蛋白激酶G磷酸化CDK4参与心衰中心肌的细胞周期调控的作用研究

The explorations of pathophysiological mechanisms involved in heart failure are always hot spots in fields of Medical researches. Though cardiomyocytes are terminal differentiated, they were found to have the potential of re-entry into cell cycle but without cell devision in heart failure samples. The function of this cell cycle re-entry is unclear. Recently, we found that inhibition of cell cycle re-entry could reduce hypertrophy in a cardiomyocyte cell model, and activation of protein kinase G (PKG) could inhibit both re-entry in cell cycle of cardiomyocytes and nuclear translocation of cyclin-dependent kinase 4 (CDK4), which is a key mediator of cell cycle. Then, bioinformatical prediction showed that CDK4 is a potential substrate of PKG. Taken together, we make this hypothesis: PKG phosphorylates CDK4 and inhibits its nuclear translocation, resulting in cell cycle exit of cardiomyocytes. Under pathological conditions, the inactivation of PKG in cardiomyocytes causes dephosphorylation of CDK4, which in turn leads to cell cycle re-entry, and finally participates in the development of hypertrophy and heart failure. We will use clinical samples, animal model and primary cardiomyocyte model of hypertrophy and heart failure as objects, then employ adeno-associated virus-mediated gene delivery to make specific interference of CDK4 phosphorylation site, and the hypothesis will be verified by techniques of morphology and molecular biology. By studying this program, we will make better understanding of the functions of cardiac cell cycle re-entry in hypertrophy and heart failure. This research will also provide valuable targets of cell cycle regulation for the treatment of heart failure.

心力衰竭的病理生理机制研究一直是医学研究热点。心肌细胞是终末分化细胞，近年发现心衰时心肌细胞出现细胞周期再进入现象（cell cycle re-entry）但不完成分裂，该现象的意义还不明确。本课题组发现，抑制细胞周期再进入减轻心肌肥厚模型病变程度，而激活蛋白激酶G（PKG）减少心肌的细胞周期再进入并抑制关键分子细胞周期素依赖性蛋白激酶4（CDK4）入核。生物信息学提示CDK4是PKG潜在磷酸化底物。综上，归纳假说：PKG磷酸化CDK4并抑制其入核，从而维持心肌细胞周期停滞。病理状态下PKG活性降低，上述机制被抑制而触发心肌的细胞周期再进入，并促进心肌肥厚/心衰发展。本项目拟从心衰临床样本、动物模型、细胞模型等层面，使用腺相关病毒介导CDK4磷酸化位点特异性干预，通过形态学、分子生物学检测来论证假说。最终，有望对细胞周期再进入在心衰中的意义形成深入认识，为探索细胞周期为靶点的干预提供思路。

心力衰竭的机制一直是医学研究热点之一。传统观点认为心肌细胞为终末分化细胞，近年来发现心衰时心肌细胞有重新进入细胞周期（cell cycle re-entry）现象，但此现象的意义尚不明确。本课题在心衰患者心脏组织及小鼠心衰模型中检测发现心肌细胞存在DNA复制、细胞周期蛋白表达增加及细胞周期激活，而磷酸二酯酶5（PDE5抑制剂）可缓解心衰并抑制细胞周期的激活。在心肌细胞系肥厚模型中同样检测到细胞周期蛋白表达增加及细胞周期的激活，并可被PDE抑制剂及细胞周期始动关键分子CDK4/6抑制剂所拮抗。在原代成年鼠心肌细胞建立肥厚模型后，流式细胞检测表明心肌细胞进入S期的数量增多，但仍未见G2及M期细胞，表明心肌细胞发生细胞周期再进入，但不能分裂。而PDE抑制剂和细胞周期关键分子CDK4/6抑制剂可改善心肌肥厚指标，提示细胞周期再进入可能参与了心肌肥厚与心衰的发生。PDE抑制剂主要引起cGMP增多、PKG活性增强。生物信息学分析提示，CDK4的S285位点可能是PKG磷酸化位点。因此本课题通过构建CDK4的野生型及S285位点突变质粒并表达融合蛋白，经体外激酶反应证明CDK4是PKG的磷酸化底物。随后，通过GST-pulldown实验又发现，CDK4的S285位点磷酸化状态影响其与细胞周期重要调控分子P21的结合能力，进而影响细胞周期的运行。因此本课题证明，通过上调PKG激酶活性、磷酸化CDK4而影响P21的结合，进而抑制细胞周期的再进入，可能是PDE5抑制剂发挥抗心肌肥厚/心衰作用的机制之一。本研究加深了对心力衰竭发生发展机制的认识，为PDE5抑制剂的作用方式提供了新的角度。