Endothelial Rap1 in the control of heart function

心血管疾病(CVD)是一种全球性的流行病，全世界有超过2600万人受到影响。对 心脏氧合和代谢的调节是心脏内皮细胞和 心肌细胞(CMS)和平滑肌细胞(SMC)。这种由局部作用、生物活性调节的串扰 心脏内皮细胞释放的物质(旁分泌功能)，特别是一氧化氮(NO)，控制着血流 和血管通透性，以及CMS的生长、收缩和节律性。然而，这些机制 心脏内皮细胞与CMS和SMC之间的功能相互作用仍然知之甚少。 我们对小GTP酶(RAS协会近端)RAP1内皮功能的开创性研究 强调其作为血管动态平衡的新调节器的作用。RAP1对于一氧化氮(NO)是至关重要的。 生产和生物利用度，因为两种Rap1亚型(Rap1A和Rap1B)的组织特异性缺失导致 严重的内皮功能障碍。我们合作中出现的数据有力地表明，两个Rap1 冠状动脉(血管)和心脏微血管(心脏)内皮细胞中的异构体可能是保存 心脏的正常收缩功能。 我们的数据表明，EC特异性的Rap1基因缺失会导致心肌收缩能力下降和即将发生 心力衰竭。从机制上讲，我们的初步数据强烈表明，通过离散但互补的方式 机制，两种Rap1亚型对内皮细胞的钙离子转运和内皮功能(NO)是必不可少的 生产)。这项建议的目的是研究两种RAP1亚型在冠状动脉和心脏中的作用。 维持心脏收缩功能所需的ECS。我们假设依赖RAP1的EC 这些功能构成了正常心脏功能所需的EC-SMC和EC-CM通讯的纽带。 相反，Rap1缺乏导致的EC功能障碍(NO释放受损，钙超载)是常见的 EC-SMC和EC-CM沟通错误导致心力衰竭的罪魁祸首。为了验证这一假设，我们将： (1)确定Rap1如何控制内皮细胞的钙稳态；我们将利用膜片钳电生理学和 体外测定钙离子浓度，观察Rap1缺乏对钙离子内流通道的影响。我们将研究 RAP1AKO内皮细胞钙稳态受损对控制旁分泌功能的细胞过程的影响 (2)研究一条涉及CalDAG-GEFIII介导的Rap1B在NO释放中激活的新的信号通路。例如 在体内，我们将测试Rap1信号和离子通道抑制对小鼠和人冠状动脉的影响 血管内皮细胞Rap1a和Rap1B对冠脉血流的影响。 (3)体外检测EC-Rap1基因敲除小鼠的心血管和心脏功能及EC-CM的旁分泌功能。 体外共培养以确定心脏EC Rap1亚型如何控制心脏收缩功能。 拟议的研究将揭示新的、以前意想不到的调控心脏内皮和心脏功能的机制 通过调控血管内皮细胞中的RAP1信号通路，可能为心功能的恢复开辟新的方向。

Cardiovascular disease (CVD) is a global pandemic with over 26 million people affected worldwide. Critical for regulation of heart oxygenation and metabolism is the cross-talk between heart endothelial cells (ECs) and cardiomyocytes (CMs) and smooth muscle cells (SMCs). This cross-talk, mediated by locally acting, bioactive substances released by cardiac ECs (paracrine function), in particular nitric oxide (NO), controls blood flow and vascular permeability, as well as CMs' growth, contractility and rhythmicity. However, the mechanisms underlying the functional interaction between cardiac ECs and CMs and SMCs are still poorly understood. Our pioneering studies on endothelial functions of the small GTPase (Ras Association Proximate) Rap1 highlight its role as novel regulator of vascular homeostasis. Rap1 is critically required for nitric oxide (NO) production and bioavailability, as tissue-specific deletion of both Rap1 isoforms (Rap1A and Rap1B) leads to severe endothelial dysfunction. Emerging data from our collaboration strongly suggest that the two Rap1 isoforms in both coronary (vascular) and heart microcapillary (cardiac) ECs may be essential to preserving normal contractile function of the heart. Our data demonstrate that EC-specific deletion of Rap1 leads to decreased cardiac contractility and impending heart failure. Mechanistically, our preliminary data strongly suggest that, via discrete yet complementary mechanisms, two Rap1 isoforms are essential for endothelial Ca2+handling and endothelial function (NO production). The goal of this proposal is to examine the role of the two Rap1 isoforms in coronary and cardiac ECs required for maintenance of cardiac contractile function. We hypothesize that Rap1-dependent EC functions form the nexus for EC-SMC and EC- CM communication required for normal cardiac function. Conversely, Rap1 deficiency-driven EC dysfunction (impaired NO release, Ca2+ overload) is the common culprit in EC–SMC and EC–CM miscommunication that leads to heart failure. To test this hypothesis, we will: (1) Determine how Rap1 controls Ca2+ homeostasis in ECs; we will utilize patch clamp electrophysiology and Ca2+ measurements in vitro to examine the effect of Rap1 deficiency on Ca2+ influx channels. We will examine the effect of impaired Ca2+ homeostasis in Rap1A KO ECs on cellular processes controlling paracrine function. (2) Examine a novel signaling pathway involving CalDAG-GEFIII-mediated Rap1B activation in NO release. Ex vivo, we will test the effect of Rap1 signaling and ion channel inhibition on mouse and human coronary vessel dilation, to determine the influence of EC Rap1A and Rap1B in the control of coronary vessel blood flow. (3) Examine vascular and cardiac function in EC-Rap1 knockout mice ex vivo and paracrine function in EC-CM co-culture in vitro to determine how cardiac EC Rap1 isoforms control heart contractile function. Proposed studies will uncover novel, previously unexpected mechanisms governing heart endothelium and may lead to a new direction in restoring cardiac function by controlling Rap1 signaling in endothelium.