Protection of rat cardiac myocytes by fructose-1,6-bisphosphate and 2,3-butanedione.

Earlier studies by our group showed that fructose-1,6-bisphosphate (FBP) enhances the hypothermic preservation of rat cardiac myocytes and the functional recovery of animal hearts after hypothermic storage. However, the mechanisms involved were not clear. We extended the cardiomyocyte studies by testing whether the FBP effects were due to chelation of extracellular calcium, leading to lower intracellular levels. We also tested effects of 2,3-butanedione monoxime (BDM), pyruvate, and adenine nucleotide precursors. Cardiomyocytes were incubated in ischemic suspension at 3 °C, and aliquots examined over 48 to 72 hours for retention of rod-shaped morphology, a measure of viability. Cytosolic Ca(2+) levels were measured in some experiments. FBP at 5 mM reduced the death rate even when added after one or two days of incubation. It caused cytosolic calcium levels that were 33% lower than controls in freshly-isolated cells and 70% lower after one day of incubation. EGTA protected against cell death similarly to FBP. These results indicated that one of the mechanisms by which FBP exerts protective effects is through chelation of extracellular calcium. BDM was strongly protective and reduced cytosolic calcium by 30% after one day of incubation. As with FBP, BDM was effective when added after one or two days of incubation. BDM may be useful in combination with FBP in preserving heart tissue. Pyruvate, adenine, and ribose provided little or no protection during hypothermia.

Characterization of the high-affinity uptake of fructose-1,6-bisphosphate by cardiac myocytes.

Previously, we reported that fructose-1,6-bisphosphate (FBP) was taken up by rat cardiac myocytes by two processes: a component that was saturable at micromolar levels and a nonsaturable component that dominated at millimolar levels. Here, we continued to characterize the saturable high-affinity component, with the aim of identifying the physiological substrate and role for this activity. ATP, ADP, and AMP inhibited the uptake of FBP with apparent affinities of 0.2-0.5 mM. Fumarate and succinate were very weak inhibitors. Several phosphorylated sugars (ribulose-1,5-phosphate, fructose-1-phosphate, ribose-5-phosphate, and inositol-2-phosphate) inhibited FBP uptake with apparent affinities of 40-500 µM. As in our previous study, no tested compound appeared to bind as well as FBP. The data suggest that the best ligands have two phosphoryl groups separated by at least 8 Å. The rates of FBP uptake were measured from 3° to 37°. The calculated activation energy was 15-50 kJ/mol, similar to other membrane transport processes. Uptake of FBP was tested in several types of cells other than cardiac myocytes, and compared to the uptake of 2-deoxyglucose and L: -glucose. While FBP uptake in excess of that of L: -glucose was observed in some cells, in no case was the uptake as high as in cardiac myocytes. The physiological substrate and role for the high-affinity FBP uptake activity remain unknown.

Fructose-1,6-bisphosphate enhances hypothermic preservation of cardiac myocytes.

BACKGROUND: Previous studies from our project found that fructose-1,6-bisphosphate (FBP) enhanced the functional recovery of animal hearts after hypothermic preservation, and that rat cardiac myocytes take up FBP at 3 degrees C. In this study we tested the effects of FBP, as well as other compounds related to glycolysis and pyruvate oxidation, on the hypothermic preservation of myocytes. METHODS: Isolated myocytes were incubated in ischemic suspensions at 3 degrees C, and aliquots examined over 72 hours for retention of rod-shaped morphology. In some experiments adenine nucleotide levels were measured by high-performance liquid chromatography (HPLC). RESULTS: FBP at 1 to 10 mmol/liter markedly reduced the death rate (65% reduction at 5 mmol/liter). Glucose at 2 to 10 mmol/liter was less beneficial (20% reduction). Insulin increased the death rate by about 25% when present alone, and it did not enhance the beneficial effects of FBP or glucose. Dichloroacetate (DCA), which stimulates pyruvate dehydrogenase, had little effect at 0.5 to 10 mmol/liter. Glucose and DCA did not increase the beneficial effects of FBP. After 6 to 24 hours of hypothermia, FBP- and glucose-treated cells had 25% to 50% higher ATP levels and 10% to 20% higher ATP:ADP ratios than untreated cells. Effects of FBP on preservation of morphology were much greater than effects on ATP levels. CONCLUSIONS: The results suggest that the effects of FBP and glucose were through glycolytic ATP production rather than through sugar oxidation via pyruvate dehydrogenase. The divergence in effects on preservation and effects on ATP suggests a role for a sub-cellular compartment of ATP in preservation.

Permeability of fructose-1,6-bisphosphate in liposomes and cardiac myocytes.

Fructose-1,6-bisphosphate (FBP) helps preserve heart and other organs under ischemic conditions. Previous studies indicated that it can be taken up by various cell types. Here we extended observations from our group that FBP could penetrate artificial lipid bilayers and be taken up by cardiac myocytes, comparing the uptake of FBP to that of L-glucose. Using liposomes prepared by the freeze-thaw method, FBP entered about 200-fold slower than L-glucose. For liposomes of either soybean or egg lipids, 50 mM FBP enhanced the permeability of FBP itself, with little effect on general permeability (measured by uptake of L-glucose). In experiments with isolated cardiac myocytes at 21 degrees C, FBP uptake exceeded the uptake of L-glucose by several fold and appeared to equilibrate by 60 min. There was both a saturable component at micromolar levels and a nonsaturable component which dominated at millimolar levels. The saturable component was inhibited by Pi and by other phosphorylated sugars, though with lower affinity than FBP. Both saturable and nonsaturable uptakes were also observed at 3 degrees C. The results indicate that FBP enters myocytes not by simple penetration through the lipid bilayer, but via at least two distinct protein-dependent processes. The uptake could lead to intracellular effects important in hypothermic heart preservation.