Oxygen-rich perfusate that is compatible with optical assessments of myocardial physiology

 描述（由申请人提供）：离体灌注心脏是研究心脏力学、代谢和电生理学的有用模型。其关键缺点是基于晶体的灌注液，例如Krebs-Henseleit溶液（KHS），不能维持足够的心肌氧合，特别是当代谢需求高时。全氟化碳（PFC）是一种流行的人造氧气载体，并积极研究临床使用，但由于安全问题，开发大多已停止。全氟化学品乳剂商业来源的丧失对科学界产生了负面影响，因为全氟化学品对灌注心脏研究和组织工程方法特别有价值。有一个未得到满足的需要，以证明个别实验室可以“在内部”制造全氟化碳乳剂，其目标是使越来越复杂的心脏病假设能够在充分充氧的灌注心脏制剂中进行测试。此外，与荧光成像兼容的富氧灌注液，包括光学映射，将大大提高这些研究的生理意义，特别是随着光学映射系统的发展，使收缩心脏的映射-具有非常高的氧气需求的准备。证明PFC灌注液维持心肌氧合并提供氧储备，同时基本上不干扰心肌功能的可见光光学评估，必将为离体心脏研究建立一个新的范式。我们的目标是使用定制的PFC乳剂在灌注心脏研究中提供氧储备并重建生理冠状动脉流速，从而可以研究心肌代谢、原位线粒体功能和电生理学，而不会出现与氧合不足相关的生理伪影，例如KATP通道激活和血管舒张。我们还将使用一种新的运动跟踪光学标测方法，以阐明PFC为基础的氧合对肌膜KATP通道激活（通过APD变化），心率和传导速度在完全工作的离体兔心脏准备的影响。我们的首要目标是 严格表明，基于PFC的灌注液和具有运动跟踪的光学标测是研究离体心脏能量学和电生理学的强大组合。第一个目的是确定PFC浓度（重量/体积），该浓度是维持肌红蛋白氧合和ETC氧化所需的，用于停止、收缩和工作的离体心脏。第二个目的是测量PFC对几种常见荧光团的荧光信号衰减的影响，包括di-4-ANEPPS，RH 237，Rhod- 2AM和BCECF。第三个目的是光学地图的APD和CV的变化之前和之后PFC灌注切除双心室工作心脏。本项目开发的方法可应用于任何灌注器官实验，对基础的全心脏研究具有具体和实质性的影响。

 DESCRIPTION (provided by applicant): The isolated perfused heart is a useful model for studying cardiac mechanics, metabolism, and electrophysiology. Its critical disadvantage is that crystalloid-based perfusate, such as Krebs-Henseleit solution (KHS), does not maintain adequate myocardial oxygenation, especially when metabolic demand is high. Perfluorocarbons (PFC) were a popular artificial O2 carrier and were actively studied for clinical use, although development has mostly ceased due to safety concerns. The loss of commercial sources of PFC emulsions detrimentally impacts the scientific community because PFC are particularly valuable for perfused heart studies and tissue engineering approaches. There is an unmet need to demonstrate that individual laboratories can make a PFC emulsion "in-house", with the goal of enabling increasingly sophisticated hypotheses of cardiac disease to be tested in adequately oxygenated perfused heart preparations. Additionally, an oxygen-rich perfusate that is compatible with fluorescence imaging, including optical mapping, will dramatically elevate the physiological significance of such studies, especially as optical mapping systems evolve to enable mapping in contracting hearts -- preparations that have a very high oxygen demand. Demonstrating that PFC perfusate maintains myocardial oxygenation and provides oxygen reserve while not substantially interfering with visible-light optical assessments of myocardial function will surely establish a new paradigm for excised heart studies. Our objective is use a custom-made PFC emulsion to provide oxygen reserve in perfused heart studies and reestablish physiological coronary flow rates, such that myocardial metabolism, in-situ mitochondrial function, and electrophysiology can be studied without physiological artifacts associated with inadequate oxygenation, such as KATP channel activation and vasodilation. We will also use a novel motion-tracking optical mapping approach to elucidate the effects of PFC-based oxygenation on sarcolemmal KATP channel activation (via APD changes), heart rate, and conduction velocity in fully working isolated rabbit heart preparations. Our primary goal is to rigorously show that a PFC-based perfusate and optical mapping with motion tracking is a powerful combination for studying energetics and electrophysiology in excised hearts. The first Aim is to determine the PFC concentration (weight/vol) that is required to maintain myoglobin oxygenation and oxidation of the ETC for excised hearts that are arrested, contracting, and working. The second Aim is to measure the effect of PFC on the fluorescence signal attenuation of several common fluorophores, including di-4-ANEPPS, RH237, Rhod- 2AM, and BCECF. The third Aim is to optically map changes in APD and CV before and after PFC perfusion in excised biventricular working hearts. Approaches developed in this project could be applied in any perfused organ experiment, having specific and substantial impact on basic whole-heart research.