Structural Changes in Isometrically Contracting Insect Flight Muscle Trapped Following a Mechanical Transient

Abstract

The structural response of isometrically contracting insect flight muscle (IFM) to rapid length-step transients was analyzed by applying multivariate data analysis to 38.7 nm repeating subvolumes (repeats) in electron tomograms of quick frozen fibers that were mechanically monitored, rapidly frozen by slamming against a liquid helium cooled copper block, freeze-substituted, sectioned and stained. IFM fibers were frozen 5.5 ms after a step stretch of 6 nm/half-sarcomere in 2 ms. In the step release experiment fibers were frozen 6.5 ms following a release of 9 nm/half-sarcomere in 2.5 ms. Tomograms sampled thin sections cut ≤6 μm below impact surface, recovering 1157 repeats from stretched fibers and 782 repeats from released fibers. Resolution of the actin helix and the stagger of troponin densities in the thin filament facilitated fitting a quasiatomic thin filament model independent of myosin positions, allowing objective recognition whether modeled cross-bridges were weak- or strong-binding. Strong myosin attachments are largely restricted to four actin subunits midway between successive troponin complexes, with a single exception in quick-stretched fibers. Significant changes in the types, distribution and structure of actin-myosin attachments were observed. Prepowerstroke, weak myosin attachments in the target zone are greatly reduced after the transient. However, myosin contacts with tropomyosin in and immediately M-ward of the target zone remain and are more frequent after a release. Weak attachments outside of the target zone remain relatively constant indicating a constant rate for formation of non-productive collision complexes. Following a stretch, there is an increase in the proportion of 2-headed cross-bridges. Myosin contacts with troponin are greatest after a release, and are reduced in frequency following a stretch. The results are interpreted in terms of the shortening cycle of stretch activated IFM. Supported by NIGMS and NIAMSD.