1.
Specific Aims : The long-term objective of this project is to learn whether transmethylation of membrane phospholipids can modulate important functions of skeletal muscle, and whether abnormalities of transmethylation can lead to muscle disorders such as Duchenne muscular dystrophy. Transmethylation of phospholipids is a process that produces important changes in the properties of membranes. Recently, the studies of Axeirod and colleagues, have suggested that membrane phospholipid methylation may modulate vital cellular processes, including calcium transport, membrane receptor insertion and function, and regulation of membrane microviscosity in many tissues. These processes are important in the function of normal skeletal muscle; disturbances of these functions are postulated as pathophysiologic mechanisms in muscle diseases. For example, several membrane abnormalities have been described in Duchenne muscular dystrophy (DMD), including decreased calcium uptake by sarcoplasmic reticulum (SR), increased cytoplasmic calcium accumulation, and ultrastructural changes in sarcolemma. Though similar processes are modulated by methylation reactions in other tissues, phospholipid transmethylation has not been examined in skeletal muscle.
The aims of the proposed study are: A) To characterize phospholipid methylation in skeletal muscle; B) To evaluate its role in certain physiologic processes in muscle; and C) To examine phospholipid methylation in Duchenne muscular dystrophy. 2. Methods: Phospholipid methylation will be characterized in normal rat and human skeletal muscle membrane fractions enriched for SR and sarcolemma by assaying incorporation of 3H-methyl groups into membrane phospholipids. The specific products are then identified and measured by thin layer chromatography. Preliminary studies have shown that the assay is quantitative and reliable. Next, in order to evaluate the potential physiologic role of transmethylation in skeletal muscle, I will study the effect of added methylation substrate and methylation inhibitors on: 1) Calcium uptake by SR in vitro (measured by a 45Ca binding method), and on 2) The rate of insertion and degradation of acetylcholine receptor (AChR) in cultured normal rat muscle (measured by an 125l-Alpha-bungarotoxin binding technique). Finally, I will study human dystrophic muscle for possible alterations in phospholipid methylation. Specific activity in defined membrane fractions will be compared in DMD and control muscle obtained at diagnostic biopsy. Co-assay of membrane markers will control for relative purity of isolated membranes. I will also study phospholipid methylation in lymphocytes from DMD patients and controls, a strategy based on the role of phospholipid methylation in lymphocyte capping and the reported reduction of lymphocyte capping in DMD.
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