Myosin 5b Deficiency Alters Liver Proliferation, Zonation and Bile Acid Composition

Background: Myosin 5b (Myo5b) is a motor protein critical for trafficking proteins to the apical surface of intestinal epithelial cells. Inactivating mutations in MYO5B cause Microvillus Inclusion Disease (MVID), a congenital diarrhea disorder that often leads to liver cholestasis. While Myo5b's role in the intestine is well characterized, its function in the liver remains unclear. Methods & Results: To define the hepatic consequences of Myo5b loss, we analyzed germline Myo5b knockout (KO) mice. Bulk RNA-seq of KO livers revealed significant transcriptomic alterations, notably downregulation of genes linked to cell proliferation. Immunostaining confirmed reduced Ki67, phospho-histone H3, and cyclin D1 expression, along with impaired growth of liver organoids in Myo5b deficient mice. Histology and lipid staining showed steatosis and enlarged lipid droplets, with gene signatures favoring lipogenesis and ketogenesis in mice lacking Myo5b. Myo5b KO livers also displayed disrupted zonated gene expression and loss of zone 1 and zone 3 markers. Bile acid profiling revealed reduced hepatic bile acid levels, decreased expression of classical pathway genes (Cyp7a1, Cyp7b1), and compensatory upregulation of Cyp27a1. In the ileum, we observed mislocalization of the apical bile acid transporter ASBT and decreased levels of basolateral OSTß, leading to impaired enterohepatic recycling and increased luminal bile acids. Conclusions: These findings reveal a previously unrecognized role for Myo5b in liver proliferation, metabolic zonation, and bile acid homeostasis, highlighting its importance in maintaining hepatobiliary function. Overall design: Neonatal CD1 mice harboring a germline deletion of Myosin 5b and control (wildtype or heterozygous) mice were euthanized between the ages of 3-8 days old. For RNAseq, the entire liver was collected in Trizol and stored at -80 ºC prior to RNA extraction. mRNA was isolated using Trizol extraction and QIAGEN RNA easy kit according to the manufacturer's instructions. mRNA was extracted from the entire liver of 4 Myo5b KO and 4 control littermates and RNAseq was performed by MedGenome. RNAseq data was used for differential gene expression analysis, pathway analysis, and principal component analysis. Differential gene expression and pathway analysis were performed with RStudio (2024.04.2+764) using the DESeq2 R package (1.46.0)

### 背景
肌球蛋白5b（Myosin 5b，简称Myo5b）是一类介导蛋白质转运至肠上皮细胞顶侧膜的关键马达蛋白。MYO5B基因的失活突变会引发微绒毛包涵体病（Microvillus Inclusion Disease，MVID），这是一种先天性腹泻疾病，常导致肝胆汁淤积。尽管Myo5b在肠道中的功能已得到充分阐明，但其在肝脏中的作用仍不明确。

### 方法与结果
为明确Myo5b缺失对肝脏的影响，本研究对生殖系Myo5b敲除（knockout，简称KO）小鼠进行了分析。对KO小鼠肝脏的批量RNA测序（bulk RNA-seq）结果显示，转录组发生显著改变，其中与细胞增殖相关的基因显著下调。免疫染色实验证实，Myo5b缺失小鼠的Ki67抗原、磷酸化组蛋白H3以及细胞周期蛋白D1的表达水平均降低，同时肝脏类器官的生长受到损害。组织学与脂质染色结果显示，Myo5b缺失小鼠出现肝脏脂肪变性及脂滴增大，且其基因表达特征偏向脂肪生成与酮体生成。Myo5b敲除小鼠的肝脏还出现了分区基因表达紊乱，以及1区与3区标志物的丢失。胆汁酸谱分析显示，Myo5b缺失小鼠的肝脏胆汁酸水平降低，经典通路基因（Cyp7a1、Cyp7b1）的表达下调，同时Cyp27a1出现代偿性上调。在回肠组织中，我们观察到顶侧胆汁酸转运体ASBT的定位异常，以及基底外侧膜上的OSTβ水平降低，这导致肠肝循环受损且肠腔胆汁酸水平升高。

### 结论
本研究结果揭示了Myo5b此前未被认知的功能：其参与调控肝脏增殖、代谢分区以及胆汁酸稳态，凸显了其在维持肝胆功能中的重要作用。

### 整体实验设计
将携带Myosin 5b生殖系敲除的新生CD1品系小鼠，以及野生型/杂合型对照小鼠，在出生后3~8日龄时进行安乐处死。用于RNA测序（RNA-seq）的肝脏组织均采集于Trizol试剂中，并于-80℃保存直至RNA提取。按照试剂盒说明书，采用Trizol提取法与凯杰（QIAGEN）RNAeasy试剂盒分离mRNA。从4只Myo5b敲除小鼠与4只同窝对照小鼠的全肝组织中提取mRNA，并由MedGenome公司完成RNA测序。RNA测序数据用于差异基因表达分析、通路分析以及主成分分析。差异基因表达分析与通路分析均通过RStudio（2024.04.2+764）及DESeq2 R包（版本1.46.0）完成。