The Actin F352S Nemaline Myopathy Mutation Disrupts Indirect Flight Muscle Structure and Function in Drosophila

Abstract

Nemaline myopathy is a congenital human muscle disorder characterized by masticatory, limb and respiratory weakness. Dominant negative disease-causing mutations have been identified in ten genes that notably encode sarcomeric thin filament proteins including α actin (ACTA1). While most of these mutations cause a loss of muscle function, the F352S actin mutation elevates myosin cross-bridge strain and steady-state isometric force production and, thereby, apparently increases contractile function in humans. To investigate the F352S-induced myopathic cascade from the level of single molecules through the level of whole muscle we generated transgenic Drosophila that express the mutation in Act88F, the indirect flight muscle (IFM) actin gene. Mutant heterozygotes were flightless. Fluorescent and electron microscopy revealed breaks in mutant IFM fibers and extensive myofibrillar disarray. For example, the highly ordered double hexagonal thin/thick filament lattice, at the periphery of myofibrils, was distorted with myofilaments occasionally losing longitudinal orientation. Z-lines showed streaming and formed “zebra bodies”. The microscopic alterations are consistent with inappropriate acto-myosin interaction, unevenly distributed force, and destructive hypercontraction. Interestingly, in vitro sliding velocities of purified IFM F-actin from wild-type (3.17±0.12 μm/s) vs. F352S mutant heterozygous (2.88±0.09 μm/s, mean±SEM) flies did not significantly differ. Similarly, no differences in maximum calcium-activated velocity (3.88±0.11 vs. 3.77±0.10 μm/s) or in cooperativity (nH = 2.13±0.27 vs. 2.62±0.46) were observed for F-actin in the presence of tropomyosin and troponin. However, F352S heterozygous filaments showed a significant (p < 0.01) increase in calcium sensitivity relative to control (pCa50 = 6.11 +/- 0.03 vs. 5.99 +/- 0.03), consistent with mutation-induced gain in molecular function. Going forward, our novel Drosophila model will be used to screen for in vivo modifiers of mutant muscle performance.Supported by NIH 2PO1 AR052354 (P.D. Allen PI; CFA Core D).