Na+ channel mRNA splicing in heart failure

描述（由申请人提供）：尽管有广泛的研究和新的治疗方法，但人类收缩性心力衰竭（HF）仍然是影响数百万美国人的重要临床问题，HF相关心律失常仍然是高发病率和死亡率的原因。最近，三个SCN 5a心脏Na+通道mRNA选择性剪接变体被发现在人类HF组织中上调。这些剪接变体由SCN 5a的末端外显子中的隐蔽剪接序列处的剪接产生（即，外显子28）和编码的心脏Na+通道在结构域IV的成孔区段之前截短。变体水平达到大于总SCN 5a mRNA的>50%。正如预期的那样，这些变体没有形成功能通道。此外，变体的存在导致全长SCN 5a mRNA丰度降低，而总SCN 5a mRNA没有改变。本申请提出建立在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的显性负性下调。初步数据表明，缺氧和血管紧张素II（AngII）可以通过诱导mRNA剪接因子CROP/hLuc 7A及其辅因子RBM 25的表达来发出病理性SCN 5a剪接调节的信号，所述mRNA剪接因子CROP/hLuc 7A及其辅因子RBM 25通过与SCN 5a外显子28（SCN 5a异常剪接发生的外显子）中的一个或多个RBM 25结合序列CGGGC（A）相互作用来改变SCN 5a剪接调节。此外，数据显示截短的Na+通道在内质网（ER）中积累并启动未折叠蛋白反应（UPR）途径，导致Na+通道翻译减少和全长SCN 5a转录物的半衰期缩短。假说.基于上述，我们假设hLuc 7A/RBM 25复合物有助于异常Na+通道mRNA剪接，并且UPR有助于异常剪接的转录物对Na+通道的显性负效应。具体目标。具体目标1：确定hLuc 7A/RBM 25剪接调控通路是否参与增加SCN 5a mRNA变体表达的机制。具体目标2：确定未折叠蛋白反应（UPR）中的三条主要途径在多大程度上负责功能性Na+通道的减少。具体目标3：证明人心力衰竭样本中hLuc 7A/RBM 25调节、未折叠蛋白反应（UPR）激活、Na+通道mRNA变体和Na+通道测量的关系。

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.