The Synaptosome Associated Protein‐23 is Necessary for Skeletal Muscle Myogenesis
Authors: Gamarra Jennifer , Engels Nichlas , Blue R Eric , Hoerr Isabel , Giudice Jimena 
Published: June 2021

Alternative splicing is a post‐transcriptional RNA processing mechanism that occurs in more than 90% of human genes and allows for many proteins to arise from one gene. Membrane trafficking genes are regulated by alternative splicing during postnatal development1. The synaptosome associated protein‐23 (Snap23) is a member of the SNARE complex and is involved in the fusion of transport vesicles with the plasma membrane before cargo release2. We have shown that the Snap23 gene is regulated by alternative splicing. Exon 6, which contains 33 nucleotides, is skipped in fetal stages and is gradually included in adulthood. This transition is exclusive to striated muscles. SNAP23 is essential for mouse viability3; however, its role during muscle development is unclear. We hypothesize that SNAP23 is necessary for maintenance of muscle cell architecture and that alternative splicing is involved as a regulatory mechanism of muscle development.

Murine C2C12 cell differentiation (myogenesis) replicates the Snap23 splicing transitions that we observed in muscle development. Thus, C2C12 cells are an appropriate model to investigate SNAP23 function and its regulation by alternative splicing. C2C12 cells were transfected with siRNAs to deplete SNAP23 protein expression in the undifferentiated stage and then differentiated for four days. Depletion of Snap23 led to a reduction in cell viability and fusion, indicating that Snap23 is required for myogenesis. Media collected from healthy differentiated cells (conditioned media) rescue the phenotype of Snap23 knock‐down cells. Immunofluorescence confirmed greater cell differentiation than those treated with standard differentiation media. This led us to hypothesize that SNAP23 mediates the secretion of a protein that is necessary for myogenesis. We coated plates with various concentrations of gelatin and observed that gelatin‐coated plates also restored cell differentiation. We are currently investigating fibronectin as a potential important protein, as it is a major component of gelatin and interacts with integrins on the plasma membrane.

In parallel, to study the implications of alternative splicing on Snap23 pre‐mRNA, we used morpholino antisense oligonucleotides (MO) to redirect endogenous splicing decisions. Cells treated with Snap23‐MO expressed exclusively the short isoform throughout myogenesis while control cells express the long isoform in differentiated stages. In Snap23‐MO treated cells, we observed more myotubes with greater nuclei per myotube compared to the controls, suggesting that Snap23 splicing contributes to cell fusion and thus myogenesis.

In conclusion, our data suggest that SNAP23 and its regulation by alternative splicing is involved in the fusion of myoblasts to form multinucleated myotubes. We propose that SNAP23 mediates the secretion of proteins necessary for myogenesis. Ongoing studies are directed to elucidate the molecular mechanisms behind the observed phenotypes.

Support or Funding Information

NIH/NIGMS award (R25 GM089569) (to J. Gamarra UNC‐Chapel Hill PREP Scholar) and start up funds from UNC‐Chapel Hill (to J. Giudice)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.