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: 10453600
• 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/10453600
• 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.

项目摘要/摘要 淋巴水肿是一种疾病，其特征是由于淋巴功能不全而导致患病组织的慢性水肿。 淋巴水肿的治疗是姑息性的，需要使用压缩绷带和手动淋巴 排水以将液体从受伤的组织中推出，这通常是通过内在抽水来完成的 淋巴收集血管（CLV）的活性。然而，来自淋巴水肿的CLV仅显示不规则 或缺乏抽水能力，因此恢复这种内在的泵活动是理想的治疗目标。 目前尚未定义推动CLV收缩起搏和启动的机制。 我最近的发现表明，小鼠，大鼠和人淋巴肌肉细胞（LMC）表现出稳定的舒张压 确定收缩频率的去极化，是CLV起搏和自律性的基础。 在鼠CLV中，这种舒张期去极化是压力依赖性的，并且是通过钙的激活而介导的 舒张期活化的氯化物通道，阳极contamin1（ANO1）。在其他自律起搏细胞中， 细胞内肉壳质网（SR）钙时钟是通过电性自律的基础 钙敏感离子通道的激活。 LMC中是否存在SR钙时钟或SR钙 通过三磷酸肌醇受体（ITPRS）或ryanodine受体（RYRS）释放，可调节ANO1和 CLV起搏是未知的。该建议旨在检验以下假设，即SR依赖性钙时钟是 对于淋巴肌肉兴奋性和压力依赖性的年度疗法至关重要，该建议利用了新颖 使用基因编码的技术方法同时监测胞质或SR钙 钙指标，GCAMP6F和GCAMP1-ER，同时记录膜 来自转基因小鼠的鼠CLV的LMC潜力。 AIM 1将确定如何内部 淋巴压通过确定频率，振幅，持续时间，调节LMC SR钙时钟 自发SR钙瞬变的扩散以及腔SR钙浓度的动力学 跨生理压力范围。此外，使用RYR2的诱导型平滑肌敲除 ITPR1除了在ITPR1和RYR2中过度活跃和活跃的敲门突变外，还将阐明 RYR2和ITPR1对舒张期间观察到的亚细胞钙瞬变的功能贡献。 AIM2 将利用这些遗传模型的新鲜分散的LMC来同时进行胞质钙 成像和穿孔贴片夹，以确定钙释放的离散电气贡献 通过与ANO1或其他钙敏感膜通道耦合，ITPR1或RYR2通道。 这些发现将提供有关如何针对的药物策略的关键知识 SR钙动力学的基础机制可以激活淋巴作用并改善淋巴管 患者的功能。