Résumé :
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The metabolic alterations occurring in DMD cells have been little studied. This metabolic aspect of the disease has an obvious interest since metabolic alterations arising in muscle cells lead to an altered energy production that may contribute to increase the severity of the disease. The goal of this work is to identify the metabolic anomalies occurring in DMD cell and characterize the molecular bases of these changes. The consequences of these changes will also be studied in terms of muscle physiology. We also will investigate whether a supplementation with L-carnitine, a key molecule in energy production, can have a positive impact on DMD cells. Our objectives were to :1- Determine the modifications in lipid composition of human muscle cell membrane in Duchenne patients. 2- Characterize the physiological consequences of these alterations in terms of membrane structure and fluidity. 3- Identify the origins of these changes by identifying the metabolic pathways that are altered in DMD patient cells. 4- Determine if a supplementation in L-carnitine is able to counteract some of the deleterious effects of Duchenne disease on these metabolic parameters Our results are : 1 - In myotube cells derived either from patient or controls, fatty acid composition was determined in whole cells and in phospholipids. Globally, the content in fatty acid is not markedly altered. In patient cells, however, an increase in saturated and omega 6 fatty acid is found as well as a significant decrease in mono unsaturated fatty acid (especially omega 7). 2 - The consequence of the changes was evaluated by looking at membrane fluidity and stability. Membrane fluidity was estimated by fluorescence anisotropy using DPH. Flip flop events were evaluated in control and patient cells at different temperatures. At the physiological temperature of 37_C, DMD membrane cells were clearly less fluid. 3 - The metabolic pathways involved in such changes were analyzed by looking for alterations in the level of mRNA encoding key enzymes of the lipid metabolism. We found a decrease in the mRNA level of both mitochondrial and peroxisomal beta oxidation enzymes. 4 L-carnitine supplementation induced an increase in both the peroxisomal and the mitochondrial beta oxidations as detected by RT QPCR, L-carnitine also induced alterations in phospholipid composition with a partial restoration in omega 6 fatty acids levels and a further increase in saturated fatty acids. A slight increase in L-carnitine treated cells was also observed. All together, these results suggest that besides the lack of functional dystrophin, muscle cells from patients exhibit also alteration in the lipid properties of the plasma membrane that is associated with modifications of the membrane function. L-carnitine exerts some effects but its potential beneficial effect on DMD cells has to be more clearly defined.
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