Improved viability and reduced apoptosis in sub-zero 21-hour preservation of transplanted rat hearts using anti-freeze proteins

Background: Freeze-tolerant fish survive sub-zero temperatures by non-colligatively lowering the freezing temperature of their body fluids using anti-freeze proteins (AFPs). We sought to evaluate and compare the effects of prolonged sub-zero cryopreservation of transplanted rat hearts using AFP I or AFP III. Methods: Two heterotopic rat heart transplantation protocols were used. In Protocol 1 (n = 104), hearts (n = 8/group) were preserved for 12, 18 and 24 hours in University of Wisconsin solution (UW) at 4°C, UW at -1.3°C, UW/AFP I at -1.3°C and UW/AFP III at -1.3°C, with and without nucleation. Post-operative evaluation consisted of visual viability scoring of the hearts after 60 minutes. Protocol 2 (n = 58) involved evaluation of 24-hour post-transplant viability, echocardiography (fractional shortening [FS], left ventricular end-systolic and -diastolic diameter [ESD, EDD] and anterior and posterior wall systolic and diastolic thickness [AWT-S, AWT-D, PWT-S, PWT-D]), TUNEL staining and electron microscopy (EM) findings for hearts preserved for 18, 21 and 24 hours in UW at 4°C or UW/AFP III at -1.3°C. Results: Hearts preserved in UW at -1.3°C with nucleation froze and died. Three of 8 hearts preserved in UW at 4°C for 24 hours died, whereas all hearts preserved at -1.3°C survived. Hearts preserved in UW/AFP for 18 and 24 hours at -1.3°C had superior viability scores compared with those in UW at 4°C. Hearts in AFP III at -1.3°C displayed greater AWT-S and AWT-D (3.5 ± 0.2 vs 2.4 ± 0.2, p < 0.05, and 3.5 ± 0.2 vs 2.2 ± 0.2, p < 0.05, respectively) after 18-hour preservation. In the 21-hour preservation group, AFP-treated hearts displayed improved echocardiographic systolic contraction indices, including: improved FS (27 ± 3.7 vs 15 ± 4, p = 0.04); diminished ESD (0.28 ± 0.57 vs 0.47 ± 0.6, p < 0.05); greater AWT-S (3.4 ± 0.18 vs 2.8 ± 0.2, p < 0.05); and fewer positively TUNEL-stained nuclei per specimen (35 ± 14 vs 5.3 ± 2.7, p = 0.04). Also, improved EM scores were noted compared with UW at 4°C. Conclusions: In prolonged sub-zero cryopreservation, AFPs protect the heart from freezing, improve survival and hemodynamics, and reduce apoptotic cell death.