Na+ channel mRNA splicing in heart failure

描述(申请人提供)：尽管有广泛的研究和新的治疗方法，人类收缩性心力衰竭(HF)仍然是一个严重的临床问题，影响着数以百万计的美国人，而且与HF相关的心律失常仍然是高发病率和死亡率的原因。最近，三个SCN5A心脏Na+通道mRNA选择性剪接变异体被发现在人心衰组织中上调。这些剪接变异体是在SCN5A末端外显子(即外显子28)的隐蔽剪接序列上剪接的结果，编码的心肌Na+通道在结构域IV的成孔片段之前被截断。变异体的水平超过SCN5A总mRNA的50%。正如预期的那样，这些变体没有形成功能通道。此外，突变体的存在导致全长SCN5A mRNA的丰度降低，但不改变总的SCN5A mRNA。本申请建议建立在HF中发生异常剪接的机制，以及截短的Na+通道变体的存在如何导致全长通道mRNA的显性负向下调。初步数据表明，缺氧和血管紧张素II(AngII)可通过诱导转录因子CROP/hLuc7A及其辅助因子RBM25的表达，通过与SCN5A外显子28上的一个或多个RBM25结合序列CGGGC(A)相互作用改变SCN5A的剪接调控，从而发出病理性的SCN5A剪接调控信号。此外，数据显示，截短的Na+通道聚集在内质网(ER)并启动未折叠蛋白反应(UPR)途径，导致Na+通道翻译减少，全长SCN5A转录本的半衰期缩短。假设。在此基础上，我们推测hLuc7A/RBM25复合体参与了Na+通道的异常剪接，UPR参与了异常剪接的转录本对Na+通道的显性负效应。具体目标。具体目的1：探讨hLuc7A/RBM25剪接调控通路是否参与了SCN5A基因表达上调的机制。具体目的2：确定未折叠蛋白反应(UPR)中的三个主要途径在多大程度上导致功能性Na+通道的减少。具体目的3：探讨心力衰竭标本中hLuc7A/RBM25调节、未折叠蛋白反应(UPR)激活、Na+通道mRNA变异和Na+通道测量之间的关系。 公共卫生相关性：心力衰竭猝死的原因尚不清楚。这一应用将探索一个潜在的原因，即钠通道处理异常，建立其机制，并可能阐明预防策略。此外，如果白细胞表现出类似的异常，这项工作可能会导致血液测试，以预测心力衰竭的猝死风险。

DESCRIPTION (provided by applicant): Despite the extensive research and novel treatments, human systolic heart failure (HF) remains a substantial clinical problem affecting millions of Americans and HF associated arrhythmia still remains a cause of the high morbidity and mortality. Recently, three SCN5a cardiac Na+ channel mRNA alternative splicing variants were found to be upregulated in human HF tissue. These splicing variants resulted from splicing at cryptic splice sequences in the terminal exon of SCN5a (i.e., exon 28) and encoded cardiac Na+ channels truncated before the pore forming segment of domain IV. Variant levels reached greater that >50% of the total SCN5a mRNA. As expected, these variants did not form functional channels. Moreover, the presence of the variants caused reduced abundance of the full-length SCN5a mRNA without alteration of total SCN5a mRNA. This application proposes to establish the mechanism whereby the abnormal splicing occurs in HF and how the presence of truncated Na+ channel variants causes a dominant negative downregulation of the full-length channel mRNA. Preliminary data suggest hypoxia and angiotensin II (AngII) can signal pathological SCN5a splicing regulation by inducing expression of the mRNA splicing factor, CROP/hLuc7A, and its co-factor RBM25, which alter SCN5a splicing regulation by interacting with one or more RBM25 binding sequences CGGGC(A) in SCN5a exon 28, the exon where abnormal splicing of SCN5a occurs. Furthermore, data show that truncated Na+ channels accumulate in endoplasmic reticulum (ER) and initiate the unfolded protein response (UPR) pathway, causing reduced Na+ channel translation and a shortened half-life of the full-length SCN5a transcript. Hypothesis. Based on the above, we hypothesized that the hLuc7A/RBM25 complex contributes to abnormal Na+ channel mRNA splicing and that the UPR contributes to the dominant negative effect the abnormally spliced transcripts have on the Na+ channel. Specific Objectives. Specific aim 1: To establish whether the hLuc7A/RBM25 splicing regulation pathway is involved in the mechanism to increase SCN5a mRNA variant expression. Specific aim 2: To determine to what extent the three major pathways in the unfolded protein response (UPR) are responsible for the reduction in functional Na+ channels. Specific aim 3: To demonstrate the relationship of hLuc7A/RBM25 regulation, the unfolded protein response (UPR) activation, Na+ channel mRNA variants, and Na+ channel measures in human heart failure samples. PUBLIC HEALTH RELEVANCE: The cause of sudden death in heart failure is unknown. This application will explore one potential cause, abnormal sodium channel processing, establishing the mechanism and possible elucidating prevention strategies. Moreover, if white cells show similar abnormalities, this work may lead to a blood test to predict sudden death risk in heart failure.