Characterization of a newly identified myosin, Myo18Aγ, in cardiac sarcomeres

• Applicant: Professor Dr. Peter Hanley
• Subject Area: Cell Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 512106520

Using various Myo18a knockout mouse models, microscopy and RNA-sequence analysis, we unexpectedly discovered Myo18Aγ as a new component of cardiac sarcomeres (published preliminary data). Myo18Aγ localizes to the sarcomeric A-band and may be a missing piece of the puzzle towards understanding sarcomere assembly and function. In unpublished preliminary data, we confirmed that the γ-isoform of Myo18A is indeed specific for striated muscle. We also found that the expression of Myo18Aγ but not Myo18Aα increases during cardiac differentiation and decreases during cardiac de-differentiation, consistent with a role for Myo18Aγ in sarcomerogenesis (myofibrillogenesis). In the current project, we plan to precisely locate Myo18Aγ on the sarcomere landscape using electron microscopy and single-molecule localization microscopy (SMLM). Preliminary data using adenoviral vectors indicate that the N-terminus of Myo18Aγ is sufficient for localization to the A-band. We also plan to knockdown Myo18Aγ (and Myo18Aα) in conditionally immortalized rat atrial myocytes and neonatal rat ventricular myocytes to explore the roles of Myo18Aγ (and Myo18Aα) in sarcomere assembly and maintenance, respectively. Western blot analyses confirmed that the rat Myo18Aγ-specific shRNAs (delivered by adenoviruses) are effective. In addition, using Myo18aexon1 deletion and inducible Myo18a knockout mouse models, we will determine whether the α-isoform of Myo18A is redundant for cardiac development and sarcomere function and whether loss of Myo18A expression causes cardiomyopathy. Finally, we will map the Myo18Aγ protein interactome using co-immunoprecipitation and BioID and validate putative interaction proteins. We anticipate that knockdown/knockout studies, together with precise localization of Myo18Aγ in the sarcomeric A-band and identification/confirmation of interaction partners, should significantly advance our understanding of the roles of class 18 myosins in sarcomere assembly and cardiac health.

• DFG Programme: Research Grants