The Regulation of Lymphatic Muscle Cell Pacemaking by Intracellular Calcium Signals

细胞内钙信号对淋巴肌细胞起搏的调节

• 批准号: 10413534
• 负责人: Scott D. Zawieja
• 金额: $ 24.89万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10413534
• 关键词: Action Potentials; Affect; American; Back; Behavior; Blood Circulation; Calcium; Calcium Signaling; Cell membrane; Cells; Chloride Channels; Chronic; Compression Bandage; Contracts; Coupling; Cytosol; Data; Diastole; Disease; Drainage procedure; Edema; Event; Exhibits; Frequencies; Genetic Models; Goals; Human; Image; Inositol; Interstitial Cell of Cajal; Intervention; Intestinal Motility; Ion Channel; Kinetics; Knock-in; Knock-in Mouse; Knock-out; Knowledge; Liquid substance; Lymph; Lymphatic; Lymphatic System; Lymphatic function; Lymphedema; Manuals; Measurement; Mediating; Membrane; Membrane Potentials; Molecular; Monitor; Mus; Muscle; Muscle Cells; Mutation; Nodal; Pacemakers; Pathway interactions; Patients; Periodicity; Pharmacologic Substance; Physiological; Potassium Channel; Pump; Rattus; Regulation; Reporting; Research Personnel; Reticulum; Role; RyR2; Ryanodine Receptors; Sarcoplasmic Reticulum; Smooth Muscle; Source; Swelling; Techniques; Testing; Therapeutic; Tissues; Training; calcium indicator; clinically relevant; defined contribution; design; event cycle; experimental study; heart cell; imaging study; improved; loss of function mutation; lymphatic insufficiency; lymphatic pump; lymphatic vessel; novel; palliative; patch clamp; pressure; receptor; response; therapeutic target; tripolyphosphate

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.

项目概要/摘要 淋巴水肿是一种以由于淋巴功能不全而导致受累组织慢性水肿为特征的疾病。 淋巴水肿的治疗是姑息性的，需要使用加压绷带和手法淋巴 引流将液体从受影响的组织中排出，这通常是通过内在泵送来完成的 淋巴集合管 (cLV) 的活动。然而，淋巴水肿患者的 cLV 仅显示不规则 或缺乏泵送能力，因此恢复这种内在的泵送活动是理想的治疗目标。 目前，驱动 cLV 收缩和起搏的机制尚未明确。 我最近的研究结果表明，小鼠、大鼠和人类淋巴肌细胞 (LMC) 表现出稳定的舒张压 去极化决定收缩频率，是 cLV 起搏和自律节律的基础。 在小鼠 cLV 中，这种舒张期去极化是压力依赖性的，并且由钙激活介导 在舒张期激活氯离子通道 Anoctamin1 (Ano1)。在其他自律起搏细胞中， 细胞内肌内质网（SR）钙时钟是电自节律的基础 激活钙敏感离子通道。 LMC 中是否存在 SR 钙时钟，或者 SR 钙是否存在 通过肌醇三磷酸受体 (Itprs) 或兰尼碱受体 (RyRs) 释放调节 Ano1 和 cLV 起搏功能未知。该提案旨在检验 SR 依赖性钙时钟的假设 对于淋巴肌兴奋性和压力依赖性变时性至关重要，该提案利用了新颖的 使用基因编码同时监测胞质或 SR 钙的技术方法 钙指示剂分别为 GCaMP6f 和 GCaMP1-ER，同时记录膜 LMC 中从转基因小鼠中收缩小鼠 cLV 的潜力。目标 1 将决定内部如何 淋巴压力通过确定频率、幅度、持续时间来调节 LMC SR 钙时钟， 自发 SR 钙瞬变的传播和传播，以及管腔 SR 钙浓度的动态 跨越生理压力范围。此外，使用 RyR2 诱导平滑肌敲除 和 Itpr1 除了 Itpr1 和 RyR2 中过度活跃和低活跃的敲入突变之外，还将阐明 RyR2 和 Itpr1 对舒张期观察到的亚细胞钙瞬变的功能贡献。目标2 将利用来自这些遗传模型的新鲜分散的 LMC 来同时进行胞质钙 成像和穿孔膜片钳以确定钙释放的离散电贡献 Itpr1 或 RyR2 通过与 Ano1 或其他钙敏感膜通道偶联而形成通道。 这些发现将提供关于如何针对该疾病的药物策略的关键知识。 SR 钙动力学的潜在机制可以激活淋巴起搏并改善淋巴 患者的功能。