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 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.
Amir
Gabriel
G
Heart Transplantation Unit, Department of Cardiac Surgery, Sheba Medical Center, Tel Hashomer, Israel. gabiamir@stanford.edu
Rubinsky
Boris
B
Basheer
Sheick Yousif
SY
Horowitz
Liana
L
Jonathan
Leor
L
Feinberg
Micha S
MS
Smolinsky
Aram K
AK
Lavee
Jacob
J
D000241:Adenosine; D000493:Allopurinol; D000818:Animals; D021301:Antifreeze Proteins / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects*; D003080:Cold Temperature; D015925:Cryopreservation / Q000379:methods*; D004452:Echocardiography; D005978:Glutathione; D006321:Heart; D016027:Heart Transplantation / Q000276:immunology / Q000502:physiology; D007328:Insulin; D008929:Mitochondria, Heart / Q000473:pathology; D009926:Organ Preservation / Q000379:methods*; D019188:Organ Preservation Solutions; D011887:Raffinose; D051381:Rats; D017207:Rats, Sprague-Dawley; D012518:Sarcomeres / Q000473:pathology; D016042:Transplantation, Heterotopic
D021301:Antifreeze Proteins; D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose