Background: Ex vivo oxygenated perfusion systems is a promising approach to extend cardiac allograft preservation beyond the typical 4 to 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 one SCS-preserved heart (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.

Disclosure, Competing Interests: Dr. Ferrari Andrew Sabin Family Foundation cardiovascular Research Laboratory received an industry-sponsored grant from XVIVO Perfusion Inc. Authors declare that they have no competing interests.

Funding: National Heart Lung and Blood Institute of the National Institutes of Health (R01 HL 170573 and R01 HL 163085) [GF] Research Agreement with xVIVO Perfusion Inc [GF] The Kibel Fund for Aortic Valve Research [GF], Andrew Sabin Family Foundation Cardiovascular Research Laboratory [GF)] The Valley Hospital Foundation Marjorie C Bunnel charitable fund [GF], German Heart Foundation (Deutsche Herzstiftung e.V.) [SJB] Ri.MED Foundation [AA].

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