Duchenne muscular dystrophy is a common genetic disorder resulting from mutations within the dystrophin gene. It is likely that the dystrophic phenotype does not result directly from alteration to the myofibrillar structures, rather it is a disruption of sarcolemmal membrane integrity that normally confers tight control of intracellular Ca homeostasis, which leads to elevated intracellular Ca and eventual muscle degeneration. The pathophysiological mechanism responsible for elevation of intracellular Ca levels is not clear, however our recent findings suggest that store-operated Ca entry (SOCE) linked to uncontrolled Ca spark activity may contribute to the aberrant Ca influx observed in dystrophic muscle. While the mechanism of SOCE activation is still a matter of intensive study, it has been recently determined that the Ca insensitive phospholipase A2 (iPLA2) is an important mediator of SOCE. The focus of this project is to test the hypothesis that aberrant Ca spark activity acts as a trigger for SOCE and thus induces a dystrophic cascade in mammalian skeletal muscle through a pathway that involves IPLA2 mediated signaling. We will test this with three specific aims:
Aim 1 : To establish induced Ca sparks as a trigger for SOCE in healthy and dystrophic muscle. We will utilize multiple methods for measurement of SOCE to examine alteration to Ca : entry in dystrophic fiber. Patch-clamp measurement and modulation of Ca influx will provide insight into SR Ca release and the activation of SOCE in dystrophic fibers.
Aim 2 : Determine the contribution of IPLA2 activity to Ca influx in muscular dystrophy. We will examine the altered characteristics of JPLA2 function in dystrophic muscle using various in vitro molecular and pharmacological methods.
Aim 3 : To elucidate if SOCE facilitates the dystrophic cascade in skeletal muscle. Due to the inability to assess changes in the dystrophic phenotype in skeletal muscle we will modulate SOCE in dystrophic animals and assay changes in dystrophic phenotypes. This project will contribute to our understanding of how muscle damage occurs in cases of Duchenne muscular dystrophy (DMD), the most common genetic disease in the United States. Our experiments will establish that defects in how calcium enters muscle cells result in the development of DMD. Such knowledge will suggest new methods to treat DMD, some of which will be tested on experimental animals in this study.
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