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1.3.5.2.1 Creatine

Increasing intramuscular PCr levels via oral Cr loading has been well documented (Harris et al., 1992; Smith et al., 1999; Op’T Eijnde et al., 2001) and has become a mainstay of athletes seeking putative performance enhancement – especially with respect to strength, speed and power. Various physiological benefits have been attributed to increases in the TCr status of muscle, including enhanced mitochondrial ATP flux via the PCr shuttle (Bessman & Geiger, 1981; Bessman & Carpenter, 1985; Kammermeier, 1987; Balsom et al., 1994), stimulation of glycolysis (Krzanowski & Matschinsky, 1969; Ceddia & Sweeney, 2004), improved buffering of both the ATP:ADP ratio via the phosphagen system (Hultman et al., 1996; Greenhaff et al., 1997; Kreider, 2003) and intramuscular pH (Walliman et al., 1992), and enhanced protein synthesis (particularly when accompanied with resistance exercise (Willoughby & Rosene, 2001). It is particularly interesting to note that each of these measures is suggestively impaired in dystrophic muscle, and thus highlights the potential benefits of such a therapy for the treatment of DMD.

As such, several studies have examined the efficacy of Cr supplementation in both human DMD (Tarnopolsky et al., 1999; 2000; 2004; Walter et al., 2000; Louis et al., 2003) and mdx muscle (Pulido et al., 1998; Passaquin et al., 2002; Louis et al., 2004). Louis et al. (2003) have demonstrated beneficial effects of a 5-week Cr supplementation protocol in measures of muscle performance in DMD patients – specifically in doubling the time to fatigue at 75% maximum voluntary contraction (MVC) and allaying the observed increase in total joint stiffness observed in controls

over the 3-month trial. The authors did not observe increases in lean body mass, reduction in serum CK levels or changes in creatinine excretion rate, and suggest that Cr may provide alternative benefits aside from enhancing cellular energetics (Louis et al., 2003). Tarnopolsky et al. (2004) have reported similar improvements in dominant handgrip strength and the maintenance of strength over time, albeit no improvement in functional task decline or measures of pulmonary function. A particularly interesting feature of this study was the finding of significantly increased fat-free mass after a four- month supplementation protocol, although this was not mirrored by a decrease in body fat percentage (Tarnopolsky et al., 2004).

Beneficial effects have also been observed consistently in mdx muscle. Louis et al. (2004) have reported a 12% increase in EDL TCr levels after a 30-day Cr supplementation protocol that effectively restored normal resting levels (as depicted by controls). This corresponded histologically, with a reduction in hypertrophic muscle mass increases and mean fibre surface area. In this study, Cr supplementation had no protective effect on susceptibility to stretch-induced fibre injury or the accumulation of centrally nucleated (regenerative) fibres in adult EDL muscle, and induced a significant increase in half relaxation time (½ RT) in both mdx and control groups (Louis et al., 2004). These findings were observed in addition to a highly significant increase in total Ca2+ _{content of Cr-supplemented mdx gastrocnemius (Louis et al., 2004) that was not}

observed in Cr-supplemented controls, indicating that Cr-supplementation failed to allay the dystrophic phenotype. In direct contrast, Pulido et al. (1998) has demonstrated the significant inhibition of dystrophy-induced [Ca2+_]

c elevation in mdx myotubes after

several days of Cr supplementation, which improved survival rates. An extension of this research by Passaquin et al. (2002) supported their findings, demonstrating delayed

onset and reduced severity of initial degenerative cycles in young mice, which was accompanied by enhanced mitochondrial oxidative function. Whilst the findings of Pulido

et al. (1998) and Passaquin et al. (2002) differ from those of Louis et al. (2004) with respect to the effects of Cr-supplementation on dystrophic [Ca2+_{], it is possible that the}

absolute gastrocnemius [Ca2+_{] ascertained by Louis et al. (2004) is misrepresentative of}

the fact that Cr supplementation might increase cell survival duration by increasing the buffering of Ca2+ _{from the sarcoplasm intro subcellular compartments and retaining it}

within. Thus whilst a greater net influx of Ca2+ _{into myofibres would occur, high [Ca}2+_] c

would be significantly decreased by the better Ca2+ _{buffering capacity afforded by Cr,}

whilst at the same time, the total (cytosolic + compartmentalised) [Ca2+_]

i would be

significantly increased. That CK is demonstrably linked to SERCA indeed indicates that improved uptake into the SR is likely (Rossi et al., 1990; Minajeva et al., 1996).

Indeed, Pulido et al. (1998) has published the only study to date investigating the effect of Cr supplementation on intracellular Ca2+ _{handling in dystrophic muscle.}

Cultured mdx myotubes were supplemented with Cr at the onset of myocyte fusion and cytoplasmic [Ca2+_{] was quantified using the Ca}2+_{-specific fluorophore Fura-2. Cr was}

shown to significantly inhibit dystrophy-induced [Ca2+_]

c elevation after several days of

supplementation, which was attributed to enhanced SR Ca2+ _{ATPase activity, albeit}

direct measurement of SR Ca2+ _{was not made and thus improvements in sarcolemmal}

(SL) and mitochondrial ATPase activity could not be ruled out. Ca2+ _{influx rates remained}

unaffected between Cr-supplemented and unsupplemented groups, indicating definite improvements in Ca2+ _{buffering capacity rather than a reduction in entry (Pulido et al.,}

The collection of studies investigating the therapeutic benefits of Cr supplementation for the treatment of DMD have thus far indicated promise and the need for further research to clearly elucidate underlying physiological mechanisms of effect. With respect to Cr, this thesis aims at (1) ascertaining whether improvement of the SR Ca2+ _{uptake mechanism is a mode of effect of Cr supplementation in dystrophic muscle;}

(2) the degree to which Cr supplementation may allay or reduce the severity of degenerative bouts in mdx skeletal muscle, and; (3) whether Cr supplementation can influence total muscle protein and/or sub-cellular compartmental protein content.

1.3.3.5.2 Whey Protein

An extract of soluble protein fractions from bovine milk, whey protein (WP) supplements (much like creatine) are becoming increasing popular as a proposed method of enhancing muscle anabolism and hence athletic performance. It has been suggested that WP offers considerable benefits over other high quality protein sources by constituting a higher concentration of essential AAs (45-55mg/100g) (Bucci & Unlu, 2000), and thus conveying a higher biological value – descriptive of its ability to provide and retain nitrogen in a balanced interplay of essential and non-essential AAs (Walzem

et al., 2002). In comparison to the other high quality dairy protein, casein, WP also claims a higher protein efficiency ratio (PER) of 2.6 versus 3.2, thus eliciting a larger rate of weight gain per gram of protein consumed over time (Walzem et al., 2002). Indeed, the unique physical properties of WP in the gastrointestinal system are likely to convey its benefits – unlike casein that clots in the stomach resulting in slowed release into the small intestine, and thus significant hydrolysis prior to absorption, WP

immediately enters the small intestine in which its progressive hydrolysis is relatively slow, providing a unique delivery of both AAs and peptides (Mahe et al., 1996; Boirie et al., 1997). Because of this difference, WP consumption elicits an acute peak in serum AA concentration (Boirie et al., 1997; Dangin et al., 2001; 2003) that when accompanied with mixed macronutrients, demonstrably induces increased rates of muscle protein synthesis and net gains in systemic protein deposition (Dangin et al., 2003). WP may also offer various other benefits specific to its high concentration of both the sulphur- containing AA cysteine for its immunological and anti-oxidant status-modulating role (Bounous & Gold, 1991), and branched-chain AA leucine for its skeletal muscle protein regulating role (Carbo et al., 1996; Anthony et al., 2001).

Whilst no study has yet examined the effect of WP (or casein) supplementation on dystrophic muscle preservation, it is clear that increasing systemic AA status would be of immense benefit, particularly if the aforementioned hypothesis of dystrophinopathy-induced AA deficit is true. Several studies have indicated the benefits of AA supplementation in DMD (Stewart et al., 1982; Granchelli, et al., 2000) and in other muscle wasting conditions including cancer cachexia (Ventrucci et al., 2004) and ischaemia-reperfusion injury (Scarabelli et al., 2004), which certainly highlights the potential in supplying a constant, combined supply of all essential and non-essential AAs to dystrophic muscle.