缝隙连接蛋白43在心衰心肌重构和凋亡中的作用及其机制

Heart failure (HF) is the end stage of many cardiovascular diseases and one of the leading causes of heart death. It has been demonstrated that connexin 43 (CX43), the dominant connexin in ventricle, is redistributed laterally from the ends of the ventricular myocytes, dephosphorylated and functional down-regulated in failure hearts. These changes due to heart failure are called CX43 remodeling and are implicated to the genesis of arrhythmias, dysfunction and apoptosis in failure heart. However, the mechanisms underlying how CX43 remodeling and involved in the pathological alterations are not clear. We have found in our previous study supported by National Nature Science Foundation that 1) in neonatal rat ventricular myocytes, serine 279/282 (S279/282) phosphorylations in CX43 C terminal upregulate the intercellular communication. Mutations at S279 and S282 cause dominant negative-effect of gap junction and even cell death; 2) in animal failure hearts, CX43 remodeling and dephosphorylations at S279/282 are found. Thus, we hypothesize that S279/282 is the key regulator of ventricular intercellular communication and cell survival. Changes in their phosphorylation status may be involved in the genesis of CX43 remodeling, downregulation of gap junction function and cell apoptosis. In this study, we investigate the role of S279/282 phosphorylations physiologically, and in the genesis and progress of heart failure, in particular, focusing on the CX43 remodeling/heart remodeling and cardiomyocyte apoptosis using molecular biological, physiological and pharmacological approaches. We hope this study may provide the molecular mechanisms underlying how CX43 remodeling occurs and CX43-induced cell apoptosis in heart failure, which may be helpful to seek potential target for the treatment and prevention of heart failure.

心衰是心血管系统疾病终末期和心源性死亡主要原因之一。研究表明心衰心室肌缝隙连接蛋白43（CX43）重构与心功能降低、心律失常和心肌细胞凋亡的发生密切相关，但确切机制尚未明了。在前一项国自然基金项目中发现：1）在正常心室肌CX43丝氨酸279/282磷酸化是细胞间通讯上调的关键位点，该双位点突变致细胞间通讯显性失活和心肌细胞凋亡；2）心衰动物心室肌CX43重构伴随丝氨酸279/282磷酸化显著减少。因此，本项目首次提出CX43丝氨酸279/282磷酸化是调控心室肌细胞间通讯和心肌存活的重要位点，并可能是心衰心肌CX43重构、细胞间通讯退减和细胞凋亡的重要途径之一。本课题拟验证丝氨酸279/282磷酸化位点的生理作用，和其磷酸化改变致心肌细胞凋亡的机制及在CX43重构，心功下调和心肌凋亡中的作用，从而，可能揭示心衰CX43重构的分子（氨基酸）机制，为心衰预防和治疗提供新的理论依据。

心衰是心血管系统疾病终末期和心源性死亡主要原因之一。研究表明心衰心室肌缝隙连接蛋白43（Cx43）重构与心功能降低、心律失常和心肌细胞凋亡的发生密切相关，但确切机制尚不清楚。在前项国自然基金项目中发现：1）在正常心室肌Cx43丝氨酸279/282磷酸化(p-S279/282)是细胞间通讯上调的关键位点，该双位点突变致细胞间通讯显性失活和心肌细胞凋亡；2）心衰动物心室肌Cx43重构伴随丝氨酸279/282磷酸化显著减少。因此，本项目进一步提出p-S279/282是调控心室肌细胞间通讯和心肌存活的重要位点，并可能是心衰心肌Cx43重构、细胞间通讯减退和细胞凋亡的重要机制之一。.研究结果：1）缺血再灌损伤大鼠心脏和阿霉素诱导损伤小鼠心脏Cx43的p-S279/282水平降低，心肌细胞凋亡和心律失常；大鼠乳鼠心肌细胞和成鼠心脏Cx43的S282位点突变去磷酸化可致心脏Cx43侧分布，心肌细胞凋亡，心律失常和心功减低; 2）心肌细胞凋亡机制与Cx43/p38相互作用增强，促p38磷酸化，进一步活化Fas/FADD和capase8/3信号通路密切相关; 3）在主动脉缩窄大鼠心脏2个月发生左室肥厚，6个月出现心衰.心脏的p-S279/282水平分别增高和减低，同时均伴有Cx43侧分布和心肌纤维化。此外，三磷酸肌醇受体(IP3R)表达在代偿和失代偿期心脏均显著增高，但与Cx43的相互作用在肥大期增加，而心衰期减少; 4）Cx43的S282去磷酸化点突变小鼠模型的表征与1-3所述相似; 5）其它扩展研究见下详细介绍。.结论：1）Cx43的2S282磷酸化下调通过激活p38/Fas/FADD/Capase8/3信号通路诱导心肌细胞凋亡并参与心肌缺血再灌损伤；2）Cx43的S282磷酸化上调与IP3R相互作用增加有关， 进而致心肌肥大和Cx43侧分布。Cx43的282磷酸化下调则与心衰阶段心肌凋亡密切相关; 3）其它扩展研究结论详见下具体介绍。