SCHIAVI, JM*; COUGHLIN, DJ; LUTZ, GJ; Widener University; Widener University; Drexel University: Cloning of Rainbow Trout Red Muscle Regulatory Myosin Light Chain and an in vivo System to Study its Function

Skeletal muscle myosin consists of the myosin light chain (MLC) and heavy chain (MHC) subunits. MLC and MHC are the primary determinants of contractile performance. Although the role of MHC in motor function is clear, the purpose of MLCs remains obscure. The regulatory MLC (RLC) is thought to be responsible for muscle sensitivity to calcium the resultant contraction kinetics. Our goal is to examine how RLC modulates contractile performance. We aim to study RLC structure and function by expressing recombinant RLC in vivo and determining contractile properties of the transgenic muscle cells. We have cloned the full-length rainbow trout RLC and assembled it into an in vivo expression vector. RLC was cloned from red muscle total RNA in two fragments using RT-PCR and RACE. Full-length RLC was then produced by PCR with a small epitope tag at its 3� end and placed into expression vector containing a CMV promoter. The epitope tag was selected to be minimally invasive (i.e., not interfere with expression) but large enough to result in a gel shift to distinguish it from the endogenous RLC on Western Blots. We are developing a system for in vivo gene transfer and expression of the trans-RLC. Red muscle was injected with cardiotoxin to cause degeneration and then injected at various stages of regeneration with a plasmid expression vector encoding green fluorescent protein (pGFP). Muscle injected with pGFP 8 days after cardiotoxin, and harvested 3 weeks later, contained GFP-positive fibers. We are currently co-injecting pGFP with tagged-RLC to determine the efficiency of this technique for the production of trans-RLC. Our next objective is to perform mechanical analysis on muscle bundles that express high levels of trans-RLC to assess contractile performance.