<i>asb5a</i>/<i>asb5b</i> Double Knockout Affects Zebrafish Cardiac Contractile Function
Ankyrin repeat and suppression-of-cytokine-signaling box (Asb) proteins, a subset of ubiquitin ligase E3, include Asb5 with six ankyrin-repeat domains. Zebrafish harbor two <i>asb5</i> gene isoforms, <i>asb5a</i> and <i>asb5b</i>. Currently, the effects of <i&g...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-11-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/22/16364 |
| _version_ | 1797459019689885696 |
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| author | Wanwan Cai Yuequn Wang Ying Luo Luoqing Gao Jian Zhang Zhigang Jiang Xiongwei Fan Fang Li Yulian Xie Xiushan Wu Yongqing Li Wuzhou Yuan |
| author_facet | Wanwan Cai Yuequn Wang Ying Luo Luoqing Gao Jian Zhang Zhigang Jiang Xiongwei Fan Fang Li Yulian Xie Xiushan Wu Yongqing Li Wuzhou Yuan |
| author_sort | Wanwan Cai |
| collection | DOAJ |
| description | Ankyrin repeat and suppression-of-cytokine-signaling box (Asb) proteins, a subset of ubiquitin ligase E3, include Asb5 with six ankyrin-repeat domains. Zebrafish harbor two <i>asb5</i> gene isoforms, <i>asb5a</i> and <i>asb5b</i>. Currently, the effects of <i>asb5</i> gene inactivation on zebrafish embryonic development and heart function are unknown. Using CRISPR/Cas9, we generated <i>asb5a</i>-knockout zebrafish, revealing no abnormal phenotypes at 48 h post-fertilization (hpf). In situ hybridization showed similar <i>asb5a</i> and <i>asb5b</i> expression patterns, indicating the functional redundancy of these isoforms. Morpholino interference was used to target <i>asb5b</i> in wild-type and <i>asb5a</i>-knockout zebrafish. Knocking down <i>asb5b</i> in the wild-type had no phenotypic impact, but simultaneous <i>asb5b</i> knockdown in <i>asb5a</i>-knockout homozygotes led to severe pericardial cavity enlargement and atrial dilation. RNA-seq and cluster analyses identified significantly enriched cardiac muscle contraction genes in the double-knockout at 48 hpf. Moreover, semi-automatic heartbeat analysis demonstrated significant changes in various heart function indicators. STRING database/Cytoscape analyses confirmed that 11 cardiac-contraction-related hub genes exhibited disrupted expression, with three modules containing these genes potentially regulating cardiac contractile function through calcium ion channels. This study reveals functional redundancy in <i>asb5a</i> and <i>asb5b</i>, with simultaneous knockout significantly impacting zebrafish early heart development and contraction, providing key insights into <i>asb5</i>’s mechanism. |
| first_indexed | 2024-03-09T16:45:30Z |
| format | Article |
| id | doaj.art-ef62712e70494a0182aa0dce34204a5b |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T16:45:30Z |
| publishDate | 2023-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-ef62712e70494a0182aa0dce34204a5b2023-11-24T14:47:20ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-11-0124221636410.3390/ijms242216364<i>asb5a</i>/<i>asb5b</i> Double Knockout Affects Zebrafish Cardiac Contractile FunctionWanwan Cai0Yuequn Wang1Ying Luo2Luoqing Gao3Jian Zhang4Zhigang Jiang5Xiongwei Fan6Fang Li7Yulian Xie8Xiushan Wu9Yongqing Li10Wuzhou Yuan11The Laboratary of Heart Development Research, College of Life Science, Hunan Normal University, Changsha 410081, ChinaThe Laboratary of Heart Development Research, College of Life Science, Hunan Normal University, Changsha 410081, ChinaThe Laboratary of Heart Development Research, College of Life Science, Hunan Normal University, Changsha 410081, ChinaThe Laboratary of Heart Development Research, College of Life Science, Hunan Normal University, Changsha 410081, ChinaThe Laboratary of Heart Development Research, College of Life Science, Hunan Normal University, Changsha 410081, ChinaThe Laboratary of Heart Development Research, College of Life Science, Hunan Normal University, Changsha 410081, ChinaThe Laboratary of Heart Development Research, College of Life Science, Hunan Normal University, Changsha 410081, ChinaThe Laboratary of Heart Development Research, College of Life Science, Hunan Normal University, Changsha 410081, ChinaThe Laboratary of Heart Development Research, College of Life Science, Hunan Normal University, Changsha 410081, ChinaThe Laboratary of Heart Development Research, College of Life Science, Hunan Normal University, Changsha 410081, ChinaThe Laboratary of Heart Development Research, College of Life Science, Hunan Normal University, Changsha 410081, ChinaThe Laboratary of Heart Development Research, College of Life Science, Hunan Normal University, Changsha 410081, ChinaAnkyrin repeat and suppression-of-cytokine-signaling box (Asb) proteins, a subset of ubiquitin ligase E3, include Asb5 with six ankyrin-repeat domains. Zebrafish harbor two <i>asb5</i> gene isoforms, <i>asb5a</i> and <i>asb5b</i>. Currently, the effects of <i>asb5</i> gene inactivation on zebrafish embryonic development and heart function are unknown. Using CRISPR/Cas9, we generated <i>asb5a</i>-knockout zebrafish, revealing no abnormal phenotypes at 48 h post-fertilization (hpf). In situ hybridization showed similar <i>asb5a</i> and <i>asb5b</i> expression patterns, indicating the functional redundancy of these isoforms. Morpholino interference was used to target <i>asb5b</i> in wild-type and <i>asb5a</i>-knockout zebrafish. Knocking down <i>asb5b</i> in the wild-type had no phenotypic impact, but simultaneous <i>asb5b</i> knockdown in <i>asb5a</i>-knockout homozygotes led to severe pericardial cavity enlargement and atrial dilation. RNA-seq and cluster analyses identified significantly enriched cardiac muscle contraction genes in the double-knockout at 48 hpf. Moreover, semi-automatic heartbeat analysis demonstrated significant changes in various heart function indicators. STRING database/Cytoscape analyses confirmed that 11 cardiac-contraction-related hub genes exhibited disrupted expression, with three modules containing these genes potentially regulating cardiac contractile function through calcium ion channels. This study reveals functional redundancy in <i>asb5a</i> and <i>asb5b</i>, with simultaneous knockout significantly impacting zebrafish early heart development and contraction, providing key insights into <i>asb5</i>’s mechanism.https://www.mdpi.com/1422-0067/24/22/16364<i>asb5a</i><i>asb5b</i>CRISPR/Cas9morpholinoRNA-seqsemi-automatic heartbeat analysis |
| spellingShingle | Wanwan Cai Yuequn Wang Ying Luo Luoqing Gao Jian Zhang Zhigang Jiang Xiongwei Fan Fang Li Yulian Xie Xiushan Wu Yongqing Li Wuzhou Yuan <i>asb5a</i>/<i>asb5b</i> Double Knockout Affects Zebrafish Cardiac Contractile Function International Journal of Molecular Sciences <i>asb5a</i> <i>asb5b</i> CRISPR/Cas9 morpholino RNA-seq semi-automatic heartbeat analysis |
| title | <i>asb5a</i>/<i>asb5b</i> Double Knockout Affects Zebrafish Cardiac Contractile Function |
| title_full | <i>asb5a</i>/<i>asb5b</i> Double Knockout Affects Zebrafish Cardiac Contractile Function |
| title_fullStr | <i>asb5a</i>/<i>asb5b</i> Double Knockout Affects Zebrafish Cardiac Contractile Function |
| title_full_unstemmed | <i>asb5a</i>/<i>asb5b</i> Double Knockout Affects Zebrafish Cardiac Contractile Function |
| title_short | <i>asb5a</i>/<i>asb5b</i> Double Knockout Affects Zebrafish Cardiac Contractile Function |
| title_sort | i asb5a i i asb5b i double knockout affects zebrafish cardiac contractile function |
| topic | <i>asb5a</i> <i>asb5b</i> CRISPR/Cas9 morpholino RNA-seq semi-automatic heartbeat analysis |
| url | https://www.mdpi.com/1422-0067/24/22/16364 |
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