抑制HDAC对心肌肥大时KChIP2表达减少和Ito电流下调的治疗作用和机制研究

The transient outward K+ current (Ito) is downregulated and contributes to the pathogenesis of ventricular arrhythmia in cardiac hypertrophy by prolonging action potential duration (APD) and increasing transmural dispersion of ventricular repolarization. It is well established that the downregulation of Ito is largely attributed to the reduction of KChIP2, the beta subunit of Ito. However, the mechanism underlying the decreased KChIP2 expression in hypertrophy remains largely unknown. Our pioneering work demonstrated that VPA, the pan-inhibitor for class I and class II histone deacetylase (HDAC), could up-regulate KChIP2 expression and Ito current densities in cardiac hypertrophy. In this project, we will identify the specific type of HDAC responsible for the therapeutic effect of inhibition of HDAC on KChIP2 expression and Ito in cardiac hypertrophy. Furthermore, we will investigate the underlying mechanisms. Here, we proposed two hypotheses based on our preliminary data. First, the expression of H3K4 demethalase was decreased by inhibition of HDAC, leading to increase in H3K4 methylation. Furthermore, the recruitment of H3K4 demethalase to the promoter of Kcnip2 (the gene encoding KChIP2) was decreased, resulting in attenuated enrichment of H3K4me3 at Kcnip2 promoter, resulting in down-regulation of Kcnip2 expression. Second, inhibition of HDAC dissociated the interaction of HDAC and Brg1 inhibitory complex with NF-kB at Kcnip2 promoter and prohibited the negative regulation of HDAC and Brg1 complex on Kcnip2 expression. In this project, we aim to elucidate novel epigenetic regulatory mechanisms for the downregulation of KChIP2 expression and Ito and try to find out new therapeutic strategies for the treatment of arrhythmia in hypertrophy.

心肌肥大引起Ito电流降低，是导致室性心律失常发生的重要原因。Ito下调与该通道beta亚基KChIP2表达降低密切相关，其机制尚待查明。我们前期工作首次发现，抑制组蛋白去乙酰化酶(HDAC)能上调肥大心肌细胞KChIP2表达和Ito电流。我们拟通过RNAi和基因敲除手段从细胞和整体水平进一步查明起作用的特异性HDAC靶点(类型)。还将应用离体和在体基因表达调控、染色质免疫沉淀（ChIP）等技术，从分子、细胞和整体水平阐明心肌肥大时抑制HDAC作用的以下可能机制: (1)降低H3K4去甲基化酶表达，及解除KChIP2基因启动子区HDAC对H3K4去甲基化酶的募集作用，增加该启动子区H3K4me3聚集，使KChIP2表达增加；(2)解除HDAC和Brg1抑制复合物与NF-kB结合，使该复合物对KChIP2表达的抑制作用解除，增加KChIP2表达。为心肌肥大时心律失常的治疗提供新靶点和思路。

心肌肥大引起瞬间外向钾电流（Ito）降低，是导致室性心律失常发生的重要原因。KChIP2是Ito电流的β调节亚基，心肌肥大时KChIP2表达降低，是引起Ito电流降低的重要原因，但其机制尚未阐明。本研究发现，在苯肾上腺素（PE）刺激引起的心肌肥大细胞模型和主动脉缩窄术（TAC）引起的压力负荷性小鼠心肌肥大模型，抑制组蛋白去乙酰化酶（ HDAC）能显著上调肥大心肌细胞KChIP2表达和Ito电流的降低。采用针对不同类型HDAC的抑制剂，我们筛选出HDAC I/II 类广谱抑制剂VPA和HDAC1/HDAC2抑制剂Romidepsin具有抑制心肌细胞肥大时KChIP2 表达下调的作用，通过RNA干扰（RNAi）手段特异性抑制HDAC1和HDAC2，发现HDAC2而非HDAC1抑制PE引起的KChIP2 表达下调。无论在离体（PE）和在体心肌肥大模型（TAC）均发现HDAC2表达增加，表明HDAC2是引起心肌肥大时KChIP2 表达降低和Ito功能下调的关键分子。进一步分子生物学研究揭示抑制HDAC2改善心肌肥大时KChIP2表达降低的机制：降低HDAC2表达使H3K4去甲基化酶（KDM5d）的泛素化水平显著增加，从而降低KDM5d的蛋白水平，引起H3K4三甲基化（H3K4me3）水平增加。同时，增加H3K4me3在Kcnip2启动子区的富集，促进Kcnip2的转录，使KChIP2表达增加。HDAC2对NF-kB所调节的KChIP2表达通路无显著影响。通过药物Romidepsin或基因调节心肌细胞HDAC2活性，显著抑制PE诱导的心肌肥大所引起的Ito电流下调和动作电位延长；在体通过小鼠尾静脉注射AAV9-HDAC2 shRNA 腺相关病毒降低心脏组织HDAC2表达，抑制TAC心肌肥大模型小鼠心肌细胞Ito电流下调和动作电位延长，发挥对Ito通道重构引起心电紊乱的治疗作用。 因此，本项目明确了心肌肥大时抑制HDAC2对KChIP2表达和Ito功能的上调作用，并阐明KChIP2表达的新的表观遗传学调节机制，提出HDAC2作为治疗心肌肥大心律失常发生的新靶点和治疗策略。