mechanistic analysis of cardiac functions by building G protein signal network

当心脏受到诸如高血压之类的压力时，它就会肥大。当压力不消除时，心脏就会发生心力衰竭，不能正常地向周围组织提供血液。心脏纤维化以细胞外基质蛋白过度沉积为特征，是导致心衰的原因之一，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厚后的继发性事件，受血管紧张素II （Ang II）和转化生长因子-β (TGF-β)信号通路调控。为了研究异三聚体G12家族G蛋白（G_<α12>/G_<α13>: Gα12/13）在心脏中的作用，我们利用p115RhoGEF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）表现出压力过载诱导的纤维化，而不影响心肌肥大。由于p115-RGS在心肌细胞中选择性表达，p115RGS是异源三聚体G_<α12/13>蛋白的选择性抑制剂，而G蛋白通常被G蛋白偶联受体（GPCR）激活，因此当心脏承受压力过载时，应该存在触发心脏纤维化的GPCR。为了确定调节纤维化的GPCR，我们对新生儿心肌细胞进行拉伸处理，因为人们认为细胞在体外拉伸处理会模拟体内压力过载。拉伸作用下G_<α12/13>下游Rho的活化是由心肌细胞释放的ATP和UDP启动的，参与ATP降解的酶或嘌呤受体阻滞剂抑制拉伸诱导的Rho活化。嘌呤受体选择性阻滞剂显示受体是P2Y6。此外，体内抑制g蛋白偶联P2Y_6受体可抑制压力过载诱导的心脏纤维化。这些结果表明，ATP和/或udp刺激的P2Y_6受体激活心肌细胞G_<α12/13>可触发压力过载诱导的心肌纤维化。百日破毒素（PTX）被广泛用作adp核糖基化Gi和Go （Gi/Go）并从Gi/Go中解偶联受体的特异性工具。为了研究心脏成纤维细胞中血管紧张素II受体（AT1R）刺激的信号通路，我们发现PTX增加了AT1R的表达并增强了AT1R介导的反应。微阵列分析显示，PTX增加了白细胞介素-1β的表达。通过敲低或抗体降低IL-1β的表达可抑制ptx治疗刺激的at1r介导的应答增强。PTX通过激活小的gtp结合蛋白Rac，刺激NADPH氧化酶依赖的活性氧的产生，从而增加IL-1β和AT1R的表达。ptx诱导的晚期而非早期的Rac激活被IL-1β的抑制所阻断。已知PYX与toll样受体4 （TLR4）结合。TLR4的敲低抑制ptx诱导的Rac激活和at1r介导的应答增强。然而，TLR4的抑制并不影响ptx介导的Gi/Go的adp核糖基化。因此，PTX至少与两个受体结合：一个是激活Rac并增强AT1R反应的TLR4，另一个是介导PTX进入细胞adp -核糖基化Gi/Go的结合位点。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