PHD-AMPK途径中PHD诱导心肌细胞内钙释放的分子机制研究

Hypoxia is the most common pathological and physiological phenomenon. Lower oxygen concentration triggers a series of oxygen adaptive reactions which is critical for cell and the whole body to survive. Recent studies demonstrate that, proline hydroxylase domain-containing enzymes (PHDs) is not only a sensitive oxygen sensors, but also the important oxygen adaptive response inducer. Our research found that, PHD-CaMKK-AMPK is a new cascade mediating hypoxic energy protection, and the sarcoplasmic reticulum (SR) Ca2+ released through RyR chanel caused by PHD inhibition is the key to the signal cascade. But the molecular mechanism about the calcium signal induction is unclear. This study, based on the latest research progress and our own research findings, we put forward a hypothesis that "PHD may interact with PDE4D directly, lead to activation of cAMP/PKA pathway, Ca2+ realease from SR. " So , we plan to research interaction of PHD and PDE4D and investgate “PHD-PDE4D-cAMP-PKA” pathway in cardiomyocytes, and tries to explore the molecualr mechanisms in PHD- Ca2+- AMPK pathway. These data would provide new clues to develop clinical strategies and approaches for the prevention and also treatment of heart hypoxic/ischemic disease.

缺氧是许多疾病条件下最常见的病生现象，氧浓度降低所触发的一系列缺氧适应性反应对细胞乃至机体的存活至关重要。现认为，脯氨酸羟化酶结构域蛋白（PHD）不仅是缺氧的感受器和传递者，还是缺氧适应性反应的启动者。我们前期研究发现，PHD-CaMKK-AMPK是一条新的缺氧能量保护途径，开放PHD信号通路所引发的心肌细胞内RyR2通道Ca2+释放是介导该途径的关键启动信号，但其分子机制目前尚不甚清楚。基于国际最新进展和前期工作基础，提出“PHD很可能通过直接作用于RyR2上的磷酸二酯酶4D（PDE4D），激活cAMP/PKA途径，使RyR2通道磷酸化，诱导SR钙库释放Ca2+”这一假说。为此，本项目拟从验证PHD-PDE4D-cAMP-PKA引发RyR2钙释放这一假说入手，力图阐明PHD-Ca2+-AMPK途径中缺氧信号向细胞内钙信号转换的分子机制，为防治心肌缺氧损害提供新的理论依据和治疗靶点。

脯氨酸羟化酶结构域（Prolyl hydroxylase domain，PHD）作为细胞的氧感受器，诱导细胞对缺氧，特别是在能量代谢动态平衡调节方面的一系列反应。开放PHD信号通路所诱发的心肌细胞内Ca2+释放是介导PHD调控能量代谢途径的关键启动信号，但其分子机制尚不清楚。本研究通过抑制剂和RNA干扰等方法激活心肌细胞中的PHD信号入手，力图探索PHD-Ca2+-AMPK钙释放的潜在机制，阐明PHD-Ca2+-AMPK途径中细胞内钙信号向转换缺氧信号的分子机制。研究证明：PHIs诱导H9C2细胞内游离Ca2+荧光增强并且激活AMPK、TRPA1、CaMKⅡ；TRPA1抑制剂HC030031(10umol/l)、钙调蛋白依赖性蛋白激酶(Ca2+/calmodulin-dependent protein kinase II，CaMKII)抑制剂KN93、兰尼碱抑制剂（Ryanodine）均能抑制PHIs诱导的胞内Ca2+增加和AMPK激活；进一步研究发现PHIs和PHD2shRNA均能有效激活CaMKII和TRPA1；然而,三磷酸肌醇受体（inositol(1,4,5)-triphosphate receptor，IP3R）抑制剂、蛋白激酶A(Protein kinase A，PKA)抑制剂H89、PDE4D抑制剂IBMX并没有明显抑制PHD诱导的胞内Ca2+释放和AMPK激活；免疫共沉淀实验证明PHD2和PDE4D的相互作用，并证明PHD2可调节胞内PDE4D的活性和表达。综上所述，PHD可通过TRPA1离子通道激活CaMKⅡ途径，诱导肌质网RyR2钙释放，从而激活AMPK，启动心肌细胞缺氧能量保护性效应。即我们证明了PHD/TRPA1/CaMKⅡ/Ca2+/AMPK途径的存在，为调节细胞Ca2+水平和适应细胞缺氧机制提供了新的视角，为开发新的治疗药物，特别是治疗缺血性和缺氧性疾病提供了新的理论依据和潜在的替代方案。