Regular articleSkeletal muscle properties in a transgenic mouse model for amyotrophic lateral sclerosis: effects of creatine treatment
Introduction
Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by degeneration and loss of upper and lower motoneurons. The identification of mutations in the cytosolic copper-zinc superoxide dismutase (SOD1) gene in a number of cases of familial ALS (Rosen et al., 1993), and the subsequent generation of transgenic mice overexpressing human mutant SOD1, have provided an adequate model to explore the pathogenic mechanisms causing the disease Gurney et al 1994, Nagai et al 2001. The precise mechanism by which SOD1 mutations induce cell death of upper and lower motoneurons is presently unclear (Brown and Robberecht, 2001), but damage to mitochondrial structures has been suggested to play a role (Beal, 2000). Recent studies on humans and animals have identified morphological abnormalities in mitochondria as (early) pathologic features of ALS Kong and Xu 1998, Borthwick et al 1999, Vielhaber et al 2000. It is hypothesized that a toxic gain of function of mutant SOD1, located in the mitochondrial intermembrane space, causes excessive generation of free radicals Julien 2001, Okado-Matsumoto and Fridovich 2001. Consequently, the oxidative damage of mitochondrial proteins, lipids and DNA causes impairment of oxidative phosphorylation and defective energy production Vielhaber et al 2000, Mattiazzi et al 2002. The resulting cellular energy depletion is thought to increase the vulnerability to indirect excitotoxicity and postsynaptic motoneuron death (Ludolph et al., 2000). A recent alternative hypothesis suggests that mutant SOD1 induces apoptosis, possibly by binding with heat shock proteins and thereby reducing their antiapoptotic function (Okado-Matsumoto and Fridovich, 2002).
Creatine plays a pivotal role in the maintenance of cellular energy homeostasis by providing a direct energy source in the form of phosphocreatine, as well as by providing an energy transfer mechanism (shuttle) between mitochondria and sites of high energy turnover (Wallimann et al., 1992). It has therefore been postulated that increased stores of creatine in neuronal and muscle tissue may provide a protective effect on cellular energy dysfunction in neurodegenerative diseases (Tarnopolsky and Beal, 2001). In order to test this hypothesis, creatine has been orally supplemented to SOD1 mutant mice. Interestingly, creatine treatment has led to reductions in cortical glutamate concentrations, reduced motoneuron loss, and increased survival in creatine versus control treated animals Klivenyi et al 1999, Andreassen et al 2001b.
SOD1 is a metalloenzyme that is ubiquitously expressed. The induction of mutant SOD1 in transgenic mice may therefore also cause abnormalities in nonneuronal tissues, which even may contribute to the disease progression. Indeed, it was recently shown that the exclusive expression of mutant SOD1 in neurons or glial cells but not in other cells of transgenic mice, does not lead to neurodegeneration, and these mice attain normal live expectancy, which indicates that mutant SOD1 expression in nonneuronal tissues contributes to disease in this model (Pramatarova et al., 2001). Interestingly, evidence for mitochondrial dysfunction which occurred independent of motoneuron loss, has also been observed in skeletal muscles of mutant SOD1 mice (Leclerc et al., 2001). Therefore, it could be hypothesized that expression of mutant SOD1 in skeletal muscle also causes muscle fiber degeneration, through a mechanism independent of motoneuron loss. Oral creatine supplementation, which is known to increase creatine content and the energy status in skeletal muscles (Harris et al., 1992), may therefore affect muscle function in SOD1 mutant mice, not only by rescue of motoneurons (Klivenyi et al., 1999), but also through a direct effect on muscle by improvement of its energy homeostasis.
A number of direct effects of oral creatine supplementation on skeletal muscle function have been observed in healthy and pathological states (Hespel et al., 2001). In healthy muscle, creatine supplementation can result in increased fuel availability in the form of glycogen and phosphocreatine, which may explain shorter relaxation times and faster recovery following muscle contractions Van Leemputte et al 1999, Op’t Eijnde et al 2001. In addition, creatine supplementation has been found to beneficially impact on McArdle’s disease and muscular dystrophy Vorgerd et al 2000, Felber et al 2000. Thus, along this line of evidence, creatine supplementation could be a symptomatic therapy for impaired skeletal muscle function in ALS.
The functional capacity and contractile characteristics of skeletal muscle in patients with ALS have been intensively investigated by R. G. Miller and coworkers Sharma et al 1995, Sharma and Miller 1996, Kent-Braun et al 1998, Kent-Braun and Miller 2000. By comparing nerve-stimulated contractions with voluntary contractions, they have shown that muscle weakness, increased fatigue and slowing of contraction and relaxation speed in ALS muscles can, to an important degree, be ascribed to upper motoneuron dysfunction Kent-Braun et al 1998, Kent-Braun and Miller 2000. At the same time, their data indicate that another part of the muscle weakness and fatigue originates from defects distal to the myoneural junction; e.g., impaired excitation-contraction coupling or metabolic changes in muscle fibers (Sharma and Miller, 1996). Recently, Krivickas et al. have investigated the contractile properties of chemically skinned single skeletal muscle fibers from ALS patients and age-matched controls (Krivickas et al., 2002). They found that during Ca2+-induced contractions, specific force was unaltered and maximal shortening velocity was even slightly increased in ALS fibers compared to controls (Krivickas et al., 2002).
The aims of the present study were to investigate in a transgenic mutant SOD1 (G93A) mouse model of ALS: 1) the changes in contractile and metabolic properties of fast-twitch and slow-twitch skeletal muscles, and 2) the effects of oral creatine supplementation hereon and on the in vivo motor function.
Section snippets
Animals
Transgenic mice carrying the G93A human SOD1 mutation (G93A) or the wild type human SOD1 (WT) transgene were obtained from Jackson Laboratories (Bar Harbor, Maine). Female littermates were mated with male G93A mice and the offspring was genotyped by PCR assay of DNA extracted from tail tissue. Comparison was made with animals with an overexpression of the wild type human SOD1 gene (WT). Thirty G93A mice and 20 WT mice were included in the study. Ten G93A mice were sacrificed for testing of
In vivo testing
Body weight was 10–15% higher in WT compared to G93A (P < 0.05) at the start of the study (72 days of age), and the difference increased to 20–25% by the age of 120 days. Rotorod performance was similar between WT and G93A at the age of 72 days (see Fig. 1). The linear slope of rotorod performance over time was −0.6 ± 2.4 in G93A, compared to 1.1 ± 1.3 in WT (P < 0.05 for G93A versus WT). This indicated that motor coordination deteriorated in G93A mice with disease progression, whereas it
Muscular phenotype of G93A mice
In this study we provide a detailed description of the functional and metabolic characteristics of skeletal muscles with varying fiber type composition in G93A mice. It is known that in this animal model of ALS, muscle weakness occurs at an earlier stage of the disease in fast-twitch than in slow-twitch muscles Chiu et al 1995, Frey et al 2000. Accordingly, we now show that at the age of 120 days, atrophy occurred in the fast-twitch hindlimb muscles (EDL) but not in slow-twitch soleus muscles.
Acknowledgements
The mice pellets were kindly provided by Degussa AG (Trostberg, Germany). The practical contribution by Monique Ramaekers is greatly appreciated. This study is supported by grants from ‘Onderzoeksraad K.U.-Leuven’ (grant #OT99/38) and from the Flemish’ Fonds voor Wetenschappelijk Onderzoek Vlaanderen’ (FWO-Vlaanderen grant #G.0255.01). B.O. obtained a research fellowship from the Onderzoeksraad K.U. Leuven (grant #OT99/38). W.D. and L.V.D.B. are recipients of a post-doctoral fellowship, and
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