In vivo Osteocyte Ca2+ Signaling in Aging Bone

摘要 骨骼对机械负荷的反应和适应能力随着年龄的增长而显著下降，这是一个主要的 导致骨骼脆弱的因素。我们不了解骨细胞机械感知的改变是如何发生的 在老化的骨骼中。鉴于骨细胞在协调骨骼中的中心作用，这是一个关键的知识鸿沟 形成和吸收反应。我导师实验室的最新突破性发现 揭示了活骨中的骨细胞如何感知和编码机械负荷信息。他们 创造了第一只以骨细胞为靶标的基因编码钙指示器的报告小鼠 允许测量骨细胞对体内负荷的钙反应。他们发现，骨细胞 活骨中的种群以数字编码负荷量，随着应变水平的增加而招募 在健康的年轻成年骨中，有更多的钙离子反应的骨细胞遵循强健的、线性的反应曲线。 初步研究进一步表明，这一骨细胞负荷反应曲线随着 系统性挑战。拟议的F31培训拨款将确定骨细胞钙离子如何响应 体内的机械负荷随年龄的变化而变化。在目标1中，我们将创建他莫昔芬诱导的GCaMP6f小鼠 在这种情况下，钙离子报告器可以在整个成年过程中选择性地打开。这将解决 在第一代中使用的长期结构性GCaMP表达所固有的挑战 本实验室前期培育的骨细胞钙报告小鼠。跖骨将被加载到 使用联合加载和多光子成像通过生理应变水平范围的活体 将测量皮质骨中骨细胞的钙离子反应。在目标2中，我们将使用这些 可诱导的GCaMP6f小鼠评估青壮年，中年， 和老年成年小鼠。最后，一系列通道(如T型和L型通道，Panx1-P2X7，Piezo 1，TRPV通道)参与了体外培养的骨细胞对钙负荷的反应。然而，体内的 人们对这些渠道的重要性知之甚少。在目标3的探索性研究中，我们将使用 验证了选择性通道拮抗剂，以测试体内调节通道功能如何影响负荷- 响应曲线。与F31拨款相关的科学和培训目标：研究：这些研究将是 第一个在成人生活中检测体内骨细胞机械感应的人。培训：建议的 团契结合了广泛的实验和分析方法(小鼠模型、骨骼 生物力学、活体研究、多光子成像、细胞钙测量、骨和骨细胞 生理学、航道生物学)，这将为研究事业的发展提供一个强大的平台。

Abstract Bone’s ability to respond and adapt to mechanical loading declines significantly with age, which is a major contributor to bone fragility. We do not understand how alterations in osteocyte mechanical sensing occur in aging bone. This is a critical knowledge gap given the central role for osteocytes in orchestrating bone formation and resorption responses. Recent breakthrough discoveries from my mentor’s laboratory revealed how osteocytes in living bone perceive and encode mechanical loading information. They created the first ever reporter mice with an osteocyte-targeted genetically encoded calcium indicator to allow measurement of osteocyte Ca2+ responses to loading in vivo. They discovered that osteocyte populations in living bone numerically encode load magnitude, with increasing strain levels recruiting more Ca2+ responding osteocytes in healthy young adult bone following a robust, linear response curve. Preliminary studies further revealed that this osteocyte loading response curve changes markedly with systemic challenge. The proposed F31 training grant will determine how osteocyte Ca2+ responses to mechanical loading in vivo change with age. In Aim 1, we will create tamoxifen inducible GCaMP6f mice in which the Ca2+ reporter can be turned on at selective times throughout adulthood. This will address challenges inherent in long-term constitutive GCaMP expression as was used in the first-generation osteocyte Ca2+ reporter mice previously created by our laboratory. Metatarsal bones will be loaded in vivo through the range of physiological strain levels using a combined loading and multiphoton imaging approach and osteocyte Ca2+ responses in cortical bone will be measured. In Aim 2, we will use these inducible GCaMP6f mice to assess osteocyte Ca2+ responses to loading in young adult, middle aged, and aged adult mice. Finally, a range of channels (eg, T-type and L-type channels, Panx1-P2X7, Piezo 1, TRPV channels) are implicated in osteocyte Ca2+ response to loading in vitro. However, the in vivo importance of these channels is poorly understood. In the exploratory studies in Aim 3, we will use validated selective channel antagonists to test how modulating channel function in vivo influences load- response curves. Relevance to F31 grant Scientific and Training goals: Research: These studies will be the first to examine osteocyte mechano-sensing in vivo throughout adult life. Training: The proposed fellowship incorporates a broad range of experimental and analytical approaches (mouse models, bone biomechanics, in vivo studies, multiphoton imaging, cell Ca2+ measurements, bone and osteocyte physiology, channel biology) that will provide a robust platform for research career growth.