Chaperone and Gene Regulatory Roles of SMYD1b during Early Skeletal Muscle Development and Heart Morphogenesis

• Author / Creator

  Prill, Kendal M

• Sarcomeres are the basic contractile units that make up the striated muscle of the heart and skeletal muscle. The specification and differentiation of muscle cells and the contractile function of the sarcomere are generally well understood, but how the sarcomere assembles remains largely unknown. To identify those factors required for sarcomere formation, we studied zebrafish embryos that were defective in sarcomere assembly. We selected zebrafish muscle mutants that showed similarities to the myosin chaperone mutant, unc45bsb60, which does not complete sarcomere assembly and exhibits paralysis of the skeletal muscle and a nonfunctional heart. We isolated the zebrafish muscle mutant herzschlag, which shows a rapid degeneration of striated muscle, and still heart, which displays paralysis in both striated tissues. We hypothesized that the herzschlag and still heart candidates were novel factors required for sarcomere assembly. Through recombinant mapping, complementation crossing and sequencing, we identified herzschlag and still heart as having mutations in titin2 and smyd1b, respectively. I examined early stages of muscle development in still heart mutants and identified SMYD1b as an assembly protein required for skeletal sarcomere assembly with respect to myosin folding and incorporation. Although fast myosin expression was normal in still heart mutants, fast myosin was never organized into the developing premyofibrils. Previous studies proposed SMYD1b as a myosin chaperone during sarcomere formation, which supported its role as an assembly factor. Smyd1bsth and unc45bsb60 mutants both exhibit nonfunctional hearts that do not complete heart morphogenesis. Our initial hypothesis was that since SMYD1b and UNC45b are required for skeletal sarcomere assembly, then SMYD1b and UNC45b are also required for sarcomere formation in heart muscle. Expression analysis of the cardiac transcriptional network in smyd1bsth and unc45bsb60 mutants demonstrated unique changes in each network. I showed that cardiac myosin thick filaments are present in the hearts of smyd1bsth and unc45bsb60 mutants, indicating that SMYD1b and UNC45b, although both being myosin chaperones in skeletal muscle, each have unique functions in heart development. Recent work has shown that SMYD1b is capable of binding myosin. However, the structure of SMYD1b is similar to that of a histone methyltransferase, suggesting SMYD1b could be transcriptionally regulating heart development. I explored the requirement of the SET domain by substituting critical amino acids necessary for the methyltransferase function and found that the SET domain is required for proper heart development, although target genes remain unknown. I initiated studies of an M-line protein, MYOMESIN1a, and the role that this protein has in sarcomere maintenance and integrity. We showed that myom1a is upregulated significantly at stages much earlier than when myosin chaperone or myosin heavy chain expression begins to increase in our smyd1bsth, unc45bsb60 and titin2hel mutants. Using myosin chaperone mutants, smyd1bsth, unc45bsb60, that do not form myosin thick filaments, I showed that myosin is required for myomesin incorporation at the M-line. Preliminary work with myom1a P0-CRISPR-injected mosaic animals suggested that myomesin is not required for myosin incorporation but muscle tissues lacking myomesin became weaker and disorganized over time. Further characterization of myomesin mutants will reveal the precise function of myomesin during sarcomere maintenance and damage. The research presented within this thesis gives support to existing models for the role of SMYD1b as a sarcomere assembly factor in skeletal muscle and as a regulatory factor during heart development. I showed that the methyltransferase function of SMYD1b is required for normal heart development, making smyd1(b) an important candidate in human heart disease and treatment. Myomesin has been suggested to be a sensitive indicator to muscle damage due to disease. We showed that in addition to myomesin acting as a biomarker for muscle disease, myomesin can be used for the early detection of myopathies that may allow time to provide treatment to patients before significant muscle deterioration occurs.

• Subjects / Keywords

  • Smyd1b
  • Muscles
  • Sarcomere
  • Zebrafish
  • Chaperones

• Graduation date

  Fall 2017

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3J38KX5F

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.