Adiponectin与内质网应激-自噬通路相互作用调节心脏肥大的机制研究

Endoplasmic reticulum stress (ERS) caused by left ventricular hypertrophy contributes to the imbalance in protein metabolism, which is characterized by Unfolded Protein Response. Autophagy system is triggered as a novel signaling pathway in response to ERS to degrade aggregated proteins. We've observed that adiponectin (APN), an endogenous ERS-autophagy regulator, protected the myocardium against the injury and apoptosis induced by serious ERS and autophagy in APN knockout mice (APNKO-/-) in cardiac hypertrophic mouse models induced by isoproterenol (ISO) subcutaneous injection and transverse aortic constriction (TAC). With the use of these cardiac hypertrophic animal models, and hypertrophic cardiomyocytes and activated cardiac fibroblasts induced by angiotensin II and isoproterenol, respectively, we further study （1）the dynamic role of APN, its receptors and dysfunction in ERS and autophagy, in the development of cardiac hypertrophy and its progression to heart failure, by detecting the change in the expression of ANP, its receptors and the key components in ERS-autophagy pathway;(2) the molecular pathways involved in regulation of ANP and its receptors by ERS-autophagy pathway, by blocking ERS and autophagy, respectively; (3)the membrane receptors and postreceptor pathways of APN involved in the regulation of ERS-autophagy pathway in protecting the heart against hypertrophy, by use of APNKO-/- mice and the methods of gene overexpression and silence. Finally, we try to understand the possible new negative regulatory loop in cardiac hypertrophy formed by APN, its receptor system, and ERS-autophagy pathway on the base of our previous work.

心脏肥大发生内质网应激（ERS），诱导心肌细胞内蛋白质代谢稳态失衡、无效蛋白质累积，活化自噬-溶酶体通路，清除ERS时错误折叠的蛋白质。我们拟在前期发现Adiponectin（APN）抑制过度ERS-自噬活性拮抗心脏肥大纤维化重塑的基础上，利用主动脉缩窄和小剂量ISO持续给药诱导的心脏肥大重塑模型，及Ang II和ISO诱导的心肌细胞肥大和成纤维细胞活化模型，进一步观察APN及其受体的表达和ERS-自噬通路关键成分的变化，明确APN系统与蛋白质代谢障碍在肥大代偿向心衰转化中的关系；分别阻断ERS和自噬，探讨ERS-自噬通路对APN及其受体的调节；利用APN基因敲除动物、基因表达和沉默技术，研究内、外源性APN调节ERS-自噬通路拮抗心脏肥大的具体受体和受体后信号通路，阐明心肌组织内源性ERS-自噬调节剂APN及其受体与ERS-自噬通路相互作用形成负性信号调节环、调节心脏肥大重塑的新机制。

交感神经系统活化是心衰发生的重要神经内分泌机制。自噬过度活化导致心肌细胞死亡。脂联素（APN）是心血管系统重要的内源性保护因子。本项目构建APN基因敲除纯合子（APN-/-）和杂合子小鼠（APN+/-），利用异丙肾上腺素（ISO）刺激模拟心衰时交感神经系统活化、诱导心脏发生肥大纤维化重塑，研究APN调节自噬保护心脏的机制。小鼠连续7天皮下注射ISO（5mg/kg/天）建立心脏肥大重塑模型，与WT小鼠相比，ISO诱导APN-/-小鼠发生心衰和心功能障碍，LDH、CK和CK-MB活性、心肌细胞横截面积、凋亡率和纤维化率、以及ANP、BNP的mRNA表达均增高。ISO处理后，与APN-/-小鼠相比，APN+/-小鼠和腹腔注射自噬抑制剂氯喹（CQ）的APN-/-小鼠均出现心功能障碍、心肌酶和肥大纤维化、凋亡指标的明显改善。ISO处理后，与WT小鼠相比，APN-/-小鼠心脏的LC-3II、Beclin-1、凋亡相关分子cleaved-caspase-9、cleaved-caspase-3和BAX的表达均升高，Bcl-2降低。ISO处理后，与APN-/-小鼠相比，APN+/-小鼠心脏的自噬和凋亡分子表达降低、Bcl-2升高，给予CQ的APN-/-小鼠心脏的凋亡分子表达及BAX/Bcl-2比值降低、Bcl-2升高。ISO（10μmol/L）处理原代大鼠心肌细胞建立肥大模型，心肌细胞面积、ANP和BNP mRNA表达、心肌酶指标、自噬和凋亡相关分子的表达均升高，而Bcl-2和细胞生存率降低。APN（30μg/mL）预保护降低ISO诱导的心肌细胞自噬，其拮抗ISO诱导的肥大损伤的作用与ATG-5 siRNA、自噬抑制剂3-MA或CQ的作用相似。利用RFP-GFP-LC3双标的腺相关病毒感染心肌细胞，可见APN预处理有效抑制ISO诱导的过强自噬流。APN预处理增加心肌细胞的Bcl-2表达及其与Beclin-1的结合。Akt/PI3K和AMPK通路的抑制有效逆转APN预保护诱导的Bcl-2表达及对自噬和凋亡的抑制。结论：APN活化心肌细胞的AMPK和Akt/PI3K通路，上调抗凋亡蛋白Bcl-2的表达并促其与自噬启动关键分子Beclin1的相互作用，有效抑制ISO诱导的过度自噬和自噬流、以及自噬诱导的细胞肥大、死亡和凋亡。提示上调内源性APN或直接抑制自噬均可以作为心衰心脏的新治疗策略。