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 degrees C, UW at -1.3 
degrees C, UW/AFP I at -1.3 degrees C and UW/AFP III at -1.3 degrees 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 degrees C or UW/AFP III at -1.3 
degrees C.
RESULTS: Hearts preserved in UW at -1.3 degrees C with nucleation froze and 
died. Three of 8 hearts preserved in UW at 4 degrees C for 24 hours died, 
whereas all hearts preserved at -1.3 degrees C survived. Hearts preserved in 
UW/AFP for 18 and 24 hours at -1.3 degrees C had superior viability scores 
compared with those in UW at 4 degrees C. Hearts in AFP III at -1.3 degrees 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 degrees C.
CONCLUSIONS: In prolonged sub-zero cryopreservation, AFPs protect the heart from 
freezing, improve survival and hemodynamics, and reduce apoptotic cell death.