核内体β1AR及其介导的信号通路在心肌肥厚中的作用及机制研究

Cardiac hypertrophy is an independent risk factor in determining the prognosis of patients with cardiovascular disease. However, the underlying molecular mechanisms of cardiac hypertrophy are still unclear. β1 adrenergic receptors (β1ARs) are G protein coupled receptors that play an important role in regulating cardiac hypertrophy and heart failure. Previous studies have pointed out that chronic sympathetic drive promotes β1AR redistribution from the plasma membrane to endosomes, which contributes to cardiac functional and structural remodeling associated with heart failure. However, the role of endosomal β1ARs in regulating cardiac hypertrophy and heart failure is still not clear. Recently, people found that GPCRs could mediate G-protein signaling not only from the plasma membrane but also from endosomal membranes. Our preliminary data show that there is a significant increase in Akt phosphorylation on endosomes compared with the plasma membrane in heart tissue after transverse aortic constriction (TAC) in wild type（WT）mice. Akt/mTOR signaling pathway was inhibited by endocytosis inhibitor in isolated adult cardiomyocytes after chronic isoproterenol (ISO) stimulation. To analyze endosomal β1AR signaling, we developed a novel mouse model, β1AR-S475A, with shifted β1AR distribution from the plasma membrane to endosomes in the heart. Compared with WT mice, β1AR-S475A mice developed cardiac hypertrophy and heart failure more quickly after TAC surgery. Accordingly, the hypothesis is that under chronic sympathetic drive, the β1AR is redistributed to endosomes, and activation ofβ1AR on endosomes promotes the PKA/Akt/mTOR signaling pathway leading to cardiac hypertrophy and heart failure. The present study aims to investigate the role and signaling transduction pathway of endosomal β1AR in cardiac hypertrophy and heart failure via the use of modern techniques including Nanobody80 adenovirus infection, fluorescent resonant energy transfer (FRET) and impermeable β blocker in adult and neonatal primary cardiomyocytes and transgenic mice. The results from the current study will be helpful to further characterize the role of endosomal β1AR signaling in regulating heart failure and provide an attractive therapeutic target for the treatment of cardiac hypertrophy and heart failure.

心肌肥厚是决定多种心血管疾病预后的危险因子，其发病机制尚不明确。β1AR为经典G蛋白偶联受体，通过多种分子机制参与调控心肌肥厚。心衰时β1AR内吞作用增强，胞膜β1AR密度降低，内吞至核内体β1AR增多，但关于核内体β1AR的作用机制尚不清楚。近来研究发现G蛋白偶联受体不仅在细胞膜介导信号转导，也在核内体产生信号转导第二时相。前期研究发现：心衰时核内体Akt活性增强；抑制核内体β1AR的增加可抑制Akt及其下游mTOR激活；核内体β1AR增多的转基因小鼠更易导致心肌肥厚和心衰发生。由此提出科学假说：心脏交感神经过度激活导致心肌细胞核内体β1AR增多并被活化，且通过PKA介导 PI3K/Akt/mTOR通路激活，促进心肌肥厚及心衰。本项目拟用模拟G蛋白80、荧光共轭能量转移技术结合不可透膜型β受体阻滞剂进行体内、外研究，阐明核内体β1AR参与调控心肌肥厚的具体机制，为心衰防治提供新的理论依据。

肾上腺素能受体在心血管疾病的发生发展中扮演者了重要的角色。其中β1AR为经典G蛋白偶联受体，通过多种分子机制参与调控心肌肥厚及心力衰竭。儿茶酚胺类物质通过经典第二信使学说，结合细胞膜上β1AR，进一步激活下游信号通路参与调控心肌细胞的收缩功能及心肌电活动。近来，前人研究发现G蛋白偶联受体也存在于细胞内部结构上，如线粒体，内质网和高尔基体等部位，同样可以被激活产生特定的作用。我们前期实验及前人的研究发现，β1AR不仅存在细胞膜上，同时也存在于细胞内结构，但是这部分β1AR作用机制暂不明确。本研究旨在探讨存在细胞内部的β1AR在心肌细胞调控中的作用。在OCT3KO基因敲除小鼠上，通过荧光共轭能量转移技术（FRET）结合透膜型、不可透膜型β受体阻滞剂以及外源性可透膜β受体激动剂、内源性不可透膜儿茶酚胺进行体内、外研究。我们研究发现，不同于外源性可透膜型β受体激动剂，内源性不可透膜的儿茶酚胺如肾上腺素（EPI）和去甲肾上腺素（NE），需要经OCT3转运才可入细胞内。不可透膜型β受体阻滞剂可选择性阻断细胞膜表面β1AR，为我们更准确的研究细胞内部结构上β1AR的作用提供保证。本研究显示，内源性儿茶酚胺在OCT3转运下进入细胞内部，结合并激活位于肌浆网SR上β1AR，进一步激活下游的PKA信号通路。将OCT3敲除后，内源性儿茶酚胺进入细胞内部受阻，细胞内位于肌浆网SR上PKA信号激活程度显著降低，但外源性β受体激动剂对细胞内部β1AR/PKA信号通路的激活不受影响，说明OCT3对于细胞内部β1AR/PKA的激活是必不可少的。另外，内源性儿茶酚胺激活细胞内部SR上的β1AR/PKA信号通路后可进一步提高PLB的磷酸化水平，同时增加了心肌细胞的收缩力，而OCT3KO小鼠中上述作用消失，说明细胞内部SR上的β1AR/PKA信号通路可进一步激活下游PLB的磷酸化，参与心肌收缩功能调节。在我们的动物实验中也发现，OCT3KO组的小鼠接受内源性儿茶酚胺的作用下，心肌收缩力增强作用相比较WT组有所减弱。因此，也更进一步说明了细胞内部结构肌浆网上的β1AR/PKA信号通路在心肌收缩力调节上起到一定的作用。