Like mammalian cardiac muscle, the flight muscle of the hawk-moth Manduca sexta is synchronous. However, it also has significant structural... Show moreLike mammalian cardiac muscle, the flight muscle of the hawk-moth Manduca sexta is synchronous. However, it also has significant structural similarities with asynchronous insect flight muscle systems, such as those of Drosophila and Lethocerus. Different physiological function depends on the underlying different molecular structures. Although Drosophila and Lethocerus have been well studied, Menduca sexta is still a newly developed research model. Many different skinning and storage methods are being used worldwide for in vitro studies of a wide variety of muscle systems. Here our goal is to develop better skinning solution and storage condition which will maintain muscle structure and function as well as intact muscle. To achieve this end, several kinds of skinning solutions and storage conditions were evaluated by laser and X-ray mechanical experiments. The solution from HAMM Lab (University of Washington) showed the best ability to destroy plasma membrane, without affecting muscle interior structure and function. For two storage conditions, 4°C without glycerol and -20°C with 50% glycerol, both laser and X-ray experiments show the latter one is better, which means muscles stored at -20°C with 50% glycerol have faster and stronger reaction for high calcium solution, and also have sharper peaks in X-ray diffraction patterns. The presence of protease inhibitors is also necessary to maintain the contraction ability of muscle. Furthermore, the X-ray experiments for muscle fibers stored at -20°C with 50% glycerol and -80°C with 75% glycerol, show the expected results, which the muscles stored at latter condition show far superior X-ray diffraction patterns, especially in meridional reflections and layer lines. The lower temperature can minimize protein degradation. Notably, incubating muscles in glycerol solutions at 4°C before storing them at -80°C, and washing muscles thoroughly after stored at 75% glycerol, are critical to ensure that all of the glycerol goes in and out of muscle fibers, respectively. The significance of these findings is that it now appears to be possible to store prepared Manduca sexta flight muscles for at least eight days at -80°C with good structural preservation enabling a large class of future experiments not requiring fresh deliveries of moth from the suppliers. M.S. in Biology, May 2012 Show less
