LENGTH DEPENDENT ACTIVATION IN MANDUCA SEXTA SKINNED FLIGHT MUSCLE
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The synchronous dorsolongitudinal muscle (DLM1) of the hawk moth, Manduca sexta has a number of characteristics similar to vertebrate cardiac muscle. These muscles constitute a large proportion of the body weight and produce most of the force to depress the wings during flight. Crossbridges, consisting of myosin heads, form between the thin and thick filaments of the muscle fiber, and it is myosin head movement that causes muscle shortening and generates force. But the cycle cannot start without calcium bound to the troponin-tropomyosin complex, which expose the myosin binding sites on actin. It has been shown that cardiac muscle, as well as other muscles, exhibit myofilament length dependent activation where larger forces would be generated for a given calcium concentration at longer the sarcomere length (SL). The main goal in this thesis is to study the relationship of force-pCa of the DLM1 fibers as a function of sarcomere length. To establish these relationships, a series of calcium concentration solutions were prepared (pCa 4-8). The force was recorded as a function of the pCa at three different SL values. One aim of the thesis is to optimize the procedure for activation of the muscle fibers. Two different experimental protocols were used and their results were compared. The force-pCa relationships were plotted and fit to the Hill equation to analyze the cooperativity. The SL giving the highest force was 3.2 μm and the force-pCa relationship showed a sigmoidal shape with pCa50 of 6.01and the Hill coefficient was 19.6. Two protocols that were tried did not yield significantly different results, but no clear trends indicating myofilament length dependent activation were seen in these experiments in Manduca sexta flight muscle. However, protocol 2 showed a clearer demonstration of the cooperativity of the force-pCa relationships. The protocols used have better calcium binding to troponin-C (TnC) and larger Fmax than previous work in Irving lab. Future studies would be adding more data points with protocol 2 and analyzing if the trend changes.