Introduction Previous studies showed that ischemia minimization (IM), accomplished by perfusing the heart xenograft during storage, prevents ‘initial cardiac xenograft dysfunction’ (ICXD) and enables prolonged survival following in vivo orthotopic cardiac xenotransplantation. Here we report initial observations in a model designed to evaluate effects associated with IM and genetic modifications on heart performance in a working ex vivo model of ICXD.

Method Hearts from genetically modified and wildtype (WT) pigs were procured after flushing with cold preservation solution (UW, 4°C) and stored for 3 hours either in cold saline (0.9%, 4°C: cold storage (CS)) or were perfused with oxygenated Steen’s solution with RBCs (IM). IM perfusion at 40 mmHg was initiated at room temperature for 20 minutes to facilitate homogeneous graft perfusion before cooling to 4°C for the remainder of the storage period. The genetically modified hearts either had combined knockouts of three specific xenogenic carbohydrate genes (GTKO, CMAHKO, b4GALNT2KO: TKO) with variable expression of human complement- and thrombo-regulatory genes (n=13); or GTKO with additional expression of hCD55 (GTKO.hCD55; n=2). Heart function and laboratory parameters were assessed at specific timepoints on a working heart rig while perfused with freshly collected heparinized whole human blood. Troponin I was used as a marker for myocardial injury. 

Results In total, 19 hearts were perfused ex vivo, ten with CS (TKO n=6, GTKO.hCD55 n=1, WT n=3), and 9 with IM (TKO n=7, GTKO.hCD55 n=1, WT n=1). Mean troponin I elaboration (ng/mL) was significantly reduced after 1 hour of ex vivo perfusion in the IM group (70.2 ng/mL vs. 279.5 ng/mL; p = .038), but not at final time points (see fig. 1a-c). Similarly, the cardiac function (measured in cardiac output (CO) in response to increased filling pressures) decreased over time after CS, whereas the IM-stored heart function remained relatively preserved (see fig. 2).

Conclusion IM significantly decreased myocardial injury during the first hour of ex vivo working heart perfusion relative to CS hearts: heart injury (troponin release) was attenuated, and graft failure was delayed in some TKO hearts treated with IM. Evaluation of whether variation in expression of human transgenes may have influenced TKO graft protection from IXD is in progress. We provisionally conclude that IM attenuates ICXD of genetically modified pig hearts during initial exposure to human blood. Whether IM is synergistic with effects associated with specific genetic modifications can be efficiently assessed in this model.