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，在这种现象中，控制性缺血的短暂发作(S) 再灌流对随后持续的缺血发作具有心脏保护作用。一个新兴的机构 大量证据表明，神经调节心率调制使心脏预适应 回应。通过模型系统了解预适应的机制将有助于我们识别 在设计未来预防缺血性心脏损伤的药物靶点时，基因和蛋白质。作为一名 光基因起搏有望取代电起搏来调节心率，不需要物理起搏 接触，具有很高的空间和时间精度，提供更具体的激励，并避免 电刺激。光遗传学领域的最新发展使非侵入性和 在动物模型中对心率的特殊光学控制，例如在果蝇中。果蝇是 一种功能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。在果蝇身上进行的研究可以提供对保守性 心脏疾病的机制，可应用于包括人类在内的高等生物。在工作中 与马萨诸塞州总医院的李艾荣博士和鲁道夫·坦齐博士合作，我们展示了 果蝇无创性光遗传起搏和并行光学相干断层扫描(OCT)成像 心脏第一次。最近，我们进一步展示了红光遗传起搏和成功的光学 在苍蝇模型中控制心动过速、心动过缓和可恢复的心脏骤停。在长达十年的基础上建设 与李博士和坦兹博士的富有成效的合作以及与阿比纳夫·迪万博士(心脏病专家)的新合作 珍妮·恩伯恩博士(心脏电生理学家)和肯尼思·谢克特曼博士(生物统计学家) 我们建议开发一种高通量的集成OCT成像和双色 光遗传起搏系统，建立研究预适应和缺氧的新研究平台 苍蝇心脏的反应能力。我们假设，一段时间的制动会使苍蝇的心脏 通过激活自噬-溶酶体途径来保护机体免受缺氧。具体目标是：1)发展 并优化了用于无创OCT成像和光遗传控制的高通量集成仪器 2)双转基因果蝇模型的建立及基于OCT成像的功能检测 描述苍蝇心脏在活体内的生理特性；3)确定对低氧反应的功能和分子变化 以及转基因果蝇模型中的光遗传预适应。如果成功，高通量光学成像和 本项目研制的双色光遗传起搏平台与强大的双转基因果蝇相结合 模型将使我们能够表征苍蝇心脏功能在应对不同应激挑战时的变化 这在以前是不可行的。这将允许我们进行一系列新的实验，提供对 低氧引起的心脏改变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.