Alternative N-terminal regions of Drosophila myosin heavy chain tune muscle kinetics for optimal power output

Abstract

We assessed the influence of alternative versions of a region near the N-terminus of Drosophila myosin heavy chain on muscle mechanical properties. Previously, we exchanged N-terminal regions (encoded by alternative exon 3s) between an embryonic (EMB) isoform and the indirect flight muscle isoform (IFI) of myosin, and demonstrated that it influences solution ATPase rates and in vitro actin sliding velocity. Because each myosin is expressed in Drosophila indirect flight muscle, in the absence of other myosin isoforms, this allows for muscle mechanical and whole organism locomotion assays. We found that exchanging the flight muscle specific exon 3 region into the embryonic isoform (EMB-3b) increased maximum power generation (P(max)) and optimal frequency of power generation (f(max)) threefold and twofold compared to fibers expressing EMB, whereas exchanging the embryonic exon 3 region into the flight muscle isoform (IFI-3a) decreased P(max) and f(max) to approximately 80% of IFI fiber values. Drosophila expressing IFI-3a exhibited a reduced wing beat frequency compared to flies expressing IFI, which optimized power generation from their kinetically slowed flight muscle. However, the slower wing beat frequency resulted in a substantial loss of aerodynamic power as manifest in decreased flight performance of IFI-3a compared to IFI. Thus the N-terminal region is important in tuning myosin kinetics to match muscle speed for optimal locomotory performance.

FIGURE 1

Location and alternative sequence choices for the exon 3 region. The exon 3 region (yellow) and the three other variable regions (7, 9, and 11) encoded by Drosophila alternative exons (shades of red) are mapped onto the chicken myosin S1 structure (gray). The exon 3 region is located near the reactive sulfhydrils (orange) and near the point about which the light-chain region (light chains in light gray) pivots in response to ADP release (Bernstein and Milligan, 1997). The blue spheres depict ADP in the active site. The two alternative amino acid sequences of the exon 3 region are shown below the molecular structure. ^*Signifies nonconserved substitutions.

FIGURE 2

IFM myofibril ultrastructure stability. All panels display longitudinal (left) and transverse (right) views of DLMs (the medial set of the two opposing sets of IFMs). Scale bar lengths are both 0.5 μm. (A) EMB-3b IFMs of 2-day-old adults exhibit ultrastructure abnormalities (Swank et al., 2003). (B) EMB-3b IFMs of <2-h-old adults. At this age the myofibrils show only very slight indications of ultrastructure abnormalities. (C) IFMs from <2-h-old adults expressing IFI myosin.

FIGURE 3

Complex stiffness and phase shift of maximally Ca²⁺-activated IFM fibers at 15°C. (A) Complex stiffness and phase as a function of frequency for IFI and IFI-3a IFM fibers from 2-day-old flies. (B) Complex stiffness and phase as a function of frequency for IFI-2h, EMB-3b, and EMB fibers from <2-h-old adults. Note the different y axis scales for complex stiffness in panels A and B as fibers from the younger flies have less myofibril area per cross section (see text).

FIGURE 4

Elastic and viscous moduli of maximally Ca²⁺-activated IFM fibers. (A) Elastic modulus (instantaneous stiffness) as a function of frequency for all five fiber types. (B) Viscous modulus as a function of frequency for all five fiber types.

FIGURE 5

Power generation by maximally activated IFM fibers at 15°C. (A) Power generated by IFI and IFI-3a muscle fibers when oscillated at 0.25% peak-to-peak strain over a frequency range of 0.5–250 Hz. Wing beat frequency (WBF) for each transgenic line (Table 4) is indicated (vertical dashed lines) at the corresponding muscle oscillation frequency. By beating their wings at a slower frequency, IFI-3a flies generate more power from their IFMs than if WBF remained at the higher IFI WBF. (B) Power generated by EMB-3b, IFI-2h, and EMB fibers as a function of frequency. EMB and EMB-3b fibers cannot generate power over the normal range of wing beat frequencies that support flight. The IFI-2h fibers (from flies <2 h old) have the same f_max as IFI, but generate only 27% as much power as adults. This is due to the young fibers having less thick and thin filaments per fiber cross section (see text). If EMB-3b power generation was normalized to IFI levels (based on IFI-2h P_max compared to IFI P_max), EMB-3b P_max would be ∼43 W/m³, which would be less than IFI-3a P_max.

FIGURE 6

Rates of tension redevelopment (r₃). IFM fibers at pCa 5.0 were subjected to a rapid lengthening step, 0.5% muscle length. Representative traces of IFI-3a, IFI, EMB, and EMB-3b are shown with tension levels normalized to maximum tension immediately after phase 3.