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Characteristics of nuclear architectural abnormalities of myotubes differentiated from LmnaH222P/H222P skeletal muscle cells

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Abstract

The presence of nuclear architectural abnormalities is a hallmark of the nuclear envelopathies, which are a group of diseases caused by mutations in genes encoding nuclear envelope proteins. Mutations in the lamin A/C gene cause several diseases, named laminopathies, including muscular dystrophies, progeria syndromes, and lipodystrophy. A mouse model carrying with the LmnaH222P/H222P mutation (H222P) was shown to develop severe cardiomyopathy but only mild skeletal myopathy, although abnormal nuclei were observed in their striated muscle. In this report, we analyzed the abnormal-shaped nuclei in myoblasts and myotubes isolated from skeletal muscle of H222P mice, and evaluated the expression of nuclear envelope proteins in these abnormal myonuclei. Primary skeletal muscle cells from H222P mice proliferated and efficiently differentiated into myotubes in vitro, similarly to those from wild-type mice. During cell proliferation, few abnormal-shaped nuclei were detected; however, numerous markedly abnormal myonuclei were observed in myotubes from H222P mice on days 5 and 7 of differentiation. Time-lapse observation demonstrated that myonuclei with a normal shape maintained their normal shape, whereas abnormal-shaped myonuclei remained abnormal for at least 48 h during differentiation. Among the abnormal-shaped myonuclei, 65% had a bleb with a string structure, and 35% were severely deformed. The area and nuclear contents of the nuclear blebs were relatively stable, whereas the myocytes with nuclear blebs were actively fused within primary myotubes. Although myonuclei were markedly deformed, the deposition of DNA damage marker (γH2AX) or apoptotic marker staining was rarely observed. Localizations of lamin A/C and emerin were maintained within the blebs, strings, and severely deformed regions of myonuclei; however, lamin B1, nesprin-1, and a nuclear pore complex protein were absent in these abnormal regions. These results demonstrate that nuclear membranes from H222P skeletal muscle cells do not rupture and are resistant to DNA damage, despite these marked morphological changes.

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Data Availability

The original data presented in the study are included in the article. Further inquiries can be directed to the corresponding author.

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Acknowledgements

We thank Mr. Keita Neishi (KEYENCE) for technical assistance with the time-lapse imaging using the BZ-X810 microscope, Dr. Gisèle Bonne (Center of Research in Myology, France) for providing the original LmnaH222P/H222P mutant mice, Dr. Helena Popiel (Tokyo Medical University) for editing the manuscript, and National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP) (Japan) for providing the lamin A (M52-1) and C (M53-1) antibodies.

Funding

This work was financially supported in part by Grants-in-Aid for Scientific Research (B) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (KAKENHI) (20H03594), Acceleration Program for Intractable Disease Research Utilizing Disease-specific iPS cells (17935395), and Practical Research Project for Rare/Intractable Diseases (23809381) from the Japan Agency for Medical Research and Development, Intramural Research Grant for Neurological and Psychiatric Disorders of NCNP (29–4, 2–5, and 5–6), and Follow-up Grant from Tokyo Medical University (2022).

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  1. Department of Pathophysiology, Tokyo Medical University, Tokyo, Japan

    Eiji Wada, Nao Susumu & Yukiko K. Hayashi

  2. Department of Physics, Graduate School of Science, The University of Tokyo, Tokyo, Japan

    Motoshi Kaya

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  1. Eiji Wada
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Correspondence to Yukiko K. Hayashi.

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Wada, E., Susumu, N., Kaya, M. et al. Characteristics of nuclear architectural abnormalities of myotubes differentiated from LmnaH222P/H222P skeletal muscle cells. In Vitro Cell.Dev.Biol.-Animal (2024). https://doi.org/10.1007/s11626-024-00915-1

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