Zebrafish Myofilaments and their Assemblies are Good Structural Models for Studying Disease Mutations

Abstract

Zebrafish have become a prominent animal model of human disease because of their genetic tractability and rapid development (Lieschke & Currie, 2007). Several muscle diseases have been studied using zebrafish, including muscular dystrophy (Johnson et al., 2013), hypertrophic and dilated cardiomyopathy, nemaline myopathy (Sehnert et al., 2002; Asnani & Peterson, 2014; Telfer et al., 2012) and distal arthrogryposis (Ha et al., 2013). Here we show that zebrafish myofilaments and their assemblies have molecular structures similar to those of higher vertebrates, demonstrating their usefulness in elucidating structural changes due to disease mutations. EM and 3D reconstruction have shown that striated muscle contraction is regulated by Ca2+-induced movement of tropomyosin on thin filaments, which uncovers myosin-binding sites on actin. Using negative staining EM and single particle reconstruction we find that tropomyosin is well resolved in zebrafish native thin filaments and undergoes the same Ca2+-induced movement as seen in other species. Zebrafish thin filaments are therefore good models for studying the impact of thin filament mutations (actin, troponin, tropomyosin, nebulin) on thin filament structure and regulation. Thick filaments are also readily isolated from zebrafish muscle and their 3D reconstruction is similar to that of mammalian filaments (González-Solá et al., 2014). We have isolated intact filament assemblies (myofibrils and A-segments) from zebrafish and find that these also closely resemble those of other vertebrates, including mammals, clearly revealing MyBP-C and M-line periodic organization. These are therefore good models for elucidating the structural impact of mutations in myosin, MyBP-C and M-line proteins. We conclude that the structures of zebrafish skeletal muscle thick and thin filaments and their native assemblies closely resemble those of higher vertebrates, making them excellent models for studying the molecular impact of disease-causing mutations on filament structure.