Sex-based differences in cardiac gene expression and function in BDNF Val66Met mice. Sex-based differences in cardiac gene expression and function in BDNF Val66Met mice

Purpose: The goal of this study was to compare whole heart transcriptomes of hBDNF trasngenic mice to ascertain underlying molecular mechanisms for differences in cardiac function among genotypes Methods: Total RNA extracted from whole hearts of 8-week-old mice, and RNA libraries were constructed using the Illumina TruSeq Stranded Total RNA kit. Libraries were sequenced using Illumina HiSeq 3000 on paired-end-150 flow cell runs at ~32M PF reads per sample. Raw reads (fastq files) were uploaded to the Partek Flow server and pre-alignment quality assessment was performed. Mean base-call quality scores were above Phred-like values of 36 in all positions of all samples, and no hard trimming of the reads was necessary. Sequences were aligned to the mm10 assembly of the mouse genome using STAR 2.5.3a and resulting summary of reads filtered (<80) and quantified at the gene level to Ensemble transcripts 83 using Partek’s expectation maximization (E/M) annotation model. Gene counts were normalized to total read count per sample and then log-transformed (with an offset of 0.0001). To identify differentially expressed genes, statistical analysis was performed using Partek’s Gene Specific Analysis (GSA) multimodal estimation algorithm, followed by SYBR Green qPCR validation. Results: We found female-specific cardiac alterations in both heterozygous and homozygous carriers, including reduction in the gene encoding SERCA2 (Atp2a2) in homozygous Met/Met mice but more profoundly in females compared to males. Enriched functions in females with the Met allele included cardiac hypertrophy in response to stress, with down-regulation of the gene encoding titin (Tcap) and upregulation of BNP (Nppb), in line with altered cardiac functional parameters. Homozygous male mice on the other hand exhibited an inflammatory profile characterized by interferon-γ (IFN-γ)-mediated Th1 immune responses. Conclusions: . These results provide evidence for sex-based differences in how the BDNF polymorphism modifies cardiac physiology, including female-specific alterations of cardiac-specific transcripts and male-specific activation of inflammatory targets. Overall design: Whole heart mRNA profiles of 8 week old wild type (Val), hterozygous (Val/Met) and homozygous (Met) mice

本研究旨在比较人脑源性神经营养因子（human Brain-Derived Neurotrophic Factor, hBDNF）转基因小鼠的全心脏转录组，以明确不同基因型小鼠心脏功能差异的潜在分子机制。

研究方法：提取8周龄小鼠的全心脏总RNA，使用Illumina TruSeq Stranded Total RNA试剂盒构建RNA文库。采用Illumina HiSeq 3000测序平台，在双端150bp读长流通池中进行测序，每个样本约获得3200万条过滤后（PF, Pass Filter）读段。将原始读段（fastq格式文件）上传至Partek Flow服务器，并进行比对前的质量评估。所有样本的所有位点的平均碱基识别质量得分均高于类Phred质量值36，因此无需对读段进行硬修剪。使用STAR 2.5.3a将序列比对至小鼠基因组mm10组装版本，随后过滤比对读段（过滤阈值设为<80），并利用Partek的期望最大化（Expectation Maximization, E/M）注释模型，针对Ensembl 83版转录本进行基因水平定量分析。基因计数先按每个样本的总读段数进行归一化，随后进行对数转换（偏移量为0.0001）。为鉴定差异表达基因，采用Partek的基因特异性分析（Gene Specific Analysis, GSA）多峰估计算法开展统计学分析，随后通过SYBR Green定量PCR进行验证。

研究结果：本研究发现杂合子与纯合子hBDNF转基因小鼠均存在性别特异性的心脏转录组改变：纯合Met/Met小鼠中编码肌浆网钙ATP酶2（SERCA2, Atp2a2）的基因表达下调，且该下调效应在雌性小鼠中较雄性更为显著。携带Met等位基因的雌性小鼠中富集的功能通路涉及应激诱导的心肌肥厚，具体表现为编码肌联蛋白的基因（Tcap）表达下调以及B型钠尿肽（BNP, Nppb）表达上调，这与心脏功能参数的改变相一致。与之相反，纯合雄性小鼠则呈现出以干扰素-γ（IFN-γ）介导的Th1免疫反应为特征的炎症表型。

研究结论：本研究结果证实，BDNF多态性调控心脏生理的过程存在性别差异，具体表现为雌性小鼠出现心脏特异性转录本的特异性改变，而雄性小鼠则出现炎症靶点的特异性激活。

整体实验设计：包含8周龄野生型（Val）、杂合型（Val/Met）及纯合型（Met）小鼠的全心脏mRNA表达谱。