mechanistic analysis of cardiac functions by building G protein signal network

当心脏受到压力时，如高血压，心脏就会肥大。当压力没有被消除时，心脏会发展为心力衰竭，不能正常地向外周组织提供血液。心脏纤维化是以细胞外基质蛋白过度沉积为特征的疾病，是心力衰竭的原因之一，导致心功能尤其是舒张能力受损。目前认为包括心脏在内的各种组织的纤维化是肥大后的继发性事件，并受到血管紧张素II（Ang II）和转化生长因子-β（TGF-β）信号通路的调节。为了研究异源三聚体G12家族G蛋白（G_<α12>/G_<α13>：G_α12/13）在心脏中的作用，我们用p115 RhoGEF的RGS结构域（p115-RGS）选择性阻断G_<α12/13>。仅在心肌细胞中表达抑制性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，而不影响心肌细胞的过度增殖。 ...更多信息 奖杯由于p115-RGS在心肌细胞中选择性表达，p115 RGS是异源三聚体G_<α12/13>蛋白的选择性抑制剂，G蛋白通常被G蛋白偶联受体（GPCR）激活，因此，当心脏处于压力超负荷时，应该存在一个GPCR触发心脏纤维化。为了鉴定调节纤维化的GPCR，我们使用新生心肌细胞的拉伸处理，因为认为体外细胞的拉伸处理模拟体内压力超负荷。牵张处理激活G_α12/13>下游的Rho是由心肌细胞释放的ATP和UDP启动的，ATP降解酶或嘌呤受体阻断剂抑制牵张诱导的Rho激活。嘌呤受体选择性阻断剂显示受体是P2 Y 6。此外，在体内抑制G蛋白偶联的P2Y_6受体可抑制压力超负荷诱导的心脏纤维化。上述结果提示，ATP和/或UDP刺激的P2 Y 6受体激活心肌细胞G_α12/13>，是压力超负荷性心肌纤维化的触发因素，百日咳毒素（PTX）是广泛使用的ADP-核糖基化Gi和Go（Gi/Go）并使Gi/Go受体解偶联的特异性工具。为了研究心脏成纤维细胞中血管紧张素II受体（AT 1 R）刺激的信号通路，我们发现PTX增加AT 1 R的表达并增强AT 1 R介导的反应。基因芯片分析显示PTX可增加IL-1 β的表达。通过敲低或抗体降低IL-1β表达可抑制PTX处理刺激的AT 1 R介导的应答增强。PTX通过激活小的GTP结合蛋白Rac（刺激NADPH氧化酶依赖性活性氧产生）增加IL-1β和AT 1 R的表达。抑制IL-1β可阻断PTX诱导的晚期而非早期Rac活化。已知PYX与Toll样受体4（TLR 4）结合。TLR 4的敲低抑制PTX诱导的Rac激活和AT 1 R介导的应答的增强。然而，TLR 4的抑制不影响PTX介导的Gi/Go的ADP-核糖基化。因此，PTX与至少两种受体结合：一种是激活Rac并增强AT 1 R应答的TLR 4，另一种是介导PTX进入细胞的结合位点-Gi/Go的ADP-核糖基化。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