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Murf1 alters myosin replacement rates in cultured myotubes in a myosin isoform–dependent manner

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Abstract

Skeletal muscle tissue increases or decreases its volume by synthesizing or degrading myofibrillar proteins. The ubiquitin–proteasome system plays a pivotal role during muscle atrophy, where muscle ring finger proteins (Murf) function as E3 ubiquitin ligases responsible for identifying and targeting substrates for degradation. Our previous study demonstrated that overexpression of Ozz, an E3 specific to embryonic myosin heavy chain (Myh3), precisely reduced the Myh3 replacement rate in the thick filaments of myotubes (E. Ichimura et al., Physiol Rep. 9:e15003, 2021). These findings strongly suggest that E3 plays a critical role in regulating myosin replacement. Here, we hypothesized that the Murf isoforms, which recognize Myhs as substrates, reduced the myosin replacement rates through the enhanced Myh degradation by Murfs. First, fluorescence recovery after a photobleaching experiment was conducted to assess whether Murf isoforms affected the GFP-Myh3 replacement. In contrast to Murf2 or Murf3 overexpression, Murf1 overexpression selectively facilitated the GFP-Myh3 myosin replacement. Next, to examine the effects of Murf1 overexpression on the replacement of myosin isoforms, Cherry-Murf1 was coexpressed with GFP-Myh1, GFP-Myh4, or GFP-Myh7 in myotubes. Intriguingly, Murf1 overexpression enhanced the myosin replacement of GFP-Myh4 but did not affect those of GFP-Myh1 or GFP-Myh7. Surprisingly, overexpression of Murf1 did not enhance the ubiquitination of proteins. These results indicate that Murf1 selectively regulated myosin replacement in a Myh isoform–dependent fashion, independent of enhanced ubiquitination. This suggests that Murf1 may have a role beyond functioning as a ubiquitin ligase E3 in thick filament myosin replacement.

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Acknowledgements

We sincerely appreciate the valuable technical support provided by Miho Ichimura (National Institute of Livestock and Grass Sciences, NARO).

Funding

This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant No. 19H03100 to KO and TN and Grant No. 23H03277 to KO.

Author information

Author notes

  1. Emi Uenaka and Koichi Ojima contributed equally to this work.

Authors and Affiliations

  1. Research Faculty of Agriculture, Graduate School of Agriculture, Hokkaido University, 9 Kita, 9 Nishi, Sapporo, Hokkaido, 060-8589, Japan

    Emi Uenaka, Ken Kobayashi & Takanori Nishimura

  2. Space Environment and Energy Laboratories, Nippon Telegraph and Telephone Corporation, Musashino, Tokyo, 180-8585, Japan

    Emi Uenaka

  3. Muscle Biology Research Unit, Division of Animal Products Research, Institute of Livestock and Grassland Science, NARO, 2 Ikenodai, Tsukuba, Ibaraki, 305-0901, Japan

    Koichi Ojima & Susumu Muroya

  4. Laboratory of Muscle and Meat Science, Department of Animal and Marine Bioresource Sciences, Faculty of Agriculture, Graduate School of Agriculture, Kyushu University, Motooka 744, Nishi-Ku, Fukuoka, 819-0395, Japan

    Takahiro Suzuki

  5. Laboratory of Meat Science and Production, Faculty of Veterinary Medicine, Kagoshima University, 1-21-24, Korimoto, Kagoshima, 890-0065, Japan

    Susumu Muroya

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  1. Emi Uenaka
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Contributions

KO and TN conceived and designed the research. EU and KO conducted the experiments; EU, KO, TS, KK, SM, and TN analyzed the data; EU, KO, and TN interpreted the results of the experiments. EU and KO prepared the figures, and EU and KO drafted the manuscript. EU, KO, TS, KK, SM, and TN edited and revised the manuscript. EU, KO, TS, KK, SM, and TN approved the final version of the manuscript.

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Correspondence to Takanori Nishimura.

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Uenaka, E., Ojima, K., Suzuki, T. et al. Murf1 alters myosin replacement rates in cultured myotubes in a myosin isoform–dependent manner. In Vitro Cell.Dev.Biol.-Animal (2024). https://doi.org/10.1007/s11626-024-00916-0

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