Endothelial Rap1 in the control of heart function

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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.