Purpose A limitation of heart transplantation is the small time period during which a heart remains viable while it is being transported on ice. Some studies have shown that fructose-1,6-bisphosphate (FBP), a glycolytic intermediate, can help preserve heart tissue while it is in this hypothermic environment. The purpose of the research project was to better understand the mechanism of this enhanced preservation and to explore other treatments that may extend heart viability during hypothermia. FBP's protective effect may come from its chelation of calcium ions and/or by providing a glycolytic intermediate that is already phosphorylated and thus is readily used to produce ATP.
Methods Using myocytes prepared from rat heart, we measured cytosolic Ca2+ fluorometrically using fura-2 AM. The leakiness of the plasma membrane, an indication of necrosis, was examined by measuring extracellular lactate dehydrogenase (LDH). To assess mitochondrial membrane leakage, we measured the penetration of nitroblue tetrazolium and its reduction via succinate dehydrogenase. Using an experimental model in which cell morphology was observed as a measure of cell survival during hypothermic incubation, three possible preservatives were tested.
Results For freshly-prepared myocytes, Ca2+ was 33 ± 7% (n = 5) lower for FBP-treated than for control cells; after 24 h of hypothermic incubation it was 70 ± 11% (n = 4) lower in FBP-treated samples. LDH release after 24 h was 25% lower for FBP-treated cells than for control cells. In initial experiments, FBP had little or no effect on the mitochondrial membrane leakage. The myosin ATPase inhibitor 2,3-butanedione monoxime (BDM) at 5 mM reduced the death rate by 70%, while the combination of BDM with FBP decreased it by 90%. Two potential treatments for increasing intracellular adenine nucleotide levels, nitrobenzylthioinosine (NBTI), a nucleoside transport inhibitor, and adenosine, an adenine nucleotide precursor, showed no significant protective effects.
Conclusions The results indicate that FBP exerts at least some of its effects via lowering cytosolic calcium, and that it helps preserve plasma membrane but not mitochondrial membrane integrity. The survival studies indicate that BDM, but not NBTI or adenosine, may be a useful addition to FBP in enhancing heart preservation. Supported by NIH HL-64186 and T35HL071486.
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