High subzero heart preservation: from zebrafish to mammals

项目总结 心脏移植是治疗终末期心脏病的唯一方法；然而， 需求和供应部门对这一救命程序的使用有限。延长心跳的时间长度 在运输过程中保持活力是一项关键的使能技术，可以解决心脏的几个限制 移植。例如，可用的心脏由于环境因素而被丢弃，例如时间 捐赠者死亡和捐赠者/接受者的位置，这可以通过延长保存时间来消除。更远，更长 保存期限有可能通过加强供者-受者匹配来改善受者结果， 减少缺血损伤，消除紧急手术的风险。因此，该项目旨在 开发一种新的方法，将移植心脏的保存时间从4-6小时延长到3天。 虽然我们已经掌握了几种临床上相关的细胞类型在悬液中的保存，但固体器官 由于不同的细胞类型对相同的损伤有不同的反应，因此取得的成功有限。 此外，用于辅助开发保存溶液的高通量实验方法也受到限制 完整的组织具有3D结构考虑因素，对成功保存和 常用的体外细胞模型不具有代表性，缺乏对功能结果的评估。 相反，我们建议战略性地利用斑马鱼转基因系来解决细胞特异性反应， 心脏完整的天然保存方法的功能后果和复合损伤 高通过率格式。在斑马鱼中开发的方法将迅速扩展到哺乳动物的心脏 促进翻译。 我们将利用这一实验平台来开发一种新的保护方法，称为 半冻住了。受自然界中耐寒的林蛙的启发，部分冻结旨在最大限度地增加存储 通过输入接近零下20°C的未探索的高温范围来持续时间。 在不同的温度范围内，我们将制定有效的策略，让心脏在冰的存在下生存。这将是 通过开发存储解决方案来实现，该存储解决方案旨在保护不同类型的心脏细胞，而不需要 不利的功能后果(具体目标1)。这些努力将因发现 激活缺氧信号的诱导物(特异性目标2)和未折叠蛋白反应(特异性目标3) 分别从缺血和温度依赖损伤中拯救心脏。这些诱导剂将被组合在一起 转化为在机器灌流过程中输送的预调液，以进一步延长保存时间。

PROJECT SUMMARY Heart transplantation is the only cure for end-stage heart disease; however, an increasing imbalance between demand and supply has limited access to this life-saving procedure. Extending the length of time hearts can remain alive during transport represents a critical enabling technology to address several limitations in heart transplantation. For example, usable hearts are discarded due to circumstantial factors, such as the timing of donor death and donor/recipient location, which can be eliminated by extended preservation. Further, longer preservation duration has the potential to improve recipient outcomes by enhancing donor-recipient matching, reducing ischemic injury, and removing the risk of emergency surgeries. Consequently, this project aims to develop a new method to extend preservation of hearts for transplantation from 4-6 hours to 3+ days. While we have mastered the preservation of several clinically relevant cell types in suspension, solid organs have been met with limited success since different cell types have different responses to the same injury. Further, high throughput experimental methods to aid development of preservation solutions are limited since intact tissue has 3D structural considerations that have a profound effect on successful preservation and commonly used in vitro cell models are not representative and lack assessment of functional outcomes. Instead, we propose to strategically leverage zebrafish transgenic lines to address cell-specific responses, functional consequences, and compounding injuries of preservation approaches with an intact, native heart in a high throughout format. Approaches developed in zebrafish will be rapidly scaled to mammalian hearts to promote translation. We will leverage this experimental platform to enable the development of a new preservation approach, termed partial freezing. Inspired by freeze-tolerant wood frogs in nature, partial freezing aims to maximize storage durations by entering unexplored high subzero temperatures ranges approaching -20°C. To enter these temperature ranges, we will develop effective strategies for hearts to live in the presence of ice. This will be achieved through the development of a storage solution designed to protect diverse cardiac cell types without adverse functional consequences (Specific Aim 1). These efforts will be complimented by the discovery of inducers that activate hypoxia signaling (Specific Aim 2) and the unfolded protein response (Specific Aim 3) to rescue hearts from ischemic and temperature-dependent injury, respectively. These inducers will be combined into a preconditioning solution that is delivered during machine perfusion to further extend preservation time.