Unlike the asynchronous flight muscles of Lethocerus or Drosophila, the flight muscles (DLM1) of the Hawkmoth Manduca sexta are synchronous,... Show moreUnlike the asynchronous flight muscles of Lethocerus or Drosophila, the flight muscles (DLM1) of the Hawkmoth Manduca sexta are synchronous, requiring a neural spike for each contraction. While the asynchronous muscles can only extend a few percent, Manduca flight muscle can reversibly extend 50% in vitro and 9% in vivo. Furthermore, the dorsal and ventral regions of the DLM1 have different power output presumably due to the temperature gradient across from the cooler dorsal to the warmer ventral subunits. Together with the observation that length-tension curves of Manduca flight muscles resemble mammalian cardiac muscle, these observations suggest that Manduca muscle might be a useful model system to study some aspects of cardiac muscle contractility. The detailed protein composition of Manduca flight muscle is not known. Here we aimed to identify proteins which might be responsible for the unique properties of Manduca muscle. We used 1% vertical SDS-agarose gel electrophoresis (VAGE) to separate the high molecular weight proteins in Manduca, Lethocerus, and Drosophila flight muscle and bovine left ventricular myocardium. The Manduca samples showed two bands around 2MDa and 1.6MDa, smaller than the two titin isoforms in bovine cardiac muscle, but larger than the largest Lethocerus proteins. Projectin and Kettin are elastic proteins found in Lethocerus and Drosophila with sequence homologies to vertebrate titin. Using western blots, the Manduca sample showed two bands cross-reacting with projectin antibodies at ~800 kDa and ~1030 kDa. Ventral (warmer) DLM1 subunits expressed higher level of both projectin isoform. The 1030 kDa projectin is the predominant expressed isoform in both dorsal and ventral subunits. Kettin antibodies cross-reacted to bands at the same position in both Lethocerus and Manduca. We also used western blots from 4-15% PAGE gels to detect a flightin –cross reacting band at around 23kDa in Lethocerus and 30 kDa in Manduca. Flightin is a thick filament associated protein that presumably helps filament assembly and stability. However, toponin I and T antibodies did not cross-react with similar molecular weight proteins from Manduca flight muscle. Thus, Manduca flight muscle has not only proteins homologous to Lethocerus projectin, kettin, flightin, but also several unknown high molecular weight proteins which might play a role in stabilizing sarcomere structure and give the muscle its unique mechanical properties. M.S. in Biology, May 2012 Show less
