The Mechanical Properties of Drosophila Jump Muscle Expressing Wild-type and an Embryonic Myosin Isoform

Abstract

Transgenic Drosophila are highly useful for muscle protein structure-function studies, particularly myosin isoform diversity. However, our ability to mechanically analyze mutant proteins in Drosophila muscle has been limited to the skinned indirect flight muscle (IFM) preparation. We have developed a new preparation using the Drosophila tergal depressor of trochanter muscle (TDT) that increases our experiments to include maximum shortening velocity (Vmax), force-velocity relations, and steady-state power generation, which are not possible using IFM fibers. As with the IFM, we can replace the native TDT myosin with our myosin of choice. When expressing its native isoform (P2), the TDT is equivalent to a very fast vertebrate muscle, with a Vmax of 6.1±0.3 muscle lengths/second at 15°C, a steep tension-pCa curve, a Hill coefficient of 11±2, a high active isometric tension of 37±3 mN/mm2, and maximum power production (Pmax) at 43% of Vmax and 42% of maximum tension. Expressing an embryonic myosin isoform (EMB) in the TDT muscle decreased Vmax, isometric tension and Pmax by 50%, and the tension-pCa Hill coefficient decreased to 6±2. Varying ATP concentration, while measuring Vmax, revealed a higher ATP affinity for EMB than P2. Increasing Pi concentration reduced isometric tension of TDT expressing either isoform. A slight decrease in TDT Vmax with increasing Pi concentration suggests TDT Vmax may be influenced by Pi release rate. TDT Vmax was not influenced by [Pi] when expressing EMB. With our advances in the TDT preparation we will now be able to test a wider speed range of myosin isoforms, including the superfast IFM myosin, to test our hypothesis that a step associated with Pi release is rate limiting for Vmax of very fast myosins, while a step associated with ADP release is limiting for slower isoforms.