A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart

Horsthemke M, Nutter LMJ, Bachg AC, Skryabin BV, Honnert U, Zobel T, Bogdan S, Stoll M, Seidl MD, Mueller FU, Ravens U, Unger A, Linke WA, Gorp PRR, Vries AAF, Bahler M, Hanley PJ

Abstract

Whereas myosin 18B (Myo18B) is known to be a critical sarcomeric protein, the function of myosin 18A (Myo18A) is unclear, although it has been implicated in cell motility and Golgi shape. Here we show that homozygous deletion (homozygous tm1a, tm1b or tm1d alleles) of Myo18a in mouse is embryonic lethal. Reminiscent of Myo18b, Myo18a was highly expressed in the embryo heart, and cardiac-restricted Myo18a deletion in mice was embryonic lethal. Surprisingly, using Western blot analysis, we were unable to detect the known isoforms of Myo18A, Myo18Aα and Myo18Aβ, in mouse heart using a custom C-terminal antibody. However, alternative anti-Myo18A antibodies detected a larger than expected protein, and RNA-Seq analysis indicated that a novel Myo18a transcript is expressed in mouse ventricular myocytes (and human heart). Cloning and sequencing revealed that this cardiac isoform, denoted Myo18Aγ, lacks the PDZ-containing N-terminus of Myo18Aα, but includes an alternative N-terminal extension and a long serine-rich C-terminus. EGFP-tagged Myo18Aγ expressed in ventricular myocytes localized to the level of A-bands in sarcomeres, and Myo18a knockout embryos at day 10.5 (E10.5) exhibited disorganized sarcomeres with wavy thick filaments. We additionally generated myeloid-restricted Myo18a knockout mice to investigate the role of Myo18A in nonmuscle cells, exemplified by macrophages, which express more Myo18Aβ than Myo18Aα, but no defects in cell shape, motility or Golgi shape were detected. In summary, we have identified a previously unrecognized sarcomere component, a large novel isoform (denoted Myo18Aγ) of Myo18A. Thus, both members of class XVIII myosins are critical components of cardiac sarcomeres.