Impaired metabolism in X-linked muscular dystrophy: experimental evaluation of potential therapies to improve calcium regulation, bioenergetics and muscle architecture
Rybalka, Emma (2007) Impaired metabolism in X-linked muscular dystrophy: experimental evaluation of potential therapies to improve calcium regulation, bioenergetics and muscle architecture. PhD thesis, Victoria University.
Duchenne Muscular Dystrophy (DMD) is a severe and progressive skeletal muscle wasting disease characterised by [Ca2+]-induced hyper-catabolism, and subsequently, a higher demand for energy production to modulate intracellular Ca2+ homeostasis, and protein degradation and synthesis pathways. The broad aim of this thesis was to elucidate potential defects in metabolism of the C57BL/10 mdx mouse model of DMD, and to determine the role of Ca2+ in any such defects. In particular, this thesis has examined the efficacy of the nutritional supplements creatine (Cr) and to a lesser extent, isolated whey protein (WP), in improving intracellular Ca2+ regulation, energy and protein balance and tissue architecture, thus alleviating a degree of the dystrophic pathology. 1. Whether the ATP-producing capacity of dystrophic mitochondria is defective remains largely contentious, and no study to date has directly quantified and contrasted ATP-production rate under the major macronutrient pathways feeding the mitochondria in either mdx hind limb or the more human DMD phenotype-like diaphragm. Chapter Three has demonstrated severely depressed mitochondrial ATP production rate (MAPR) of mdx diaphragm across all macronutrient substrate pathways, but to a lesser extent under protein metabolism, and in tibialis anterior (TA) across the sum of all macronutrient substrate pathways. Function of the electron transport chain complex II was not impaired in dystrophic diaphragm, but was notably depressed in TA. Citrate synthase activity was comparable to controls in both muscles, as was mitochondrial protein content. However, the susceptibility of dystrophic mitochondrial to mechanical damage during the mitochondria isolation process was significantly greater in mdx compared to controls, and in diaphragm compared to TA. It is postulated that mdx skeletal muscle has an intrinsic inability to utilise macronutrient substrates leading to substrate “back-up” and inhibition/down-regulation of key Krebs’ cycle enzymes, thus creating an intracellular “starvation” scenario. That protein metabolism was less affected than the other macronutrient substrate pathways suggests that a portion of the muscle hyper-catabolism observed in DMD may occur due to autophagy to increase amino acid funnelling to mitochondria for ATP production. 2. Supplementation of the high-energy storage nutrient Cr is demonstrably beneficial in maintaining muscle function, energy status and cell survival rate in human DMD and dystrophic mdx skeletal muscle. Long-term chronic dose supplementation regimes, however, have been associated with down-regulation of creatine transporter (CreaT) expression thus making such regimes futile in maintaining consistently high intramuscular PCr stores. Chapter Four has demonstrated successful prevention of CreaT mRNA down-regulation by a chronic dose in utero and life-long Cr supplementation protocol, and subsequently persistently elevated [PCr] compared to unsupplemented mdx skeletal muscle. This was associated with a drastically reduced amount of muscle damage as depicted by Evan’s blue dye (EBD) uptake into myofibres, and a lesser degree of damage to those fibres that were permeant to EBD. This unequivocally demonstrates that muscle wasting occurs secondary to metabolic compromise and failure to maintain ATP supply to intracellular mechanisms that promote cell survival. 3. Effective intracellular Ca2+ regulation by the sarcoplasmic reticulum (SR) is integral to muscle function and becomes of paramount importance to the maintenance of cell survival in conditions of increasing [Ca2+]i such as that evident in DMD. Chapter Five details an optimised method for the fluorometric quantitation of SR Ca2+ flux kinetics currently utilised by our laboratory. It was demonstrated that the SR Ca2+ ATPase (SERCA) preferentially utilises ATP produced by a linked creatine kinase (CK) system over both exogenously administered ATP and ATP produced by SR-linked glycolytic enzymes, and as such, SR Ca2+ uptake rate was considerably faster under these conditions. It was also demonstrated that high [ADP] proximal to the SR vesicles impairs the binding of Ca2+ to Fura-2 and that the presence of 25mM PCr and low [ADP] drastically reduces passive Ca2+ leak from the SR. Thus, this study provides sound rationale for the use of Cr supplementation to improve intracellular Ca2+ handling by the SR subsequent to increasing PCr stores and the buffering of rising [ADP] during metabolic compromise. 4. It has been suggested that the beneficial effects observed in human DMD and dystrophic mdx skeletal muscle following Cr supplementation result from an improved capacity for intracellular Ca2+ handling by the SR and, therefore, delayed degenerative progression. Chapter Six has demonstrated no direct modulatory effect for Cr supplementation on SERCA or RyR function such to increase/decrease SR Ca2+ uptake, leak or release rates, but postulates that the benefit imparted by Cr supplementation is in maintaining maximum uptake (and subsequently release) velocity secondary to buffering rising [ADP] and collapse of the ATP:ADP ratio proximal to the SR. This study has also investigated the effects of supplementation with WP and a Cr+WP combination. As with Cr supplementation, WP induced no direct modulation of SR Ca2+ flux kinetics. Both supplements were shown to modulate differential expression of specific intracellular protein pools, although not necessarily net protein accretion. It is speculated that modification of expressed intracellular protein pools permits the “switching” of dystrophic mdx skeletal muscle from a “functional” phenotype to a “cell survival” phenotype, and that this is inhibited in unsupplemented muscle by a lack of amino acid and energy resources. It was also apparent that Cr and WP supplementation exerted different effects on tissue architecture maintenance. Cr supplementation increased the proportional area of functional muscle tissue and decreased non-muscle “gap” areas thus suggesting a role in muscle hypertrophy, where as Cr+WP combined increased active/recent degeneration and regeneration and the proportion of centrally-nucleated previously damaged fibres compared to peripherally-nucleated undamaged fibres in functional muscle tissue, thus indicating a potentially damaging effect. Collectively, the studies comprising this thesis indicate that the progressive skeletal muscle wasting evident in DMD is closely related to dissipating energy stores, and that the primary disease pathology may, therefore, be an intrinsic metabolic defect caused by the DMD genotype that subsequently induces Ca2+ dys-regulation. Cr supplementation was shown to provide several benefits to dystrophic mdx skeletal muscle architecture, including reduced severity of degenerative cycles, maintenance of muscle tissue and reduced proportional area of non-muscle tissue secondary to increased intramuscular [PCr]. The findings of Chapter Five suggest that Cr supplementation modulates its effect by maintaining normal [PCr] and a high ATP:ADP and PCr:Cr proximal to the SR, such to maintain maximum Ca2+ uptake velocity and reduce passive Ca2+ leak. Both Cr and WP supplementation also seem to modulate intracellular protein synthesis such to increase the capacity for myofibre survival. Thus, these supplements could be of benefit in the adjunct treatment of DMD, and warrant further investigation as to their long-term and mechanistic efficacy.
|Item Type:||Thesis (PhD thesis)|
|Uncontrolled Keywords:||ResPubID16265, Duchenne muscular dystrophy, metabolism, calcium, bioenergetics, muscle architecture, nutritional supplements, creatine, whey protein|
|Subjects:||RFCD Classification > 320000 Medical and Health Sciences
Faculty/School/Research Centre/Department > School of Biomedical and Health Sciences
|Depositing User:||Ms Lyn Wade|
|Date Deposited:||22 Oct 2008 04:23|
|Last Modified:||23 May 2013 16:40|
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