Five Alternative Myosin Converter Domains Influence Muscle Power, Stretch Activation, and Kinetics

Abstract

Muscles have evolved to power a wide variety of movements. A protein component critical to varying power generation is the myosin isoform present in the muscle. However, how functional variation in muscle arises from myosin structure is not well understood. We studied the influence of the converter, a myosin structural region at the junction of the lever arm and catalytic domain, using Drosophila because its single myosin heavy chain gene expresses five alternative converter versions (11a-e). We created five transgenic fly lines, each forced to express one of the converter versions in their indirect flight muscle (IFM) fibers. Electron microscopy showed that the converter exchanges did not alter muscle ultrastructure. The four lines expressing converter versions (11b-e) other than the native IFM 11a converter displayed decreased flight ability. IFM fibers expressing converters normally found in the adult stage muscles generated up to 2.8-fold more power and displayed up to 2.2-fold faster muscle kinetics than fibers with converters found in the embryonic and larval stage muscles. Small changes to stretch-activated force generation only played a minor role in altering power output of IFM. Muscle apparent rate constants, derived from sinusoidal analysis of the chimeric converter fibers, showed a strong positive correlation between optimal muscle oscillation frequency and myosin attachment kinetics to actin, and an inverse correlation with detachment related cross-bridge kinetics. This suggests the myosin converter alters at least two rate constants of the cross-bridge cycle with changes to attachment and power stroke related kinetics having the most influence on setting muscle oscillatory power kinetics.

Figure 1

Drosophila myosin S-1 fragment and five converter sequences. (A) Structure of the Drosophila myosin S-1 region (Drosophila EMB isoform, Protein Data Bank: 4QBD). The converter domain is shown in orange, the essential light chain (ELC) in green, and the rest of the MHC S-1 domain in gray. The regulatory light chain is not part of this crystal structure. (B) Sequences of the five Drosophila converters. Bold amino acids are identical. To see this figure in color, go online.

Figure 2

Small amplitude work and power production by IFM fibers from the five converter lines. (A) Work and (B) power generation from 0 to 300 Hz measured by small amplitude, 0.125% ML, sinusoidal analysis at pCa 5.0 and 15°C. Error bars are mean ± SE. Brackets indicate number of fibers tested. To see this figure in color, go online.

Figure 3

SA characteristics of all fiber converter lines. (A) Representative tension traces from fibers stretched by 1% ML over 0.5 ms at pCa 5.0. Numbers and arrows indicate the classic four phases of the response to a rapid step increase. (B) Representative tension traces from fibers stretched by 1% ML over 3.5 ms at pCa 5.0, and (C) over 0.5 ms at pCa 8.0 (relaxed tension traces). (D) Corrected tension traces after subtracting the 0.5 ms, pCa 8.0 relaxed tension traces from the 0.5 ms, pCa 5.0 active tension traces. To see this figure in color, go online.

Figure 4

Representative IFM Nyquist plots fitted with the complex modulus equation. Each fiber was oscillated through frequencies from 0.5 to 650 Hz at a ML change of 0.125%. The resultant force and length traces were used to generate the viscous and elastic modulus. A three-term equation was fit to each fiber’s Nyquist plot to determine muscle apparent rate constants and amplitudes (see Materials and Methods and Table 5). To see this figure in color, go online.