转录抑制因子DEC1在病理性心肌肥厚中的作用及机制研究

Pathological cardiac hypertrophy is an independent risk factor of cardiovascular adverse events. Molecularly, an imbalance between pro-hypertrophic genes and anti-hypertrophic genes causes cardiac hypertrophy, which, in turn, could be recognized as a disorder of gene regulation. Differentiated embryo chondrocyte 1 (DEC1) is known as a transcription repressor and acts through binding to the E-box elements in the promoter of target genes. Though abundant expression has been found in heart tissues, the role of DEC1 in cardiac biology has never been elucidated. Our preliminary study found that loss of Dec1 exaggerated pressure overload-induced cardiac hypertrophy in mice. Moreover, there is an E-box element in the promoter of Gata4 gene, which has been regarded to play a central role in cardiac hypertrophy. Thus, we hypothesized DEC1 repressed the expression of Gata4 by binding to its E-box element. Furthermore, we found that the expression of GATA4 was elevated in pressure overload-stressed hearts of Dec1 knockout mice. The following questions will be answered in the current study: to determine whether DEC1 gain-of-function could inhibit cardiac hypertrophy, and could be a potential therapeutic target in this maladaptive process; to dissect the molecular mechanisms by which DEC1 regulates cardiac hypertrophy; whether DEC1 binds to the E-box element of Gata4 gene and represses its transcription. Therapies targeting upstream signaling cascades might cause severe side effects due to extensive cross-talk between intracellular signalings. Developing drugs that selectively affect transcriptional factors as converging targets during the onset of cardiac hypertrophy have a better specificity and can be of great therapeutic interest.

病理性心肌肥厚是多种心血管不良事件发生的独立危险因素，是心肌组织转录调控紊乱的综合体现。分化型胚胎软骨发育基因1（DEC1）作为转录抑制因子，通过特异性结合于E-box转录调控元件并抑制下游基因表达，其在心脏中表达丰富但功能未知。本项目前期研究发现，Dec1基因敲除显著加重了小鼠压力过负荷诱导的心肌肥厚。心肌肥厚关键基因Gata4启动子区域含有E-box转录调控元件，由此申报者猜想，DEC1可能结合至Gata4基因E-box元件并抑制其表达，从而发挥对心肌肥厚的调控作用。进一步研究发现GATA4在Dec1敲除的肥厚心肌组织中表达升高。本项目拟进一步从DEC1功能增强的角度明确其对于心肌肥厚的调控及治疗作用，并在分子及在体水平解析DEC1是否通过Gata4调控心肌肥厚。本项目以转录因子为研究靶点，试图在心肌肥厚发病机制的末端给予精准干预，从而避免上游机制干预带来的细胞活动紊乱和广泛性副作用。

病理性心肌肥厚是心力衰竭极为重要的独立危险因素，分化型胚胎软骨发育基因1（Differentiated embryo chondrocyte 1，DEC1）在病理性心肌肥厚过程中的作用仍不清楚，本研究旨在深入探究DEC1在病理性心肌肥厚中的作用与并试图解析潜在的分子机制。利用去氧肾上腺素刺激大鼠心脏成肌细胞系H9C2细胞建立体外病理性心肌细胞肥大模型，通过互作蛋白质谱技术，我们发现DEC1能与GATA盒结合蛋白4（GATA binding protein 4，GATA4）结合并抑制其活性。体外细胞实验表明，DEC1能明显抑制去氧肾上腺素刺激诱导的新生大鼠原代心肌细胞和H9C2细胞病理性心肌细胞肥大。进一步研究提示，DEC1对心肌细胞肥大的抑制作用依赖于其对GATA4蛋白活性的抑制。机制研究表明，DEC1并不影响Gata4基因转录和GATA4蛋白核质定位，而能促进GATA4蛋白发生K6和K63类型的泛素化和泛素-蛋白酶体途径的降解。进一步的，利用互作蛋白质谱技术，我们发现DEC1通过招募E3连接酶PRP19促进GATA4蛋白的泛素化。GATA4蛋白赖氨酸位点突变实验结果表明，DEC1和PRP19主要促进GATA4蛋白第256号赖氨酸位点发生泛素化修饰。最后，细胞功能实验提示，过表达PRP19能抑制GATA4功能增强后对心肌细胞肥大的促进作用。我们的研究结果为病理性心肌细胞肥大和心肌肥厚预防和治疗靶点的探索提供了理论依据和分子基础，靶向DEC1-PRP19-GATA4轴可能作为病理性心肌肥厚预防和诊断的新策略。