The Regulation of Lymphatic Muscle Cell Pacemaking by Intracellular Calcium Signals

项目摘要/摘要 淋巴水肿是一种疾病，其特征是由于淋巴供应不足而导致受累组织的慢性水肿。 淋巴水肿的治疗是姑息性的，需要使用压迫绷带和手动淋巴。 引流将液体从受影响的组织中排出，这通常是通过固有的泵来完成的 淋巴集合管(CLV)活性。然而，淋巴水肿患者的cLV仅显示为不规则的。 或缺乏泵送能力，因此恢复这种固有的泵送活动是理想的治疗目标。 目前，驱动CLV收缩的起搏和启动的机制尚未确定。 我最近的发现表明，小鼠、大鼠和人类的淋巴肌细胞(LMC)表现出稳定的舒张期 决定收缩频率的去极化，是CLV起搏和自律性的基础。 在小鼠的cLV中，这种舒张期的去极化是压力依赖性的，并且是由钙离子激活所介导的。 舒张期激活氯通道，AnocTamin1(Ano1)。在其他自律性起搏细胞中， 细胞内肌浆网(SR)钙钟通过 激活钙敏感离子通道。LMC中是否存在SR钙时钟，或者SR钙是否存在 通过三磷酸肌醇受体(Itprs)或兰尼定受体(RyRs)释放调节Ano1和 CLV起搏尚不清楚。这一建议试图检验一种假设，即依赖SR的钙钟是 淋巴肌肉兴奋性和压力依赖的变时性至关重要，这一建议利用了新的 使用基因编码同时监测胞浆或SR钙的技术方法 钙指示剂分别为GCaMP6f和GCaMP1-ER，同时记录膜 转基因小鼠收缩小鼠cLV在LMCs中的潜力。目标1将决定内部如何 淋巴压力通过决定频率，幅度，持续时间， 和自发肌浆网钙瞬变的扩散，以及管腔内肌浆网钙浓度的动态 在生理压力范围内。此外，RyR2可诱导的平滑肌基因敲除的使用 而ITPR1除了ITPR1和RyR2的过度活跃和非活跃敲入突变外，还将阐明 RyR2和ITPR1在舒张期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来同时进行胞内钙 成像和穿孔膜片钳确定钙释放的离散电贡献 ITPR1或RyR2通道通过与Ano1或其他钙敏感膜通道偶联。 这些发现将提供有关药物战略如何针对 肌浆网钙动力学激活淋巴起搏和改善淋巴循环的机制 在病人身上起作用。

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.