The Regulation of Lymphatic Muscle Cell Pacemaking by Intracellular Calcium Signals

项目总结/摘要 淋巴水肿是一种疾病，其特征是由于淋巴功能不全导致受累组织的慢性水肿。 水肿的治疗是姑息性的，需要使用压迫绷带和手动淋巴 排出以将流体推出患病组织，这通常通过内在泵送来实现 淋巴收集管（cLV）的活性。然而，水肿患者的cLV仅显示不规则的 或缺乏泵送能力，因此恢复这种固有的泵活性是理想的治疗目标。 目前，驱动起搏和启动cLV收缩的机制尚未确定。 我最近的研究结果表明，小鼠，大鼠和人类淋巴肌细胞（LMC）表现出稳定的舒张功能， 去极化决定收缩频率，是cLV起搏和自律性的基础。 在小鼠cLV中，这种舒张期去极化是压力依赖性的，并且由钙通道的激活介导。 激活氯离子通道，Anoctamin 1（Ano 1）。在其他自主节律起搏细胞中， 细胞内肌内质网（SR）钙时钟是电自律性基础， 激活钙敏感离子通道。LMC中是否存在SR钙时钟，或者SR钙 通过肌醇三磷酸受体（ItlR）或兰尼碱受体（RyR）的释放调节Ano 1和 cLV起搏未知。该建议旨在检验SR依赖性钙时钟是 对于淋巴肌兴奋性和压力依赖性变时性至关重要， 使用遗传编码的方法同时监测胞质或SR钙的技术方法 钙指示剂GCaMP 6 f和GCaMP 1-ER，同时记录膜 LMCs中从转基因小鼠感染鼠cLV的可能性。目标1将决定如何在内部- 淋巴压力通过确定LMCSR的频率、幅度、持续时间 自发SR钙瞬变的扩散，以及腔SR钙浓度的动态 在生理压力范围内。此外，使用RyR 2的可诱导平滑肌敲除， 和Itpr 1以及Itpr 1和RyR 2中的过度活跃和活性不足的敲入突变将阐明 RyR 2和Itpr 1对在阿托洛尔过程中观察到的亚细胞钙瞬变的功能贡献。AIM2 将利用来自这些遗传模型的新鲜分散的LMC， 成像和穿孔膜片钳，以确定钙释放的离散电贡献， Itpr 1或RyR 2通道通过与Ano 1或其他钙敏感性膜通道偶联。 这些发现将提供关键知识，关于如何针对药物策略， SR钙动力学的潜在机制可以激活淋巴起搏， 在患者中发挥作用。

Project Summary/Abstract Lymphedema is a disease characterized by chronic edema of the afflicted tissue due to lymphatic insufficiency. Treatment for lymphedema is palliative and requires the use of compression bandages and manual lymph drainage to push fluid out of the afflicted tissue, which is normally accomplished by the intrinsic pumping activity of lymphatic collecting vessels (cLV). cLVs from lymphedema patients, however, display only irregular or absent pumping ability and therefore restoring this intrinsic pump activity is an ideal therapeutic goal. Currently the mechanisms that drive the pacemaking and initiation of cLV contraction have not been defined. My recent findings show that mouse, rat, and human lymphatic muscle cells (LMCs) exhibit a steady diastolic depolarization that determines contraction frequency, and is the basis of cLV pacemaking and autorhythmicity. In murine cLVs, this diastolic depolarization is pressure-dependent and is mediated by activation of a calcium activated chloride channel, Anoctamin1 (Ano1) during diastole. In other autorhythmic pacemaking cells, an intracellular sarcoendoplasmic reticulum (SR) calcium clock underlies electrical autorhythmicity through activation of calcium sensitive ion channels. Whether an SR calcium clock is present in LMCs or if SR calcium release through either inositol triphosphate receptors (Itprs) or ryanodine receptors (RyRs) regulates Ano1 and cLV pacemaking is unknown. This proposal seeks to test the hypothesis that a SR dependent calcium clock is critical for lymphatic muscle excitability and pressure dependent chronotropy, This proposal utilizes novel technical approaches to simultaneously monitor either cytosolic or SR calcium using genetically encoded calcium indicators, GCaMP6f and GCaMP1-ER respectively, while simultaneously recording membrane potential in LMCs of contracting murine cLVs from genetically modified mice. Aim 1 will determine how intra- lymphatic pressure regulates the LMC SR calcium clock by determining the frequency, amplitude, duration, and spread of spontaneous SR calcium transients, and the dynamics of the luminal SR calcium concentration across a physiological pressure range. Additionally, the use of inducible smooth muscle knockouts of RyR2 and Itpr1 in addition to over-active and under-active knock-in mutations in Itpr1 and RyR2 will elucidate the functional contribution of RyR2 and Itpr1 to the subcellular calcium transients observed during diastole. Aim2 will utilize freshly dispersed LMCs from these genetic models to perform simultaneous cytosolic calcium imaging and perforated patch clamp to determine the discrete electrical contribution of calcium release from either Itpr1 or RyR2 channels through coupling with Ano1 or other calcium sensitive membrane channels. These findings will provide critical knowledge regarding how a pharmaceutical strategy targeting the mechanisms underlying SR calcium dynamics could activate lymphatic pacemaking and improve lymphatic function in patients.