X-Ray Diffraction “Movie” Of Complete Oscillatory Work Cycles Myogenically Produced In Glycerinated Insect Flight Muscle (IFM)

Abstract

When slightly calcium-activated (pCa ∼5.7, gives ∼0.2 peak isometric force), glycerinated Lethocerus insect flight muscle (IFM) can be mechanically stretch-activated at constant [Ca2+] to give a delayed active rise to peak force, a myogenic response typical of asynchronous IFM. Continuous sine-wave length-oscillations elicit sinusoidally cycling force traces, delayed ∼45° behind length cycles. Force-length x-y plots therefore follow anti-clockwise Lissajous loops of elliptical form, enclosing an area proportional to oscillatory work output per cycle, which peaked at ∼2%X∼2Hz for 10-20-fiber bundles. A Pilatus 100K detector collected 64 synchrotron-x-ray fiber-diffraction frames per full cycle (8ms time resolution), throughout an 11-cycle run (704 frames). Summing successive cycles and adjacent frames produced a 16-frame movie (32ms time resolution) showing weaker details. The movie shows clear within-cycle peak-to-valley intensity changes in multiple reflections, some signaling crossbridge mass shifts toward (and away from) thin-filament lattice positions, others crossbridge shifts between binding to and detaching from actin target zones, still others signaling crossbridge shifts between tilt angles mostly near 90° versus mostly dispersed to non-90° angles. Surprises include: 1) Maximum 90° angles occur near force peak in Drosophila but near force valley in Lethocerus. 2) Although the force sine-wave varies smoothly, two structural signals of crossbridge attachment show biphasic profiles as force rises and again as force falls, as if outer and inner myosin heads (AL-Khayat etal model) attach/detach in separate cohorts during the 2% (26 nm/half-sarcomere) length changes. 3) Structural signals of crossbridge action are variably phase-coupled to the force sine-wave; some x-ray signals even differ in phase lag between force peak and valley. Maximum tropomyosin shift spans ∼5 frames around force peak. Overall results strongly constrain possible mechanisms of stretch-activation, suggesting complementary approaches for revealing it. (Support: NIH, DOE).