mechanistic analysis of cardiac functions by building G protein signal network

当心脏暴露在高血压等压力下时，它就会肥大。当压力不被消除时，心脏就会发生心力衰竭，不能适当地为周围组织提供血液。心肌纤维化以细胞外基质蛋白过度沉积为特征，是导致心功能尤其是松弛能力受损的心力衰竭的原因之一。到目前为止，包括心脏在内的各种组织的纤维化被认为是肥厚后的继发性事件，并受血管紧张素II(AngII)和转化生长因子-β(TGFR-β)信号通路的调节。为了研究异源三聚体G12家族G蛋白(G_&lt；α12&gt；/G_&lt；α13&gt；：Gα12/13)在心脏中的作用，我们利用p115-RGS的RGS结构域选择性地阻断了G_&lt；α12/13&gt；仅在心肌细胞表达抑制性p115-RGS的转基因小鼠(p115-TG小鼠)显示压力超负荷诱导的纤维化，而不影响高…更多的奖杯。由于p115-RGS选择性地表达于心肌细胞，p115RGS是异源三聚体G_&lt；α12/13&gt；蛋白的选择性抑制剂，而G蛋白一般由G蛋白偶联受体(GPCRs)激活，因此当心脏暴露于压力超负荷时，应该存在G蛋白偶联受体，从而触发心肌纤维化。为了确定调节纤维化的GPCR，我们使用了新生儿心肌细胞的拉伸处理，因为人们认为细胞在体外的拉伸处理模拟体内的压力超负荷。牵张处理引起的Rho激活是由心肌细胞释放的三磷酸腺苷和二磷酸尿苷启动的，因为参与α降解的酶或嘌呤受体阻断剂抑制牵张诱导的Rho激活。嘌呤受体选择性阻滞剂显示该受体为P2Y6。此外，在体内抑制G蛋白偶联的P2Y6受体可抑制压力超负荷所致的心肌纤维化。这些结果表明，三磷酸腺苷和/或二磷酸腺苷激活心肌细胞G_(α)12/13&gt；在压力超负荷诱导的心肌纤维化中触发纤维化。百日咳毒素被广泛用作腺苷二磷酸核糖基化GI和GO(GI/GO)和解偶联GI/GO受体的特异性工具。为了研究血管紧张素II受体(AT1R)刺激心脏成纤维细胞的信号通路，我们发现PTX增加AT1R的表达并增强AT1R介导的反应。基因芯片分析显示，甲状旁腺素可增加白细胞介素1β的表达。通过基因敲除或抗体降低IL-1β的表达，可抑制PTX刺激的AT1R介导的反应增强。PTX通过激活一个小的GTP结合蛋白Rac来增加IL-1β和AT1R的表达，Rac可以刺激NADPH氧化酶依赖的活性氧的产生。抑制IL-1β可阻断PTX诱导的晚期而不是早期的RAc激活。已知PYX与Toll样受体4(TLR4)结合。TLR4基因敲除可抑制PTX诱导的RAC激活和AT1R介导的反应增强。然而，抑制TLR4并不影响PTX介导的GI/GO的ADP核糖化。因此，PTX至少与两个受体结合：一个是TLR4，它激活RAC并增强AT1R反应；另一个是PTX进入细胞的结合部位，介导GI/GO的ADP核糖化。较少

The heart is hypertrophied when it is exposed to stresses such as hypertension. When the stress is not removed, the heart develops heart failure that does not properly provide the blood to peripheral tissues. Cardiac fibrosis is characterized by excessive deposition of extracellular matrix proteins, and is one of the causes of heart failure that contributes to the impairment of cardiac function, especially relaxation ability. So far, it is believed that fibrosis of various tissues including the heart is a secondary event following hypertrophy, and is regulated by the signaling pathway of angiotensin II (Ang II) and transforming growth factor-β (TGF-β). In order to examine the roles of heterotrimeric G12 family G protein (G_<α12>/G_<α13> : Gα12/13) in the heart, we used RGS domain of p115RhoGEF (p115-RGS) to selectively block G_<α12/13>. Transgenic mice (p115-Tg mice) expressing inhibitory p115-RGS only in cardiac myocytes shows pressure overload-induced fibrosis without affecting hyper … More trophy. As p115-RGS is selectively expressed in cardiac myocytes, p115RGS is selective inhibitor of heterotrimeric G_<α12/13> protein, and G protein is generally activated by G protein-coupled receptors (GPCRs), there should be a GPCR that triggers cardiac fibrosis when the heart is exposed to pressure overload. To identify a GPCR that regulates fibrosis, we used stretch treatment of neonatal cardiomyocytes, as it is thought that stretch treatment of cells in vitro mimics in vivo pressure overload. The activation of Rho downstream of G_<α12/13> by stretch treatment is initiated by ATP and UDP released from cardiac myocytes, as enzymes involved in ATP degradation or blockers of purine receptor inhibit stretch-induced Rho activation. Purine receptor-selective blockers reveal that the receptor is P2Y6. Furthermore, inhibition of G-protein-coupled P2Y_6 receptors in vivo inhibits pressure overload-induced cardiac fibrosis. These results suggest that activation of G_<α12/13> in cardiomyocytes by ATP and/or UDP-stimulated P2Y_6 receptor triggers fibrosis in pressure overload-induced cardiac fibrosis.Pertussis toxin (PTX) is widely used as a specific tool that ADP-ribosylates Gi and Go (Gi/Go) and uncouples receptors from Gi/Go. To examine signaling pathways of angiotensin II receptor (AT1R) stimulation in cardiac fibroblasts, we found that PTX increases the expression of AT1R and enhances AT1R-mediated response. Microarray analysis shows that PTX increases the expression of interleukin (IL)-1β. Decreased expression of IL-1β by knockdown or antibody inhibits the PTX-treatment-stimulated enhancement of AT1R-mediated response. PTX increased the expression of IL-1β and AT1R through activation of a small GTP-binding protein Rac that stimulates NADPH oxidase-dependent production of reactive oxygen species. PTX-induced late but not an early phase of Rac activation is blocked by inhibition of IL-1β. It is known that PYX binds to Toll-like receptor 4 (TLR4). Knockdown of TLR4 inhibits PTX-induced Rac activation and enhancement of AT1R-mediated responses. However, inhibition of TLR4 does not affect PTX-mediated ADP-ribosylation of Gi/Go. Thus, PTX binds to at least two receptors: one is TLR4 that activates Rac and enhances AT1R responses, and another is the binding site that mediates entry of PTX into cells ADP-ribosylation of Gi/Go. Less