High subzero heart preservation: from zebrafish to mammals

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.