ANALYSIS OF SMYD1 FUNCTION AND THE MOLECULAR MECHANISM OF ACTION IN SKELETAL AND CARDIAC MUSCLE CELL DIFFENCIATION
Creators: name: Xu, Rui
Publisher: name: Maryland Shared Open Access Repository
Year: 2024

description: Smyd1, a key lysine methyltransferase, is crucial for myofibrillogenesis in both skeletal and cardiac muscles. The zebrafish orthologue, smyd1b, encodes two isoforms, Smyd1b_tv1 and Smyd1b_tv2, differing by 13 amino acids due to alternative splicing. It remains unclear whether the Smyd1b_tv1 and Smyd1b_tv2 isoforms exhibit distinct expression patterns and functions. The Janus face of Smyd1 in muscle cell development is perplexing and controversial. Some lines of evidence have suggested that Smyd1 functions as a chromatin modifier to regulate gene expression through a common post-translational mechanism. However, other lines of evidence indicate that Smyd1 may act as a chaperone protein in the cytosol to stabilize sarcomeric-associated proteins. The factors that trigger the enhanced protein degradation in muscles upon loss of Smyd1 function remain unknown. To determine whether Smyd1b_tv1 and Smyd1b_tv2 may have distinct function in muscle development, we first generated transgenic zebrafish models that express only Smyd1b_tv1 or Smyd1b_tv2 isoform in a zebrafish mutant that lacks the endogenous smyd1b gene. Functional analysis revealed that expression of Smyd1b_tv1 is essential for cardiomyocyte differentiation and fish viability, while Smyd1b_tv2 is dispensable for heart development and fish survival. Targeted deletion of Smyd1b_tv1 using CRISPR/Cas9 to produce smyd1b mutants that only express endogenous smyd1b_tv2, referred to as Smyd1bTV2. Surprisingly, Smyd1bTV2 fish were viable, and showed no apparent phenotypes. However, upon on the cardiac injury in adult fish, Smyd1bTV2 fish showed poor heart regeneration compared to WT siblings, emphasizing the critical role of Smyd1b_tv1 in cardiac regeneration. To elucidate the subcellular functionality of Smyd1b in orchestrating muscle development in zebrafish, multiple zebrafish transgenic lines expressing nuclear (Smyd1bNLS) and cytosolic (Smyd1bNES) Smyd1b were established. We showed that cytosolic Smyd1b (Smyd1bNES) completely complements Smyd1b-deficient mutants, while nuclear Smyd1b (Smyd1bNLS) exacerbates heart defects, highlighting the importance of Smyd1b cytosolic localization. Furthermore, comprehensive RNA-seq analysis revealed that smyd1b exhibits strikingly similar transcriptional profiles to unc45b, a chaperone mutant. In smyd1b-/- mutants, misfolded Myosin forms aggregate positive for ubiquitin during myogenesis, indicating Smyd1b primarily acts as a chaperone for Myosin folding, and the ubiquitin-proteasome system (UPS) may play an important role in deconstructing misfolded Myosin. Further investigation into specific UPS components, including CHIP and UBA1, showed that their loss did not ameliorate the sarcomeric defects or prevent sarcomeric protein from degradation in smyd1b-/- mutants. These findings imply the involvement of multiple E3 ligases in this process and the complexity of the protein quality control mechanisms in maintaining sarcomere integrity. Overall, Smyd1b primarily functions as a chaperone in the cytoplasm, aiding in myosin folding and sarcomere assembly. The degradation of misfolded myosin in smyd1b-/- mutants may be related to the ubiquitin-proteasome system (UPS). The two isoforms encoded by Smyd1b are not functionally equivalent, with Smyd1b_tv1 playing a more critical role in cardiac muscle development and regeneration.