Drosophila jump muscle myofibrils: A new tool for investigating activation and relaxation

Published: 18 June 2026| Version 1 | DOI: 10.17632/fvx7g8g3jp.1
Contributors:
Axel Fenwick,
,
,

Description

Drosophila models have proven invaluable for studying skeletal and cardiac muscle diseases. While permeabilized indirect flight muscle (IFM) and jump muscle fibers from Drosophila yield insightful mechanical data, these larger fiber preparations cannot resolve the kinetics of activation and relaxation because calcium diffusion into the fiber core is rate-limited at this scale. A single myofibril preparation, which is ideal for measuring physiologically relevant activation and relaxation rates, would therefore be a valuable addition to the Drosophila toolkit. However, previous attempts to prepare IFM myofibrils have failed to produce active force. Here, we developed a method to isolate myofibrils from the Drosophila jump muscle. By applying brief, low-amplitude sonication to permeabilized jump muscles, we isolated myofibrils that produced 19.8 ± 10.5 mN/mm2 net active tension with an activation rate of 8.2 ± 4.0 s-1. Jump muscle myofibrils exhibited the typical biphasic relaxation seen in vertebrates: an initial slow, linear phase lasting 82 ± 9 ms, followed by a fast exponential decay with a rate constant of 19.7 ± 9.6 s-1. We further characterized myofibrils from flies expressing an alternative myosin isoform (EMB) known to have slower actin-binding and detachment kinetics. EMB myofibrils produced ~1.6-fold higher active tension and a 38% slower activation rate compared to controls, while relaxation parameters remained unchanged. These results suggest that activation rate is influenced by myosin attachment kinetics, whereas myosin detachment from actin is not likely the rate-limiting step for relaxation. To our knowledge, this is the first report of active myofibril mechanics from an insect muscle, representing a significant addition to the Drosophila experimental toolkit.

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Steps to reproduce

For mechanical experiments, 5–10 µL of the myofibril suspension was added to a 500 µL bubble of relaxing solution in the temperature-controlled chamber of the myofibril apparatus. The preparation was left undisturbed for at least 5 minutes to allow myofibrils to settle onto the chamber bottom. We then gently replaced the solution bubble with fresh relaxing solution to remove excess debris. Individual myofibrils were mounted between two glass microtools (a piezoelectric length controller and a cantilever force probe) and stretched to a sarcomere length of 3.6 µm. Activation and relaxation were induced by rapidly switching between two continuously flowing solution streams (relaxing solution, pCa 8; activating solution, pCa 4) delivered through a double-barreled pipette attached to a fast step motor (Warner Instruments). Data were collected and analyzed with custom LabView software (National Instruments). Active force (FMAX) was calculated as the difference between the baseline tension in relaxing solution and the peak tension in activating solution just prior to the onset of relaxation. Tension values were obtained by normalizing force to the myofibril’s cross-sectional area. The rate constant of tension development (kACT) was determined by fitting the force rise during activation to a single-exponential function (Levenberg–Marquardt nonlinear least squares). The fast phase of relaxation was analyzed similarly: the final, rapid exponential force decay was fit to obtain a rate constant kREL. The duration of the initial slow relaxation phase (tLIN) was measured from the start of solution exchange (digital trigger signal from the switch to relaxing solution) to the onset of the fast exponential drop in force, detected with an automated linear-fit residual algorithm. The rate of force decline during this slow relaxation phase (kLIN) was calculated as the slope of a linear fit to the force trace during tLIN, normalized to the maximum tension.

Institutions

  • Rensselaer Polytechnic Institute Biological Sciences
    NY, New York
  • Johns Hopkins University Department of Medicine
    MD, Baltimore

Categories

Drosophila, Myofibril, Muscle Mechanics

Funders

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