SYNAPTOPODIN-2, AN ACTIN-BINDING PROTEIN, IS A PROMYOGENIC FACTOR FOR MYOBLAST FUSION AND MYOFIBRILLOGENESIS IN MOUSE AND ZEBRAFISH

Abstract

Myogenesis is a differentiation-dependent process involving migration and fusion of uninucleated myoblasts to form multinucleated myotubes, the building blocks of striated, contractile muscle fibers. Several actin-binding proteins are involved in remodeling the actin cytoskeleton and one such protein is synaptopodin-2 (synpo2). Synpo2 binds and polymerizes actin and is upregulated during myogenesis, however, its functional role in the multistep myogenic program is unknown. My objective was to use cell culture and in vivo models of myogenesis to determine the roles of SYNPO2 isoforms during myogenesis. The first model used ectopic expression of the three mouse SYNPO2 isoforms (SYNPO2A, SYNPO2B and SYNPO2As) in stably transduced mouse C2C12 myoblasts or shRNA knockdown of endogenous synpo2, and the effects of these isoforms on migration and fusion was assessed using various approaches. Results indicated that only SYNPO2As is upregulated following differentiation and that knockdown of endogenous SYNPO2As inhibits myotube formation. Ectopic overexpression of SYNPO2As increased myotube formation and pharmacological inhibition of the Rho effector kinase ROCK resulted in loss of the enhanced fusion phenotype. Conversely, ectopic expression of SYNPO2A or SYNPO2B inhibited myotube fusion, consistent with the lack of upregulated expression of these isoforms following differentiation. All three isoforms increased C2C12 migration independent of ROCK inhibition indicating SYNPO2As utilizes two different pathways to promote myoblast migration and fusion. To understand the function of synpo2 in vivo, studies were carried out using zebrafish embryos. Zebrafish Synpo2 expression was restricted to the musculature of developing embryos and inhibiting Synpo2 expression using morpholino knockdown or CRISPR knockout resulted in abnormal muscle development. Morpholino-injected embryos showed a dramatic curved tail phenotype and immunostaining and electron microscopy revealed disorganized actin fibers, reduced myotube formation and disorganized Z-disk filaments. CRISPR knockout embryos did not show a curved tail phenotype but electron microscopy revealed immature myofilaments and reduced I band width. Loss of the curved tail phenotype and abnormal myotube formation did not reflect upregulated genes that could be compensating for the absence of Synpo2, as assessed by RNAseq analysis, but numerous genes associated with myofibril organization were significantly downregulated. Together, these results identify synpo2 as a new promyogenic factor.