al. 2003a,b). However, these results do not exclude the possibility of increased transcriptional changes or enhanced activation of satellite cells when creatine intake and physical activity are combined (Rennie et al. 2004). Recent findings have supported the idea of a facilitating effect of creatine on skeletal muscle growth with training. Myogenin and MRF-4 mRNA and protein expression increased more after creatine supplementation compared to training alone after 12 weeks of resistance training (Willoughby & Rosene, 2003). These myogenic regulatory factors (MRFs) are thought to regulate muscle heavy chain (MHC) expression at the transcriptional level, and therefore up-regulation of MRF may lead to muscle accretion, which was supported by correlations between increase in myofibrillar protein and increased mRNA expression of Myo-D and myogenin (Willoughby & Nelson, 2002), although data on muscle size were not reported (Willoughby & Rosene, 2001, 2003).
It has been suggested that the enhanced muscle size gain observed when strength training is combined with creatine supplementation could be caused by a rise in training quality and/or greater total training load (Volek et al. 1999), which was supported by a higher total resistance load lifted by creatine supplemented subjects as reported by Steenge (1999). Such effect of increased work output during creatine supplementation could cause a greater than normal stimulus to muscle anabolism (Louis et al. 2003a). However, it is not obvious how increased hypertrophy should result merely from a marginally greater training intensity or volume in a progressive training programme, as the acute stimulation of muscle protein synthesis does not seem affected by the intensity of the preceding contractile activity (M. Rennie, personal communication; manuscript in preparation). Accordingly, in the present study total training load was not greater in subjects supplemented by creatine compared to the subjects supplemented by protein and placebo.
In conclusion, the present study is the first to demonstrate that creatine supplementation and to a lesser extent protein supplementation in combination with strength training augment the training-induced increase in the number of satellite cells and myonuclei in human skeletal muscle, resulting in enhanced muscle fibre growth. Furthermore, creatine supplementation appears to induce an early accelerated adaptation of satellite cells and myonuclei, which peaked at week 4 and 8 followed by a return of satellite cells to baseline levels at week 16 of training, while myonucleus number and myofibre area remained elevated.
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