Alternative Splicing of Cav1.2 in ARVC Patients
Alternative Splicing of Cav1.2 in ARVC Patients
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ARVC 患者中 Cav1 2 的选择性剪接
DOI：
10.1016/j.bpj.2019.11.366
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发表时间：
2020
影响因子：
3.4
通讯作者：
Hammer
中科院分区：
文献类型：
--
作者：
Bourjau;DiBiase;Campiglio;Giglberger;Schober;Stauber;Pietrzyk;Baessler;Fischer;Wagner;Hammer
致心律失常性右心室心肌病是一种罕见的遗传性疾病，包括桥粒蛋白的各种突变。第一个明显的症状通常是严重的心律失常，如心脏性猝死（SCD）。我们发现了钠电流和钙循环的改变，但导致心律失常的确切分子机制仍不完全understood.We产生诱导多能干细胞从ARVC患者生存后SCD与功能丧失PKP 2突变和她的无症状同胞相同的突变，并分化成心肌细胞（iPSC-CM-index和iPSC-CM-sibling，分别）。我们发现，iPSC-CM-index（-25%）和iPSC-CM-sibling（-38%）中的L型Ca电流密度（ICa）均降低。CaV 1.2的表达水平不变，表明ICa的降低可能依赖于通道门控调节的改变。然而，晚期钠电流和蛋白质印迹数据没有提供证据的上调CaMKII或PKA活性，可以解释的监管ICa。有趣的是，两名患者的钙瞬变都有所减缓（延长了26%），但幅度仅在iPSC-CM指数中降低（-30%）。为了测试细胞中I Ca和Ca循环的失调是否取决于Ca V 1.2剪接变体的变化，我们通过qPCR测量了Ca V 1.2外显子9 β（E9 β）相对于外显子9和外显子33（E33）的相对表达，外显子9和外显子33都参与肥大和心力衰竭。E33在iPSC-CM-指数和iPSC-CM-同胞中均增加，而E9 β仅在iPSC-CM-指数中上调。我们产生了具有E9缺失或E33缺失或两者的GFP标记的Ca V 1.2构建体。所有Ca V 1.2构建体与RyR共定位，表明成功的通道靶向Ca释放单位。
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare hereditary disease comprising various mutations of desmosomal proteins. The first overt symptoms are often severe arrhythmias such as sudden cardiac death (SCD). We found alterations in sodium currents and Ca cycling but the exact molecular mechanisms leading to arrhythmias are still not fully understood.We generated induced pluripotent stem cells from an ARVC patient after survived SCD with a loss-of-function PKP2 mutation and her asymptomatic sibling with the same mutation and differentiated them into cardiac myocytes (iPSC-CM-index and iPSC-CM-sibling, respectively). We found that L-type Ca current densities (I Ca) are reduced in both iPSC-CM-index (− 25%) and iPSC-CM-sibling (− 38%). The expression level of Ca V 1.2 is unchanged, indicating that the reduction in I Ca might depend on altered regulation of channel gating. However, late sodium currents and Western Blot data provide no evidence for upregulated CaMKII or PKA activity that could account for the regulation of I Ca. Interestingly, Ca transients were slowed in both patients (∼ 26% longer) but the amplitude was reduced only in the iPSC-CM-index (− 30%). To test whether the dysregulation of I Ca and Ca cycling in the cells depend on changes of Ca V 1.2 splice variants, we measured the relative expression of Ca V 1.2 exon 9∗(E9∗) vs. exon 9 and exon 33 (E33) who are both involved in hypertrophy and heart failure by qPCR. E33 is increased in both iPSC-CM-index and iPSC-CM-sibling whereas E9∗ is upregulated solely in iPSC-CM-index. We generated GFP tagged Ca V 1.2 constructs with either E9∗ or deletion of E33 or both. All Ca V 1.2 constructs co-localize with RyR, indicating successful channel targeting to Ca release units.