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

 描述(申请人提供)：分离的灌流心脏是研究心脏力学、新陈代谢和电生理学的有用模型。它的主要缺点是以晶体为基础的灌流液，如Krebs-Henseleit溶液(KHS)，不能维持足够的心肌氧合，特别是当代谢需求较高时。全氟化碳(PFC)是一种流行的人工O2载体，临床应用研究很活跃，尽管出于安全考虑，开发大多已停止。PFC乳剂商业来源的丧失对科学界造成了不利影响，因为PFC对于灌流心脏研究和组织工程方法特别有价值。还有一种尚未得到满足的需求，那就是证明各个实验室可以“内部”制造PFC乳剂，目标是使日益复杂的心脏病假说能够在充分充氧的灌流心脏制剂中进行测试。此外，与荧光成像兼容的富氧灌流液，包括光学测绘，将极大地提高此类研究的生理意义，特别是随着光学测绘系统的发展，能够对收缩的心脏进行测绘--这是一种具有非常高氧气需求的制剂。证明PFC灌流液在维持心肌氧合和提供氧气储备的同时，不会实质上干扰对心肌功能的可见光光学评估，这肯定会为体外心脏研究建立一个新的范式。我们的目标是使用定制的PFC乳剂在灌流心脏研究中提供氧气储备，并重建生理冠脉流量，以便可以研究心肌代谢、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性伪影，如KATP通道激活和血管扩张。我们还将使用一种新的运动跟踪光学标测方法来阐明基于PFC的氧合对充分工作的离体兔心脏标本的肌膜KATP通道激活(通过时程变化)、心率和传导速度的影响。我们的主要目标是 严谨地表明，基于PFC的灌流和带有运动跟踪的光学标测是研究离体心脏的能量学和电生理学的强大组合。第一个目标是确定PFC浓度(重量/体积)，对于停搏、收缩和工作的离体心来说，维持肌红蛋白的氧合和ETC的氧化是必要的。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、RH2 37、Rhod-2AM和BCECF。第三个目的是光学标测切除的双心室工作心脏在PFC灌注前后的时程和心动过速的变化。本项目开发的方法可应用于任何灌流器官实验，对基础全心研究具有特定和实质性的影响。

 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.