High-throughput integrated live imaging and optogenetic pacing platform to assess hypoxia responsiveness in the fly heart

项目摘要 缺血预适应是一种公认的现象，其中短暂的受控缺血发作 并且再灌注使心脏免受随后的持续缺血发作。一个新兴机构 有证据表明，神经调节的心率调制赋予心脏预适应 应答通过预处理的模型系统来理解这一机制将有助于我们识别 基因和蛋白质，设计未来的药物靶点，以预防缺血性心脏损伤。作为 有希望的替代电起搏来调节心率，光遗传起搏不需要物理 接触，具有高的空间和时间精度，提供更具体的激励，并避免伪影 电刺激光遗传学领域的最新发展使得非侵入性和 在动物模型中，如在黑腹果蝇中，对心脏节律的特定光学控制。果蝇是 一个强大的遗传模型系统，自20世纪初以来一直用于表征相关基因， 人类疾病，包括心脏病。在苍蝇中进行的研究可以提供对保守的 心脏疾病的机制，这可以应用于高等生物，包括人类。工作 我们与来自马萨诸塞州总医院的李爱荣和鲁道夫·坦齐博士合作， 果蝇的非侵入性光遗传学起搏和同步光学相干断层扫描（OCT）成像 心第一次。最近，我们进一步证明了红光光遗传学起搏和成功的光学 在果蝇模型中控制心动过速、心动过缓和可诱发的心脏骤停。在长达十年的 与李博士和Tanzi博士的富有成效的合作，以及与Abhinav Diwan博士（心脏病专家）的新合作 Jeanne Nerbonne博士（心脏电生理学家）和Kenneth Schechtman博士（生物统计学家）， 华盛顿大学，我们建议开发一种高通量的集成OCT成像和双色 光遗传学起搏系统，建立一个新的研究平台，研究预适应和缺氧 苍蝇心脏的反应。我们假设，心动过缓的周期将预处理苍蝇的心脏， 通过激活自噬-溶酶体途径防止缺氧。具体目标是：（1）发展 并优化用于非侵入性OCT成像和光遗传学控制的高通量集成仪器， 建立双转基因果蝇模型和基于OCT成像的功能检测 描述果蝇体内心脏生理学特征; 3）定义缺氧反应的功能和分子变化 和转基因果蝇模型中的光遗传学预处理。如果成功，高通量光学成像和 本项目开发的双色光遗传学起搏平台结合强大的双转基因果蝇 模型将使我们能够描述苍蝇心脏功能在应对不同压力挑战时的变化 这在以前是不可行的。这将使我们能够进行一系列新的实验， 缺氧诱导的心脏变化和预适应的保守分子机制。

Project Summary Ischemic preconditioning is a well-established phenomenon, in which a brief episode(s) of controlled ischemia and reperfusion renders cardioprotection from a subsequent sustained episode of ischemia. An emerging body of evidence demonstrated that neural regulated heart rate modulation confers cardiac preconditioning responses. Understanding the mechanism through model systems of preconditioning would help us identify the genes and proteins when designing future drug targets for the prevention of ischemic cardiac injury. As a promising alternative to electrical pacing to modulate heart rate, optogenetic pacing does not require physical contact, has high spatial and temporal precision, offers more specific excitation, and avoids artifacts from electrical stimulation. Recent developments in the field of optogenetics make it possible for non-invasive and specific optical control of the heart rhythm in animal models, such as in Drosophila melanogaster. Drosophila is a powerful genetic model system that has been used since the early 1900s to characterize genes associated with human diseases, including cardiac diseases. Studies performed in flies can provide insights into conserved mechanisms in cardiac diseases, which can be applied to higher organisms, including humans. Working in collaboration with Drs. Airong Li and Rudolph Tanzi from the Massachusetts General Hospital, we demonstrated non-invasive optogenetic pacing and concurrent optical coherence tomography (OCT) imaging of the Drosophila heart for the first time. Recently, we further demonstrated red-light optogenetic pacing and successful optical control of tachycardia, bradycardia, and restorable cardiac arrest in fly models. Building on the decade-long productive collaboration with Drs. Li and Tanzi and new collaborations with Dr. Abhinav Diwan (cardiologist) and Dr. Jeanne Nerbonne (cardiac electrophysiologist) and Dr. Kenneth Schechtman (biostatistician) at Washington University, we propose to develop a high-throughput integrated OCT imaging and dual-color optogenetic pacing system and establish a novel research platform to study preconditioning and hypoxia responsiveness in the fly heart. We hypothesize that periods of bradypacing will precondition the fly heart to protect against hypoxia, via activation of the autophagy-lysosome pathway. The specific aims are: 1) Develop and optimize a high-throughput integrated instrument for non-invasive OCT imaging and optogenetic control of fly heart function in vivo; 2) Develop double transgenic fly models and functional assays based on OCT imaging to characterize fly heart physiology in vivo; 3) Define functional and molecular changes in response to hypoxia and optogenetic preconditioning in transgenic fly models. If successful, the high-throughput optical imaging and dual-color optogenetic pacing platform developed in this program combined with powerful double transgenic fly models will enable us to characterize changes of the fly heart function in response to different stress challenges that is not feasible before. This will allow us to perform a series of new experiments, providing insights into conserved molecular mechanisms on hypoxia-induced cardiac changes and preconditioning.