Improved Viability and Reduced Apoptosis in Sub-Zero 21-hour Preservation of Transplanted Rat Hearts Using Anti-Freeze Proteins

J Heart Lung Transplant. 2005 Nov;24(11):1915-29. doi: 10.1016/j.healun.2004.11.003. Epub 2005 Oct 3.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine
  • Allopurinol
  • Animals
  • Antifreeze Proteins / pharmacology*
  • Apoptosis / drug effects*
  • Cell Survival / drug effects*
  • Cold Temperature
  • Cryopreservation / methods*
  • Echocardiography
  • Glutathione
  • Heart Transplantation* / immunology
  • Heart Transplantation* / physiology
  • Heart*
  • Insulin
  • Mitochondria, Heart / pathology
  • Organ Preservation / methods*
  • Organ Preservation Solutions
  • Raffinose
  • Rats
  • Rats, Sprague-Dawley
  • Sarcomeres / pathology
  • Transplantation, Heterotopic

Substances

  • Antifreeze Proteins
  • Insulin
  • Organ Preservation Solutions
  • University of Wisconsin-lactobionate solution
  • Allopurinol
  • Glutathione
  • Adenosine
  • Raffinose