CaMKII-dependent mechanisms in RBM20 cardiomyopathy

扩张型心肌病（DCM）是心衰的主要原因之一。所有DCM病例中有30-50%是家族性的，在3-7%的家族性DCM病例中，rna结合基板蛋白20 （RBM20）的突变似乎是原因。RBM20突变通常导致临床侵袭性DCM，并与进行性心功能障碍、心脏Ca2+处理紊乱和恶性室性心律失常的风险增加有关。我们之前已经确定错剪接的CaMKIIδ作为一个候选靶点，可能导致RBM20突变携带者的钙处理紊乱和心律失常负担增加，但目前尚不清楚错剪接的CaMKIIδ在多大程度上以及如何导致这种表型。本项目的总体目标是进一步揭示RBM20突变携带者引起心律失常和心功能障碍的分子机制。在WP1中，我们将Rbm20-/-小鼠与CaMKIIδ-/-小鼠杂交，以证明CaMKIIδ确实是Rbm20的关键剪接靶点。同时，为了研究剪接变体CaMKIIδ a导致钙处理紊乱和心律失常的具体原因，我们将使用无偏ip质谱法和磷酸化蛋白质组学来确定不同心脏CaMKIIδ剪接异构体（WP2）的特定目标谱。第三，作为一种转化方法，我们将测试使用维拉帕米和吉利德最近披露的新的CaMKII抑制剂GS-680降低细胞内Ca2+的潜在治疗策略，以减少Rbm20-/-小鼠（WP3）的心律失常负担。此外，我们将研究Rbm20-/-小鼠（WP4）的转录和代谢失调。最后，我们将探讨camkii介导的HDAC4转录调控的作用，以及它如何影响Rbm20-/-小鼠（WP5）的心律失常和心功能障碍。

Dilated cardiomyopathy (DCM) is one of the major causes of heart failure. 30-50% of all DCM cases are familial, and in 3-7% of familial DCM cases a mutation in RNA-binding motif protein 20 (RBM20) seems to be the cause. RBM20 mutations often lead to a clinically aggressive form of DCM, and are associated with progressive cardiac dysfunction, disturbed cardiac Ca2+ handling, and an increased risk of malignant ventricular arrhythmias. We have previously identified misspliced CaMKIIδ as a candidate target that may contribute to the disturbed calcium handling and increased arrhythmic burden of RBM20 mutation carriers, but it is yet unknown to what extent and how misspliced CaMKIIδ contributes to this phenotype. This projects overall aim is to further unravel the molecular mechanisms that cause arrhythmias and cardiac dysfunction in RBM20 mutation carriers. In WP1, we will cross Rbm20-/- mice to CaMKIIδ-/- mice to provide proof of concept that CaMKIIδ indeed serves as a critical splicing target of RBM20. In parallel, in order to investigate why specifically the splice variant CaMKIIδA causes disturbed calcium handling and arrhythmias, we will perform unbiased IP-mass spectrometry and phospho-proteomics to identify the specific target profiles of the different cardiac CaMKIIδ splice isoforms (WP2). Third, as a translational approach, we will test the potential therapeutic strategy of lowering intracellular Ca2+ with the use of verapamil and the recently disclosed new CaMKII inhibitor GS-680 from Gilead to reduce the arrhythmia burden Rbm20-/- mice (WP3). Furthermore, we will investigate transcriptional and metabolic dysregulation in Rbm20-/- mice (WP4). Lastly, we will interrogate the contribution of CaMKII-mediated HDAC4 transcriptional regulation and how this impacts on arrhythmias and cardiac dysfunction in Rbm20-/- mice (WP5).