Supplement to Serotonin Signaling in Mitral Valve Homeostasis, Maintenance and Restoration

摘要/概要 这个为期一年的补充项目将调查Piezo1，一个机械传感的新关系。 之前从未研究过与二尖瓣脱垂（MVP）或二尖瓣返流（MR）相关的5-羟色胺通道 (5HT)我们以前证明，二尖瓣关闭不全是由于二尖瓣关闭不全引起的。 间质细胞（MVIC）5-羟色胺转运体（SERT）活性被饮食药物芬氟拉明抑制 或氟西汀，一种选择性5HT再摄取抑制剂（SSRI），因此导致5HT清除率降低， 增强5-HT受体（HTR）活性。此外，我们在MR手术患者的临床研究中发现， SSRI的使用与年轻时的MR手术显著相关， 与正常人二尖瓣（MV）瓣叶相比，瓣叶在MR中显示SERT活性降低。 Piezo1和5-HT分泌增加：先前的研究报告称，肠道Piezo1激活导致 增加5 HT，激发我们的研究兴趣。本提案中提出的可行性研究表明， Piezo1激动剂增加MVIC中的HTR信号传导。 假设：MR进展是由于SERT表达减少导致HTR2B信号传导增加所致 以及由机械敏感Ca ++通道Piezo 1激活引起的5 HT分泌增加。 具体目的1：研究Piezo1激活和抑制对人体5-HT机制的影响 MVIC和氟西汀诱导MR的小鼠模型中。我们将使用特异性Piezo 1激动剂Yoda 1来建模， 机械刺激MVIC中的Piezo1，我们还将研究抑制Piezo1，HTR2B， 色氨酸羟化酶-1（TPH1）以减少HTR信号传导。MVIC终点包括细胞外 基质（ECM）的产生、5-HT相关基因、胶原蛋白1和3以及Piezo 1和2的表达。我们的研究 氟西汀小鼠二尖瓣瓣膜病模型将研究HTR2B和HTR2B对瓣膜病的缓解。 抑制剂和Piezo1抑制剂Dooku1，终点包括： 超声心动图、心室和瓣膜形态学以及5HT相关基因和Piezo 1和2的qRTPCR研究。 具体目标2：研究人类和绵羊二尖瓣（MV）瓣叶，来自MR（仅人类）和 正常MV（绵羊和人）在循环拉伸研究与我们的机械生物反应器系统。我们将比较 静态和循环拉伸条件下，暴露于Piezo1，HTR2B和TPH 1的抑制剂，与 未处理的，评估ECM蛋白、5HT相关基因和Piezo 1和2的表达差异。RNA 将在机械刺激前后对生物反应器样品进行测序研究， 信息学分析，以确定基因表达模式和途径相比，我们的人类MV数据。 本补充材料的拟定研究响应了NOT-HL-23 - 078，因为它们解决了 导致MVP进展为MR的纤维化的潜在基本机制，并利用相关动物 模型

Abstract/Summary This one-year Supplement project will investigate the novel relationship of Piezo1, a mechanical sensing channel never studied previously concerning mitral valve prolapse (MVP) or mitral regurgitation (MR) to serotonin (5HT) mechanisms that contribute to the progression of MR. We previously demonstrated that mitral valve interstitial cell (MVIC) serotonin transporter (SERT) activity was inhibited by either the diet drug, Fenfluoramine or Fluoxetine, a selective 5HT reuptake inhibitor (SSRI), thus resulting in diminished 5HT clearance and enhanced 5HT receptor (HTR) activity. In addition, we showed in clinical studies of MR surgical patients that SSRI use was significantly associated with MR surgery at a younger age, and qRTPCR studies of human MR leaflets, compared to normal human mitral valve (MV) leaflets showed diminished SERT activity in MR. Piezo1 and increased 5HT secretion: Prior research has reported that intestinal Piezo1 activation results in increased 5HT, stimulating our research interest. Feasibility studies presented in this proposal demonstrate that Piezo1 agonists increase HTR signaling in MVIC. Hypothesis: MR progression results from increased HTR2B signaling due to diminished SERT expression and increased 5HT secretion, caused by activation of the mechanically sensitive Ca++ channel, Piezo1. Specific Aim 1: To study the effects of Piezo1 activation and inhibition on 5HT mechanisms in human MVIC, and in a mouse model of Fluoxetine induced MR. We will use the specific Piezo1 agonist, Yoda1, to model mechanical stimulation of Piezo1 in MVIC, and we will also study the effects of inhibition of Piezo1, HTR2B, and tryptophan hydroxylase-1 (TPH1) to reduce HTR signaling. MVIC endpoints include differences in extracellular matrix (ECM) production, expression of 5HT related genes, collagens 1&3, and Piezo1 and 2. Studies of our Fluoxetine mouse mitral valvulopathy model will investigate mitigation of the valvulopathy with both a HTR2B inhibitor and the Piezo1 inhibitor, Dooku1, with endpoints including: Ventricular remodeling per echocardiography, ventricular and valve morphology, and qRTPCR studies of 5HT related genes, and Piezo1&2. Specific Aim 2: To study human and sheep mitral valve (MV) leaflets, from both MR (human only) and normal MV (sheep and human) in cycle stretch studies with our mechanical bioreactor system. We will compare static and cyclic-stretch conditions, with exposure to inhibitors of Piezo1, HTR2B, and TPH1 compared to untreated, assessing differences in expression of ECM proteins, 5HT related genes and Piezo1 and 2. RNA sequencing studies will be carried out on bioreactor samples before and after mechanical stimulation with informatics analyses to identify gene expression patterns and pathways, compared to our human MV data. The proposed studies for this Supplement are responsive to NOT-HL-23-078 because they address the underlying basic mechanisms of fibrosis responsible for progression of MVP to MR, and utilize a relevant animal model.