Titin splicing. Mus musculus

Titin is a striated muscle-specific giant elastic protein and largely responsible for the generation of the diastolic force in the cardiac myocyte. Cardiac titin undergoes developmental changes in isoform expression during the course of cardiac development. At present, at least five size classes of titin isoforms (N2B and N2BA-A1, A2, N1, N2) have been identified using SDS agarose gel electrophoresis. The larger titin isoform N2BAs gradually decreased with ages, in contrast, the smaller titin isoform N2B increased in normal cardiomyocyte, and cardiac myocytes containing a higher proportion of the smaller titin isoform N2B have stronger passive tension than that with a lower proportion of the larger titin isoform N2BAs. Recently we found an autosomal dominant mutation which caused totally opposite cardiac titin isoform expression as compared to developmental stages. The larger total titin isoform N2BA increased in mutant rat cardiac myocytes with ages instead of the smaller cardiac titin isoform N2B. For the moment, mechanism of titin isoform switch is still unknown, therefore, the mutant rats will give us nevol sight to elucidate the titin splicing mechanism. Keywords: Titin isoforms, autosomal dominant mutation Overall design: Total RNA was extracted using TRIzol according to manufacturer instruction and further purified by the RNeasy mini kit (Qiagen, Valencia, CA). Double stranded cDNA was synthesized from total RNA (SuperScript II system; Invitrogen). An in vitro transcription reaction was then performed to obtain biotin-labeled cRNA from the double-stranded cDNA (Enzo BioArray High Yield RNA Transcript Labeling kit; Enzo Diagnostics, Farmingdale, NY). The cRNA was fragmented before hybridization, and then mixed in a hybridization mixture containing probe array controls, BSA, and herring sperm DNA. A cleanup procedure was performed on the hybridization cocktail using an RNeasy spin column (Qiagen), after which it was applied to the Affymetrix Rat 230 2.0 probe array. Total eighteen hybridization experiments were performed in which each stage (1-day, 20-day and 49-day) was represented by three normal and three mutant individual ventricular RNA extracts. Hybridization was allowed to continue for 16 h at 45°C in a GeneChip 640 hybridization oven, after which the arrays were washed and stained with phycoerythrin-conjugated streptavidin (Molecular Probes, Eugene, OR). Images were scanned using a GeneArray scanner (Agilent Technologies, Palo Alto, CA) and GeneChip cel files were subsequently processed by the log scale robust multi-array analysis (RMA). The log scale robust multi-array analysis (RMA) estimates are based upon a robust average of log2 (B (PM)), where B (PM) are background corrected perfect match intensities. three replicates were available for these conditions: three wild types and three homozygotes for 1-day, three wild types and three homozygotes for 20-day, and three wild types and three homozygotes for 49-day.

肌联蛋白（Titin）是一类横纹肌特异性巨型弹性蛋白，在心肌细胞中主要负责舒张张力的产生。心肌肌联蛋白在心脏发育进程中会发生同工型表达的动态变化。目前，通过SDS琼脂糖凝胶电泳（SDS agarose gel electrophoresis）已至少鉴定出五种尺寸类别的肌联蛋白同工型，包括N2B以及N2BA-A1、A2、N1、N2。在正常心肌细胞中，较大尺寸的肌联蛋白同工型N2BA会随年龄增长逐渐减少，而较小尺寸的肌联蛋白同工型N2B的表达量则逐步升高；且相较于含有较高比例较大尺寸肌联蛋白同工型N2BA的心肌细胞，含有较高比例较小尺寸肌联蛋白同工型N2B的心肌细胞具有更强的被动张力。

近期我们发现了一种常染色体显性突变，该突变会导致心肌肌联蛋白同工型的表达模式发生与发育阶段完全相反的改变：在突变大鼠的心肌细胞中，随年龄增长，原本应升高的较小尺寸同工型N2B未出现表达上调，反而较大尺寸的肌联蛋白同工型N2BA的表达量持续上升。目前，肌联蛋白同工型转换的分子机制仍不明晰，因此该突变大鼠模型将为阐明肌联蛋白的剪接机制提供全新视角。

关键词：肌联蛋白同工型，常染色体显性突变

整体实验设计：按照厂商说明书，使用TRIzol试剂提取总RNA（total RNA），并通过RNeasy迷你试剂盒（RNeasy mini kit，Qiagen，美国加利福尼亚州巴伦西亚）进一步纯化总RNA。采用SuperScript II反转录系统（SuperScript II system，Invitrogen）以总RNA为模板合成双链cDNA。随后通过体外转录反应，利用Enzo BioArray高产量RNA转录标记试剂盒（Enzo BioArray High Yield RNA Transcript Labeling kit，Enzo Diagnostics，美国纽约州法明代尔）从双链cDNA中制备生物素标记的cRNA。杂交前先对cRNA进行片段化处理，随后将其与含有探针阵列对照、牛血清白蛋白（BSA）以及鲑鱼精DNA的杂交混合液混匀。使用RNeasy离心柱（Qiagen）对杂交混合液进行纯化后，将其加载至Affymetrix大鼠230 2.0探针阵列（Affymetrix Rat 230 2.0 probe array）中。

本研究共开展18次杂交实验，每个实验阶段（1日龄、20日龄和49日龄）均设置3份野生型心室RNA提取物与3份突变型心室RNA提取物作为生物学重复。将杂交体系置于GeneChip 640基因芯片杂交炉中，于45℃条件下杂交16小时，随后对探针阵列进行洗涤，并使用藻红蛋白偶联链霉亲和素（phycoerythrin-conjugated streptavidin，Molecular Probes，美国俄勒冈州尤金）进行染色。使用GeneArray扫描仪（GeneArray scanner，Agilent Technologies，美国加利福尼亚州帕洛阿尔托）扫描芯片图像，后续通过对数尺度稳健多阵列分析（log scale robust multi-array analysis, RMA）处理GeneChip cel文件。对数尺度稳健多阵列分析（RMA）的估值基于log₂(B(PM))的稳健平均值，其中B(PM)为经背景校正后的完美匹配信号强度。

各实验条件均设置3次生物学重复：1日龄组包含3份野生型样本与3份纯合突变型样本，20日龄组与49日龄组同理。