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 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.
Highlights
Whereas myosin 18B (Myo18B) is known to be a critical sarcomeric protein, the function of myosin 18A (Myo18A) is unclear, it has been implicated in cell motility and Golgi shape
We started out with heterozygous (HET) Myo18a knockout first mice, obtained from the Knockout Mouse Project (KOMP), the targeting strategy of which is delineated in Fig. 1 (A and B)
We investigated the expression of the unconventional myosin Myo18A and the phenotypes of various Myo18a knockout mouse models, which led to the identification of a novel cardiac isoform of Myo18A (Myo18A␥) as a critical sarcomere component
Summary
We started out with heterozygous (HET) Myo18a knockout first (denoted Myo18aϩ/tm1a) mice, obtained from the Knockout Mouse Project (KOMP), the targeting strategy of which is delineated in Fig. 1 (A and B). To investigate alternative exon expression in the heart, we isolated RNA from mouse ventricular myocytes and performed next-generation sequencing (Fig. 4A). This explains why our custom antibody directed against the last 18 amino acids of Myo18A␣ (and Myo18A), as indicated, was unable to detect the cardiac isoform of Myo18A, Myo18A␥. Transmission EM of E10.5 embryonic hearts from homozygous Myo18a knockout (Myo18atm1b/tm1b) mice revealed, compared with age-matched WT controls, overt disorganization of developing myofibrils, sarcomeric disarray, and disruptions of the A-bands (Fig. 7). Thick myosin filaments together with I-Z-I brushes were widespread, and a few short sarcomeric formations with loosely packed A-band formations could be detected (Fig. 7C)
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