<i>asb5a</i>/<i>asb5b</i> Double Knockout Affects Zebrafish Cardiac Contractile Function

<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...

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Bibliographic Details
Main Authors: Wanwan Cai, Yuequn Wang, Ying Luo, Luoqing Gao, Jian Zhang, Zhigang Jiang, Xiongwei Fan, Fang Li, Yulian Xie, Xiushan Wu, Yongqing Li, Wuzhou Yuan
Format: Article
Language:English
Published: MDPI AG 2023-11-01
Series:International Journal of Molecular Sciences
Subjects:
<i>asb5a</i>
<i>asb5b</i>
CRISPR/Cas9
morpholino
RNA-seq
semi-automatic heartbeat analysis
Online Access:https://www.mdpi.com/1422-0067/24/22/16364
Description
Summary: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.
ISSN:1661-6596
1422-0067

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