ION CHANNEL DETERMINANTS OF MAST CELL ACTION IN THE HEART

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在我们的Cobre项目中，我们将研究离子型大麻素受体TRPV1，它是钙进入和肥大细胞激活的关键决定因素，对大麻素和物理化学信号都有反应。我们的数据表明，驱动肥大细胞释放糜酶的多元分泌物通过TRPV1通过Gi/o偶联途径发挥作用。TRPV1的表达是必要的，也是充分的，使促分泌剂介导的钙内流进入天生对促分泌剂无反应的细胞。最近，肥大细胞的一项特殊功能涉及到促分泌剂的应用，以及暴露在酸性环境中：释放激活心脏和肺等组织中肾素-血管紧张素系统(RAS)的蛋白酶。TRPV1介导的钙内流促进肥大细胞分泌促分泌剂释放的糜酶。这些数据表明，TRPV1促进肥大细胞释放促肥大介质。此外，我们的数据表明，TRPV1整合了促肥厚和抗肥厚的信号。ANP受体鸟苷酸环化酶偶联A(GC-A，NPR1)与TRPV1物理结合，我们已经证明GC-A介导cGMP/PKG介导的TRPV1活性抑制。这些数据表明，TRPV1对促进和反对肥大细胞分泌促肥大介质的刺激有反应。 虽然肥大细胞在皮肤和粘膜免疫系统中得到了最好的研究和理解，但它们存在于心肌中，在那里它们被认为在炎症中发挥作用。这一提议的中心前提是肥大细胞是细胞因子、基质活性酶和促炎因子的来源，这些有助于维持健康心脏的动态平衡，但有助于在衰竭的心脏中进行不适当的组织重建。所有肥大细胞激活的一个常见机制是通过TRPV1或CRACM1的钙内流，以响应非免疫刺激或免疫刺激。我们的主要假设是，在衰竭的心脏中，是TRPV1负责肥大细胞中的钙内流。因此，我们计划用我们的三个具体目标来研究肥大细胞TRPV1和CRACM1在衰竭心脏的组织建模和病理学中的贡献程度。 目的1.确定肥大细胞、TRPV1和CRACM1在心力衰竭中的作用。 我们将在主动脉缩窄所致心力衰竭模型中评估肥大细胞信号。将使用组织学、功能和转录分析，对正在接受这些治疗的TRPV1、CRACM1和肥大细胞缺陷(KitW-sh/W-sh)小鼠的心脏进行基本心脏生理学评估。 目的2.观察通过重建肥大细胞缺陷小鼠来恢复肥大细胞对心力衰竭的贡献。 为了控制TRPV1和CRACM1基因敲除对大多数非肥大细胞组织的影响，我们将用TRPV1和CRACM1基因敲除小鼠的骨髓重建致死性照射的小鼠。重建后，心力衰竭的模型将如目标1所示。 目的3.检测TRPV1、CRACM1的药物修饰和肥大细胞活化是否能改善压力超负荷所致的心力衰竭。 肥大细胞激活的调节可能影响心力衰竭的重塑。有一本广泛的药典规范了TRPV1，我们计划在压力诱导的心力衰竭模型中测试这些药物对解剖、功能和组织学进展的影响。我们希望确定目前的小分子TRPV1和ICRAC/CRACM1拮抗剂是否对心力衰竭具有治疗作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In our COBRE project we will study the ionotropic cannabinoid receptor, TRPV1, which is a critical determinant of calcium entry and mast cell activation in response to both cannabinoids and physico-chemical signals. Our data show that polybasic secretagogues, which drive chymase release from mast cells, act via TRPV1 using a Gai/o-coupled pathway. Expression of TRPV1 is necessary and sufficient to confer secretagogue-mediated calcium entry on cells that are natively unresponsive to secretagogue. Secretagogue application, and exposure to an acidified environment, have recently been implicated in a specialized function of mast cells: the release of proteinases that activate the Renin-Angiotensin System (RAS) in tissues such as the heart and lung. TRPV1-mediated calcium entry drives chymase release from mast cells in response to secretagogues. These data implicate TRPV1 in the promotion of pro-hypertrophic mediator release from mast cells. Moreover, our data suggest that TRPV1 integrates both pro- and anti-hypertrophic signals. The ANP receptor guanylyl cyclase-coupled A (GC-A, NPR1) physically associates with TRPV1 and we have shown that GC-A mediates cGMP/PKG-mediated suppression of TRPV1 activity. These data implicate TRPV1 in responses to stimuli that both promote, and oppose, the secretion of pro-hypertrophic mediators from mast cells. Although mast cells are best studied and understood in dermal and mucosal immune systems, they are present in the myocardium where they are thought to play a role in inflammation. The central premise of this proposal is that mast cells are sources of cytokines, matrix-active enzymes and pro-inflammatory factors that help maintain homeostasis in the healthy heart, but contribute to inappropriate tissue re-modeling in the failing heart. A common mechanism of all mast cell activation is calcium influx through either TRPV1, in response to non-immunological stimulation, or CRACM1, in response to immunological stimulation. It is our overarching hypothesis that it is TRPV1 that is responsible for calcium influx in mast cells in the failing heart. We therefore plan to investigate the degree to which mast-cell TRPV1 and CRACM1 contribute to tissue modeling and pathology in the failing heart with our three specific aims. Aim 1. Determine the role of mast cells, TRPV1, and CRACM1 in the failing heart. We will evaluate mast cell signaling in a model of heart failure induced by aortic constriction. Assessment of basic cardiac physiology will be performed, using histological, functional, and transcriptional assays, of hearts from TRPV1, CRACM1 and mast cell deficient (KitW-sh/W-sh) mice undergoing these treatments. Aim 2. Examine the effects of restoring the mast cell-related contribution to the failing heart by reconstitution of mast cell-deficient mice. To control for the effects of TRPV1 and CRACM1 knockout in the majority of non-mast cell tissues, we will reconstitute lethally irradiated mice with bone marrow from TRPV1 and CRACM1 knockout mice. After reconstitution, heart failure will be modeled as in Aim 1. Aim 3. Test whether pharmacologic modification of TRPV1, CRACM1 and mast cell activation can ameliorate pressure overload-induced cardiac failure. Modulation of mast cell activation may influence remodeling in heart failure. There is an extensive pharmacopeia that regulates TRPV1 and we plan to test the effects of these drugs on anatomic, functional, and histological progression in a model of pressure-induced cardiac failure. We expect to determine whether current small molecule TRPV1 and ICRAC/CRACM1 antagonists could have therapeutic utility in heart failure.