Abnormalities in the mechanisms of calcium regulation and excitation-contraction (EC) coupling that may be linked to the degeneration of skeletal muscle fibers in Becker Muscular Dystrophy (BMD) and in Duchenne Muscular Dystrophy (DMD) will be investigated using isolated muscle fibers from mdx mice. Cells from this animal model, like those of dystrophic patients, have deficiencies in the expression of the protein dystrophin. Although there is substantial biochemical evidence demonstrating the association of the dystrophin-glycoprotein complex with transmembrane- and membrane-bound muscle proteins, little is known about its specific role in the physiological aspects of a muscle fiber. The main goal of this proposal is to obtain critical experimental evidence linking the absence of dystrophin with specific alterations in the electrical propagation in the transverse tubular system and calcium signaling machinery. Several possibilities that may explain these observations will be explored experimentally. Changes in intracellular calcium concentration triggered by electrical activity of the muscle fibers will be recorded with the aid of low affinity calcium sensitive fluorescent indicators and membrane potential changes in the transverse tubules will be monitored with potentiometric indicators. The investigations will be carried out using high-resolution optical methods that permit to assess the functional state of these critical steps of the EC coupling process, not only at the cellular level, but also within sub-regions of the muscle fiber and even within a single sarcomere. We will perform these measurements across three different age groups of the mdx mouse in order to understand the progression of the disease with time. We will also test if muscle fibers from a utrophin/dystrophin-lacking double mutant mouse, which exhibits a harsher pathology (similar to DMD), show signs of more pronounced defects in EC coupling. These types of experiments are necessary to unravel the mysterious role that dystrophin may play in the normal regulation of calcium metabolism in skeletal muscle. The knowledge gained in the proposed studies will help to elucidate the functional role of dystrophin in mammalian skeletal muscle, to this date the most fundamental and elusive problem in muscular dystrophy research. The enhanced methods proposed to detect defective steps in the EC coupling mechanisms within localized submicroscopic regions of mammalian muscle fibers may become the optimal choice for the future evaluation of genetic therapeutic procedures in sub-regions of a single muscle cell. ? ?
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