Novel Isolation and Characterization of Myofibrils from Drosophila Jump Muscle

Drosophila models have proven themselves invaluable for the study of both skeletal and cardiac muscle due to their short generation times, reduced genetic redundancy, and an extensive genetic toolbox. While isolated permeabilized muscle fibers from Drosophila have provided insightful mechanical data, this cell-level approach is unable to characterize the kinetics of muscle activation and relaxation because calcium diffusion is rate-limiting at this scale. For these reasons, a myofibril preparation would be an extremely useful addition to the Drosophila muscle testing portfolio. Myofibrils, the contractile organelles of striated muscle, can be used to acquire, with high accuracy, muscle activation and relaxation kinetics. Despite their potential utility, myofibril measurements from Drosophila have been elusive, primarily due to difficulties in their isolation or, as is the case with the indirect flight muscle, very low force generating ability. To overcome these problems, we developed and employed a novel protocol for the isolation of myofibrils from the Drosophila tergal depressor of the trochanter (TDT), commonly known as the jump muscle. By applying brief, low amplitude sonication to permeabilized jump muscles, we were able to consistently isolate myofibrils suitable for comprehensive measurements of resting and active tension, as well as the kinetics of activation and relaxation. In vertebrate myofibrils, relaxation occurs in two phases: an initial slow, linear phase, primarily mediated by the rate of thin-filament deactivation, followed by a fast, exponential phase mediated by passive elements such as titin. We show that Drosophila jump myofibrils also exhibit bi-phasic relaxation, demonstrating an initial linear phase consistent with the vertebrate mechanism of troponin-tropomyosin steric regulation/deactivation of the thin filament. Preliminary data indicate that the duration of the slow linear relaxation phase for jump muscle myofibrils is ~ 82 ± 9 ms. Compared to vertebrate myofibril kinetics acquired using the same system, this duration is similar to that observed in murine fast-twitch myofibrils (81 ± 10 ms), while shorter than that of cardiac myofibrils from murine and porcine ventricular tissue (115 ± 6 and 194 ± 15 ms, respectively). These data and the new technique represent significant additions to the experimental toolkit available for Drosophila muscle phenotyping, and the first ever mechanical measurements on an insect muscle myofibril. This will enable even better application of Drosophila’s genetic and experimental power to investigate muscle mechanics, including activation and relaxation parameters, ultimately contributing to a deeper understanding of muscle physiology, sarcomeric proteins, and associated diseases. National Institutes of Health (R01HL124091) American Heart Association and DC Women’s Board (915659) Johns Hopkins University catalyst award This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.