The comparative evolutionary genomics analysis was used to study the functions of novel Ka/Ks-predicted human exons in a zebrafish model. The Yulink (MIOS, Entrez Gene: 54,468), a conserved gene from zebrafish to human with WD40 repeats at N-terminus, was identified and found to encode an 875 amino acid in human. The biological function of this Yulink gene in cardiomyocytes remains unexplored. The purpose of this study is to determine the involvement of Yulink in the functions of cardiomyocytes and to investigate its molecular regulatory mechanism. Knockdown of Yulink was performed using morpholino or shRNA in zebrafish, mouse HL-1 cardiomyocytes, and human iPSC-derived cardiomyocytes. The expression levels of mRNA and protein were quantified by qPCR and western blots. Other methods including DNA binding, ligand uptake, agonists treatment and Ca2+ imaging assays were used to study the molecular regulatory mechanism by Yulink. Statistical data were shown as mean ± SD or mean ± standard error. The knockdown of yulink with three specific morpholinos in zebrafish resulted in cardiac dysfunctions with pericardial edema, decreased heart beats and cardiac output. The Yulink knockdown in mouse HL-1 cardiomyocytes disrupted Ca2+ cycling, reduced DNA binding activity of PPARγ (peroxisome proliferator-activated receptor gamma) and resulted in a reduction of Serca2 (sarcoplasmic reticulum Ca2+ ATPase 2) expression. Expression of Serca2 was up-regulated by PPARγ agonists and down-regulated by PPARγ-shRNA knockdown, suggesting that Yulink regulates SERCA2 expression through PPARγ in mouse HL-1 cardiomyocytes. On the other hand, YULINK, PPARγ or SERCA2 over-expression rescued the phenotypes of Yulink KD cells. In addition, knockdown of YULINK in human iPSC-derived cardiomyocytes also disrupted Ca2+ cycling via decreased SERCA2 expression. Overall, our data showed that Yulink is an evolutionarily conserved gene from zebrafish to human. Mechanistically Yulink regulated Serca2 expression in cardiomyocytes, presumably mediated through PPARγ nuclear entry. Deficiency of Yulink in mouse and human cardiomyocytes resulted in irregular Ca2+ cycling, which may contribute to arrhythmogenesis.

中文翻译：

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根据进化分析预测，Yulink 与心脏功能有关

比较进化基因组学分析用于研究斑马鱼模型中新型 Ka/Ks 预测的人类外显子的功能。Yulink (MIOS, Entrez Gene: 54,468) 是一种从斑马鱼到人类的保守基因，在 N 末端具有 WD40 重复，已被鉴定并发现在人类中编码 875 个氨基酸。该 Yulink 基因在心肌细胞中的生物学功能仍未得到探索。本研究的目的是确定Yulink参与心肌细胞功能并探讨其分子调控机制。在斑马鱼、小鼠 HL-1 心肌细胞和人 iPSC 衍生的心肌细胞中使用吗啉或 shRNA 对 Yulink 进行敲除。通过qPCR和蛋白质印迹定量mRNA和蛋白质的表达水平。其他方法包括 DNA 结合、配体摄取、Yulink 使用激动剂处理和 Ca2+ 成像分析来研究分子调控机制。统计数据显示为平均值 ± SD 或平均值 ± 标准误差。在斑马鱼中用三种特定的吗啉基敲除 yulink 导致心包水肿、心跳和心输出量减少的心脏功能障碍。小鼠 HL-1 心肌细胞中的 Yulink 敲低破坏了 Ca2+ 循环，降低了 PPARγ（过氧化物酶体增殖物激活受体 γ）的 DNA 结合活性，并导致 Serca2（肌质网 Ca2+ ATPase 2）表达减少。Serca2 的表达被 PPARγ 激动剂上调，被 PPARγ-shRNA 敲低下调，表明 Yulink 通过 PPARγ 在小鼠 HL-1 心肌细胞中调节 SERCA2 表达。另一方面，YULINK，PPARγ 或 SERCA2 过表达挽救了 Yulink KD 细胞的表型。此外，人类 iPSC 衍生的心肌细胞中 YULINK 的敲低也会通过降低 SERCA2 表达来破坏 Ca2+ 循环。总体而言，我们的数据表明，Yulink 是从斑马鱼到人类的进化保守基因。Yulink 机械调节心肌细胞中 Serca2 的表达，可能是通过 PPARγ 核进入介导的。小鼠和人类心肌细胞中 Yulink 的缺乏导致不规则的 Ca2+ 循环，这可能导致心律失常。Yulink 机械调节心肌细胞中 Serca2 的表达，可能是通过 PPARγ 核进入介导的。小鼠和人类心肌细胞中 Yulink 的缺乏导致不规则的 Ca2+ 循环，这可能导致心律失常。Yulink 机械调节心肌细胞中 Serca2 的表达，可能是通过 PPARγ 核进入介导的。小鼠和人类心肌细胞中 Yulink 的缺乏导致不规则的 Ca2+ 循环，这可能导致心律失常。