Inhibition of ubiquitin‐specific protease 2 causes accumulation of reactive oxygen species, mitochondria dysfunction, and intracellular ATP decrement in C2C12 myoblasts
Abstract Ubiquitin‐specific protease 2 (USP2) is considered to participate in the differentiation of myoblasts to myotubes, however, its functions in myoblasts under growth conditions remain elusive. In this study, we analyzed the physiological roles of USP2 in myoblasts using Usp2 knockout (KO) C2C...
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| Format: | Article |
| Language: | English |
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Wiley
2019-07-01
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| Series: | Physiological Reports |
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| Online Access: | https://doi.org/10.14814/phy2.14193 |
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| author | Mayuko Hashimoto Natsuko Saito Haru Ohta Kumiko Yamamoto Asuka Tashiro Kosuke Nakazawa Osamu Inanami Hiroshi Kitamura |
| author_facet | Mayuko Hashimoto Natsuko Saito Haru Ohta Kumiko Yamamoto Asuka Tashiro Kosuke Nakazawa Osamu Inanami Hiroshi Kitamura |
| author_sort | Mayuko Hashimoto |
| collection | DOAJ |
| description | Abstract Ubiquitin‐specific protease 2 (USP2) is considered to participate in the differentiation of myoblasts to myotubes, however, its functions in myoblasts under growth conditions remain elusive. In this study, we analyzed the physiological roles of USP2 in myoblasts using Usp2 knockout (KO) C2C12 cells as well as a USP2 specific inhibitor. In addition to the disruption of differentiation, clustered regularly interspaced short palindromic repeats/Cas9‐generated Usp2KO cells exhibited inhibition of proliferation compared to parental C2C12 cells. Usp2KO cells reduced the accumulation of intracellular adenosine triphosphate (ATP) content and oxygen consumption. Moreover, Usp2KO cells had fragmented mitochondria, suggesting that mitochondrial respiration was inactive. The deficiency of Usp2 did not affect the enzymatic activities of respiratory chain complexes I, III, IV, and V. However, mitochondrial membrane permeability—evaluated using calcein AM‐cobalt staining—was increased in Usp2KO cells. The membrane potential of Usp2KO cells was clearly decreased. Usp2KO cells accumulated reactive oxygen species (ROS) in the mitochondria. The USP2‐selective inhibitor ML364 also increased the levels of mitochondrial ROS, and modulated the membrane potential and morphology of the mitochondria. These effects were followed by a decrement in the intracellular content of ATP. Based on these findings, we speculate that USP2 may be involved in maintaining the integrity of the mitochondrial membrane. This process ensures the supply of ATP in myoblasts, presumably leading to proliferation and differentiation. |
| first_indexed | 2024-12-18T11:03:00Z |
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| id | doaj.art-c922ea8db5c845c485c67d12f572c1e9 |
| institution | Directory Open Access Journal |
| issn | 2051-817X |
| language | English |
| last_indexed | 2024-12-18T11:03:00Z |
| publishDate | 2019-07-01 |
| publisher | Wiley |
| record_format | Article |
| series | Physiological Reports |
| spelling | doaj.art-c922ea8db5c845c485c67d12f572c1e92022-12-21T21:10:11ZengWileyPhysiological Reports2051-817X2019-07-01714n/an/a10.14814/phy2.14193Inhibition of ubiquitin‐specific protease 2 causes accumulation of reactive oxygen species, mitochondria dysfunction, and intracellular ATP decrement in C2C12 myoblastsMayuko Hashimoto0Natsuko Saito1Haru Ohta2Kumiko Yamamoto3Asuka Tashiro4Kosuke Nakazawa5Osamu Inanami6Hiroshi Kitamura7Laboratory of Veterinary Physiology, School of Veterinary Medicine Rakuno Gakuen University Ebetsu JapanLaboratory of Veterinary Physiology, School of Veterinary Medicine Rakuno Gakuen University Ebetsu JapanLaboratory of Veterinary Physiology, School of Veterinary Medicine Rakuno Gakuen University Ebetsu JapanLaboratory of Radiation Biology, Graduate School of Veterinary Medicine Hokkaido University Sapporo JapanLaboratory of Veterinary Physiology, School of Veterinary Medicine Rakuno Gakuen University Ebetsu JapanLaboratory of Veterinary Physiology, School of Veterinary Medicine Rakuno Gakuen University Ebetsu JapanLaboratory of Radiation Biology, Graduate School of Veterinary Medicine Hokkaido University Sapporo JapanLaboratory of Veterinary Physiology, School of Veterinary Medicine Rakuno Gakuen University Ebetsu JapanAbstract Ubiquitin‐specific protease 2 (USP2) is considered to participate in the differentiation of myoblasts to myotubes, however, its functions in myoblasts under growth conditions remain elusive. In this study, we analyzed the physiological roles of USP2 in myoblasts using Usp2 knockout (KO) C2C12 cells as well as a USP2 specific inhibitor. In addition to the disruption of differentiation, clustered regularly interspaced short palindromic repeats/Cas9‐generated Usp2KO cells exhibited inhibition of proliferation compared to parental C2C12 cells. Usp2KO cells reduced the accumulation of intracellular adenosine triphosphate (ATP) content and oxygen consumption. Moreover, Usp2KO cells had fragmented mitochondria, suggesting that mitochondrial respiration was inactive. The deficiency of Usp2 did not affect the enzymatic activities of respiratory chain complexes I, III, IV, and V. However, mitochondrial membrane permeability—evaluated using calcein AM‐cobalt staining—was increased in Usp2KO cells. The membrane potential of Usp2KO cells was clearly decreased. Usp2KO cells accumulated reactive oxygen species (ROS) in the mitochondria. The USP2‐selective inhibitor ML364 also increased the levels of mitochondrial ROS, and modulated the membrane potential and morphology of the mitochondria. These effects were followed by a decrement in the intracellular content of ATP. Based on these findings, we speculate that USP2 may be involved in maintaining the integrity of the mitochondrial membrane. This process ensures the supply of ATP in myoblasts, presumably leading to proliferation and differentiation.https://doi.org/10.14814/phy2.14193USPmyoblastmitochondriarespiratory chainoxidative phosphorylation |
| spellingShingle | Mayuko Hashimoto Natsuko Saito Haru Ohta Kumiko Yamamoto Asuka Tashiro Kosuke Nakazawa Osamu Inanami Hiroshi Kitamura Inhibition of ubiquitin‐specific protease 2 causes accumulation of reactive oxygen species, mitochondria dysfunction, and intracellular ATP decrement in C2C12 myoblasts Physiological Reports USP myoblast mitochondria respiratory chain oxidative phosphorylation |
| title | Inhibition of ubiquitin‐specific protease 2 causes accumulation of reactive oxygen species, mitochondria dysfunction, and intracellular ATP decrement in C2C12 myoblasts |
| title_full | Inhibition of ubiquitin‐specific protease 2 causes accumulation of reactive oxygen species, mitochondria dysfunction, and intracellular ATP decrement in C2C12 myoblasts |
| title_fullStr | Inhibition of ubiquitin‐specific protease 2 causes accumulation of reactive oxygen species, mitochondria dysfunction, and intracellular ATP decrement in C2C12 myoblasts |
| title_full_unstemmed | Inhibition of ubiquitin‐specific protease 2 causes accumulation of reactive oxygen species, mitochondria dysfunction, and intracellular ATP decrement in C2C12 myoblasts |
| title_short | Inhibition of ubiquitin‐specific protease 2 causes accumulation of reactive oxygen species, mitochondria dysfunction, and intracellular ATP decrement in C2C12 myoblasts |
| title_sort | inhibition of ubiquitin specific protease 2 causes accumulation of reactive oxygen species mitochondria dysfunction and intracellular atp decrement in c2c12 myoblasts |
| topic | USP myoblast mitochondria respiratory chain oxidative phosphorylation |
| url | https://doi.org/10.14814/phy2.14193 |
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