6.6 TALLERES
6.6.4 TALLER N° 4 Conociendo nuestros cuerpos
proteins
The present study is the first to demonstrate that taurine supplementation in the mdx
mouse alters the expression of contractile and E-C coupling proteins, as well as the SR Ca2+ binding protein CSQ. In fact, this is the first study to examine actin and myosin protein expression and E-C coupling proteins in untreated mdx mice during the acute phase of degeneration, and then during the stabilised phase of muscle damage/repair. Investigation of contractile protein and E-C coupling expression during these phases is important, as the acute phase of damage in the mdx model more closely mimics the human DMD condition, while at day 70 compensatory mechanisms that result in functional recovery in the mdx mouse are likely to have occurred, and thus protein expression may be altered as part of this process.
Treatment with taurine appears to be protective of contractile protein expression, as it prevented the decline in myosin expression observed in MDX mice at day 70, while significantly increasing actin expression such that it was significantly increased compared to MDX at day 28 and 70 (see Figures 4.5 and 4.6). Contractile proteins are thought to be damaged as a result of increased proteolysis in dystrophic tissue, and derangement of motor protein structure and activation have been observed in dystrophic muscle (Friedrich et al., 2010). The protection of contractile filaments with taurine supplementation in mdx muscles may explain, in part, why taurine has been shown to have beneficial effects on contractile function and force output in the mdx mouse (Cozzoli et al., 2011b, De Luca et al., 2003).
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While taurine supplementation increases contractile proteins in the mdx mouse, E-C coupling proteins were variably affected by taurine supplementation. DHPR expression was unaltered by taurine treatment at both day 28 and day 70The limited studies available in the literature on DHPR protein expression in mdx skeletal muscle confirm results from the present study, namely that DHPR expression is unaltered in dystrophic skeletal muscle (Desnuelle et al., 1986, Pereon et al., 1997).
RyR protein expression was altered, with the already significant decline in RyR expression at day 28 in mdx muscle being exacerbated by taurine treatment, resulting in significantly less RyR protein expression within the TAU group. The finding that dystrophic muscle displays a decrease in RyR protein expression is as expected, as both structural and functional deficits in RyR have been reported within dystrophic cardiac and skeletal muscle, although there is less information available on protein expression (Bellinger et al., 2009, Fauconnier et al., 2010, Morel et al., 2004). Bellinger et al. (2009) identified alterations to RyR structure and function that contributed to the increased cytosolic Ca2+ concentration observed in mdx muscle, due to increased leak from RyR channels resulting from nitric oxide-mediated modifications causing increased S-nitrosylation of cystine residues on the RyR. Reductions in RyR protein expression in
mdx mice may explain the decrease in force production reported during the acute damage phase at day 28, although the effect of taurine treatment causing further reductions to RyR expression is unexpected. It is possible that RyR protein expression is reduced in untreated mdx at day 28 as a protective mechanism, as at this time point there is significant muscle degeneration due to a cascade of events such as increased protease activity, ROS production and inflammation, all of which are driven by excessive Ca2+ accumulation in skeletal muscle (Bakker et al., 1993, Emery, 2003, Fong et al., 1990, Williams et al., 1990, Yeung et al., 2005). Perhaps decreasing the expression of RyR at day 28 limits Ca2+ entry into dystrophic muscle that occurs due to increased Ca2+ leak from the RyR, which has been shown to be exacerbated under conditions of increased oxidative stress, as would be the case at this time point (Bellinger et al., 2009, Brookes et al., 2004, Whitehead et al., 2006). While reduced RyR expression, and thus less capacity for Ca2+ release from the SR, would also decrease force production, due to the extensive muscle damage and necrosis that occurs in the 3rd to 4th week of life in the mdx mouse this reduction in contractile activity may again be protective, limiting
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damage to muscle fibres that are known to have increased susceptibility to contraction induced damage (Brussee et al., 1997, Selsby, 2011, Vilquin et al., 1998). Moreover, this loss of contractile function may also be compensated for by the increased expression of contractile proteins. Thus, taurine is potentially increasing this adaptive response during this stage of the mdx lifespan to further protect skeletal muscle from damage. The fact that there is no significant difference in RyR expression between CON, MDX or TAU by day 70 where muscle damage and necrosis is decreased, supports this theory.
Previous investigations within the mdx mouse have demonstrated no significant difference in SERCA expression (Dowling et al., 2004), although there is some suggestion that the activity of SERCA is reduced in dystrophic muscle causing impaired Ca2+ handling and contractile function (Divet et al., 2005, Kargacin and Kargacin, 1996). For example, Divet et al. (2005) found that within fast-twitch muscles of the mdx mouse, there was a switch to the slower isoform of SERCA leading to reduced Ca2+ uptake. Moreover, a recent investigation by Morine et al. (2010) showed that SERCA overexpression in the mdx DIA significantly improved Ca2+ handling and decreased muscle damage. The present study found no significant difference inSERCA expression in association with taurine treatment at day 28 or day 70 in the mdx mouse, while no difference in SERCA expression was found between CON and the untreated MDX groups. Four weeks of taurine treatment in rats also had no effect on SERCA expression (Goodman et al., 2009). Given that there is no significant muscle degeneration occurring due to Ca2+-dependant processes in the rat, the mdx model at day 70 would be more comparable to the rat model employed by Goodman et al. (2009), as skeletal muscle damage is stabilised to a low level at this time, thus yielding similar results.
Decreases in Ca2+ binding proteins within dystrophic tissue have previously been reported within the literature, causing drastic reductions in the Ca2+ buffering capacity of the SR (Culligan et al., 2002, Dowling et al., 2004). It is proposed that this alteration to Ca2+ binding proteins in dystrophic skeletal muscle is a significant contributor to the development of pathogenesis in DMD, as increasing the expression of Ca2+ binding proteins reduces muscle necrosis. For example, examination of the effect of two Ca2+ channel blockers on skeletal and cardiac muscle damage in the mdx mouse found an attenuation of the rise in intracellular Ca2+ concentration in mdx mice, and increased CSQ expression, which was associated with reduced degree of muscle damage
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(Matsumura et al., 2009). Moreover, Ferretti et al. (2009) found that the relatively spared intrinsic laryngeal muscles of the mdx mouse naturally contain higher expression of CSQ, in addition to greater expression of SERCA.
In the present study, CSQ expression was not significantly different at day 28 between CON, MDX or TAU groups, however, at day 70, there was a significant decrease in CSQ protein expression in mdx mice, while taurine supplementation significantly increased CSQ expression, such that there was no significant difference between the control and taurine-supplemented mdx. This increase in CSQ protein expression at day 70 improves the capacity of the SR to store Ca2+, which could improve intracellular Ca2+ handling in dystrophic skeletal muscle and limit Ca2+ driven damage. Elevated CSQ expression in association with taurine treatment could also result in improved contractile function. Indeed, Goodman et al. (2009) found a 49% increase in CSQ protein content in rats after 2 weeks of taurine supplementation via drinking water. Associated with this reported increase in CSQ was increased peak tetanic and twitch force, as well as improved recovery after a high-frequency continuous stimulation to fatigue (Goodman et al., 2009).
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4.6 Conclusions
In summary, this is the first study to demonstrate that taurine supplementation is able to significantly increase skeletal muscle taurine content during peak degeneration, through to recovery in the mdx mouse to a normal healthy control level. Taurine has been shown to alter skeletal muscle development in the mdx mouse, resulting in less muscle mass when compared to non-dystrophic control mice, although this may result in better protection of skeletal muscle from damage. TauT protein expression was found to be lower in dystrophic mdx mice, and as hypothesised expression was unaltered in association with taurine supplementation, suggesting that some of the impairment in retaining normal skeletal muscle taurine concentration may be, in part, due to alterations in TauT expression in dystrophic skeletal muscle.
E-C coupling and contractile proteins are also affected by taurine supplementation, with actin and CSQ all found to be significantly altered in association with taurine treatment. Due to the large increase in CSQ expression, it is possible that taurine may be having positive effects on muscle function via improvements to Ca2+ handling. Thus, there is a need for the functional properties of taurine supplemented mdx mice to be examined.
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