The effect of active YAP in neonatal heart of Xinβ knockout mice. The effect of active YAP in neonatal heart of Xinβ knockout mice

Purpose: The goals of this study are 1) to define the transcriptome changes in the heart in the absence of intercalated-disc protein Xinβ, and 2) to define the effect of active Yap overexpression on the cardiac transcriptome in the absence of Xinβ. Methods: Total RNA from heart apex of postnatal day (P) 7.5 Xinβ KO or WT mice were profiled by bulk-RNA sequencing (50bp SE). Total RNA from heart apex of P7.5 Xinβ KO or WT mice injected with AAV9 carrying cardiac troponin T promoter driven active Yap (S127A) or GFP (control) at P1 were profiled by bulk-RNA sequencing (150bp PE). FASTQ files were extracted, and the TruSeq sequencing adapters and low-quality reads were removed from FASTQ files with Cutadapt v.2.3. The cleaned FASTQ files were quality checked using FastQC (Babraham Bioinformatics), then aligned to the mouse genome (Esembl GRCm38 genome obtained from GENCODE) using HISAT2 (v.2.1.0). Subsequently, transcript assembly was performed using StringTie (v.1.3.4) with the annotated transcriptome as a reference. The assembled transcriptomes were quantified using prepDE.py script provided by the StringTie developer to generate gene matrix files. EdgeR (v.3.26.1) was used to compute Counts Per Million (CPM) as a normalized measurement for gene expression. Differentially expressed genes were tested using the Fisher’s exact test, and multiplicity correction is performed with the Benjamini-Hochberg method on the p-values, to control the false discovery rate (FDR). Results: We find that Xinβ knockout heart have changes on the transcriptome with hallmark of decrease proliferation etc. and subsets of the transcriptome alteration induced by the absence of Xinβ could be rescued by overexpressing active Yap in the neonatal mice. Overall design: Total RNA from heart apex of postnatal day (P) 7.5 Xinβ KO or WT mice (n=4 for each group) were profiled by bulk-RNA sequencing (50bp SE). Total RNA from heart apex of P7.5 Xinβ KO or WT mice injected with AAV9 carrying cardiac troponin T promoter driven active Yap (S127A) or GFP (control) at P1 (n=4 for each group) were profiled by bulk-RNA sequencing (150bp PE).

研究目的：本研究旨在实现两项目标：1）明确缺失嵌合盘蛋白Xinβ时心脏转录组（transcriptome）的变化特征；2）阐明在缺失Xinβ的背景下，活性Yap过表达对心脏转录组的影响。

研究方法：针对出生后第7.5天（P7.5）的Xinβ敲除（knockout, KO）小鼠与野生型（wild type, WT）小鼠的心尖组织总RNA，采用批量RNA测序（bulk-RNA sequencing）进行分析，测序模式为50bp单端测序（single-end, SE）。对于在出生后第1天（P1）注射了携带由心肌肌钙蛋白T启动子驱动的活性Yap(S127A)或GFP（对照）的腺相关病毒9型（AAV9）载体的P7.5 Xinβ KO与WT小鼠，提取其心尖组织总RNA，采用批量RNA测序进行分析，测序模式为150bp双端测序（paired-end, PE）。

测序数据预处理：使用Cutadapt v.2.3从FASTQ文件中移除TruSeq测序接头与低质量reads；利用Babraham生物信息学团队开发的FastQC对清理后的FASTQ文件进行质量校验。随后使用HISAT2 v.2.1.0将过滤后的reads比对至小鼠参考基因组（从GENCODE数据库获取的Ensembl GRCm38基因组）。

转录组组装与定量：以已注释的转录组为参考序列，使用StringTie v.1.3.4完成转录本组装；通过StringTie开发者提供的prepDE.py脚本对组装后的转录组进行定量分析，生成基因矩阵文件。

差异表达基因分析：使用EdgeR v.3.26.1计算每百万reads计数（Counts Per Million, CPM）作为基因表达的标准化衡量指标；采用Fisher精确检验筛选差异表达基因，并通过Benjamini-Hochberg方法对p值进行多重校正，以控制错误发现率（false discovery rate, FDR）。

研究结果：本研究发现，Xinβ敲除小鼠的心脏转录组发生显著改变，其特征包括增殖能力下降等；且缺失Xinβ所诱导的部分转录组异常，可通过在新生小鼠中过表达活性Yap得到挽救。

实验整体设计：本实验包含两类测序样本：① P7.5的Xinβ KO与WT小鼠（每组n=4）的心尖总RNA，采用50bp单端测序的批量RNA测序进行分析；② P1时注射了携带心肌肌钙蛋白T启动子驱动的活性Yap(S127A)或GFP（对照）的AAV9载体的P7.5 Xinβ KO与WT小鼠（每组n=4）的心尖总RNA，采用150bp双端测序的批量RNA测序进行分析。