Ex Vivo Hypothermic Perfusion Enables 48-Hour Heart Preservation and Bench-Top Functional Recovery via Normothermic Reperfusion in a Porcine Model
Background: Ex vivo oxygenated perfusion systems is a promising approach to extend cardiac allograft preservation beyond the typical 4–6h limit allowed by static-cold-storage (SCS).  Hypothermic oxygenated perfusion (HOPE) has been proven to safely preserve donor hearts, yet its underlying molecular mechanisms have not been extensively evaluated.   Objectives: The aim of the study is to characterize cardiomyocyte viability, transcriptomic and metabolomic responses, and functional recovery of porcine hearts preserved with HOPE for up to 48h, including evaluating their ability to regain sinus rhythm following bench-top normothermic reperfusion (NMP).  Methods: Seventeen Yorkshire pigs underwent donor cardiectomy. In the first arm, ten hearts were preserved for up to 48h using either SCS (n=5) or HOPE (n=5). Endomyocardial biopsies were collected at 0, 12, 24, and 48h for histology, RNA sequencing, flow cytometry, and metabolomics. In the second arm, six HOPE-preserved hearts (3h, 24h, 48h) and two SCS-preserved heart (3h and 24h) underwent 2h NMP to simulate transplantation and assess reanimation.  Results: HOPE preserved cardiomyocyte viability and structural integrity for 48h, in contrast to SCS in both arms of the study. RNA sequencing and untargeted metabolomics revealed conserved energy-substrate profiles in HOPE and progressive ischemic metabolite accumulation in SCS. All HOPE hearts regained stable sinus rhythm.  Conclusions: HOPE enables 48h ex vivo heart preservation while maintaining cardiomyocyte integrity, normal gross and microscopic architecture, and rapid functional recovery on bench-top reperfusion in a preclinical model. These findings establish a foundation for redefining clinical preservation times and widening geographic donor access. Overall design: RNA-seq profiling of swine myocardial tissue biopsied (1) at T0h, T12h, T24h and T48h of either SCS or HOPE preservation, and (2) after  T3h, T24h and T48h of HOPE or T24h of SCS, followed by 2h of Normothermic Machine Perfusion.