The mechanical properties of Drosophila jump muscle expressing wild-type and embryonic Myosin isoforms

Abstract

Transgenic Drosophila are highly useful for structure-function studies of muscle proteins. However, our ability to mechanically analyze transgenically expressed mutant proteins in Drosophila muscles has been limited to the skinned indirect flight muscle preparation. We have developed a new muscle preparation using the Drosophila tergal depressor of the trochanter (TDT or jump) muscle that increases our experimental repertoire to include maximum shortening velocity (V(slack)), force-velocity curves and steady-state power generation; experiments not possible using indirect flight muscle fibers. When transgenically expressing its wild-type myosin isoform (Tr-WT) the TDT is equivalent to a very fast vertebrate muscle. TDT has a V(slack) equal to 6.1 +/- 0.3 ML/s at 15 degrees C, a steep tension-pCa curve, isometric tension of 37 +/- 3 mN/mm(2), and maximum power production at 26% of isometric tension. Transgenically expressing an embryonic myosin isoform in the TDT muscle increased isometric tension 1.4-fold, but decreased V(slack) 50% resulting in no significant difference in maximum power production compared to Tr-WT. Drosophila expressing embryonic myosin jumped <50% as far as Tr-WT that, along with comparisons to frog jump muscle studies, suggests fast muscle shortening velocity is relatively more important than high tension generation for Drosophila jumping.

Figure 1

(A) Anatomy and location of the TDT muscle. The TDT is represented in dark gray for visibility among the other major muscles of the Drosophila thorax, the DLM (horizontal IFM fibers, yellow) and DVM (vertical IFM fibers, yellow). The TDT is translucent and difficult to observe during dissection. The red dotted lines mark the location of the two cuts through a half thorax that are made to free the TDT from the thoracic cuticle. (B) The free TDT is cut just above the end of the tendon and down the side to remove the four smaller diameter fibers. (C) The TDT is composed of two fiber layers so that the vertical cut allows it to open up into one layer with a thickness of a single fiber. (D) The TDT is split parallel to the fibers producing a preparation consisting of 8–10 fibers. (E) Each end of the TDT is clipped in laser cut T-clips, which is then mounted on the muscle mechanics rig using hooks extending from the force transducer and servo motor. (F) A Tr-WT TDT preparation mounted on the mechanics rig, in relaxing solution, showing good alignment of fibers between the T-clips. This preparation produced excellent force-velocity data. (G) An EMB preparation that produced poor quality data as the fibers were twisted while T-clipping the preparation. Both Tr-WT and EMB TDT muscle fiber bundles produce equally good data if the preparations are as good as the one in F. (H) A differential interference contrast image of a Tr-WT TDT muscle that was dissected from a 2-day old fly and imaged in Ringer's solution without staining or fixation. (I) Same as F, except this TDT expresses EMB. Both EMB and control fibers display normal sarcomere patterns. Scale bars = 30 μm.

Figure 2

Isometric tension-pCa relationships. Tension generated by TDT fibers was measured at a sarcomere length of 3.6 μm. Tension was normalized to equal 1 at pCa 5.0 and fit with a standard Hill curve. There is a significant leftward shift of the bottom of the curve when expressing EMB myosin as seen by the significant tension difference at pCa 5.7 (p < 0.05, t-test) and significant differences in the Hill coefficient and pCa₅₀ values (Table 2). n = 6 for each fiber type. Inset: Isometric tension of EMB TDT muscle, at saturating calcium, pCa 5.0, was significantly higher than Tr-WT TDT fibers (p < 0.05, t-test). n = 21 for Tr-WT and n = 23 for EMB. All values are mean ± SE.

Figure 3

Unloaded shortening velocity (V_slack) was measured using the slack test. (A) Raw data from TDT muscle expressing EMB showing the time to force redevelopment for three different shortening amounts, expressed as percent of muscle length. Arrows indicate the points at which force redevelopment starts. (B) Slack test data from Tr-WT TDT fibers showing the earlier time points at which force redevelopment starts compared to EMB. The slope of percent length change against time was determined to calculate the shortening velocities shown in Table 2. Time 0 equals the start of shortening.

Figure 4

TDT tension-velocity and power measured using the force-clamp technique. (A) Raw position traces from a Tr-WT TDT showing the percent ML slope required to achieve the tension level written at the end of the trace. Tension values are percent of maximum isometric tension. Vertical dashed lines show the approximate range over which shortening velocity was measured. (B) The corresponding tension traces from the same muscle preparation. (C) Velocities, calculated from the slope of the traces in A, were plotted against tension that was normalized to equal 1 at maximum isometric tension. The Tr-WT curve was generated from the data shown in A and B, whereas the EMB curve is from raw data not shown. Both curves were fit with the Hill curve. (D) Mechanical power calculated from multiplying average tension and velocity values, and fit with curves generated from multiplying the tension and velocity from the average Hill curves. Although the maximum power generated by Tr-WT is higher than EMB when tension is normalized, maximum power generation is not significantly different between the fiber types when actual tension values in units of mN/mm² are used in the power calculation (Table 3).