The goal of this research is to develop a long-term freezing method to storage the cardiac explant for transplantation purpose. Current storage methods, which preserve the human donor heart for about 4 hours, severely limit the organ availability and restrict the number of end-stage patients who can benefit from cardiac transplantation. Ideally, storage duration of weeks or months can be establish and the donor organ pool can be increased by many fold. The most likely approach to achieve such time frame would be freezing preservation. Previous attempts to store cardiac organ by freezing all failed. The major problem stemmed from the low temperatures (-20 to -196 degrees C) involved. Thus high and cytotoxic levels of synthetic cryoprotectants have to be used to avoid the lethal intracellular ice formation. To circumvent this apparent dilemma, the present project proposes to investigate freezing preservation of the cardiac explant with slow freezing rate at high subzero temperatures (-8 degrees C or higher) employing modest levels of natural cryoprotectants (glucose, glycerol, sorbitol, etc.). This approach emulates the overwintering strategies used by terrestrially hibernating amphibians and reptiles. These freeze tolerant animals use glucose or glycerol in the order of 20 to 200 mM and survive a frozen state at temperatures ranging from -2 to -8 degrees C for periods up to 2 weeks. Preliminary study from our laboratory demonstrated the feasibility of this approach and achieved successful reanimation of the isolated rat heart frozen at -1.2 degrees C for 5 hours. Specifically in this proposal we will 1) study in the isolated rat heart, the relationship between the extent of freezing at -1, -3, -5, and -7 degrees C as measured by tissue ice content and the recovery of cardiac function after reperfusion. Post-freezing recovery of hemodynamic function will be assessed by isolated working heart perfusion. Tissue damage will be assessed by enzyme leakage during reperfusion, the myocardial high energy phosphate contents, and morphologically using light and electron microscopy. This experiment will establish the minimal ice content that allows full recovery and the lethal ice content that kills the heart 2) select freezing conditions which permits about 50% return in function to study the effects of rate of freezing and thawing and the effect of modulating ice content by cryoprotectants or pro-freezing tissue dehydration on organ preservation, 3) investigate the effect of changing the extracellular milieu, i.e., the composition of the cardioplegic flush solution by optimizing the concentration of the electrolytes, pH buffer and species, osmolality and osmotic agents, and the oncotic agents, and 4) evaluate the impact of freezing on myocardial glycolytic and energy metabolism using NMR spectroscopic and radioactive tracer techniques. Understanding the cryobiology of freezing the heart-at high subzero temperatures may have wide implications. It would provide knowledge applicable to the preservation of other solid organs. This research may benefit not only the cardiac patients but other organ transplantation patients.
