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

Abstract

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.

Figure 1. Effects of FBP addition after one day of hypothermic incubation.

Cardiac myocytes were incubated at 3°C for the time indicated on the x-axis, with additions of 5 mM fructose-1,6-bisphosphate prior to the beginning of the incubation (triangles) or after one day (squares); controls (circles) had no addition. The percentage of rod-shaped cells at each time point is shown. Results are means from two experiments. Error bars indicate the individual values (in some cases the bars lie within the symbols).

Figure 2. Effects of calcium and EDTA on hypothermic preservation of cardiac myocytes.

Cells were incubated with no additions or with 100 μM calcium, 5 mM FBP, 0.5 mM EDTA, or various combinations of additions. Death rates for treated cells were normalized to those of control cells. The averages of the relative death rates for two experiments are plotted, with error bars indicating S.E.M.

Figure 3. Effects of EGTA on hypothermic preservation.

In this figure and in Figure 5, death rates for treated cells were normalized to those of control cells in the same experiment, and the relative death rates averaged over all experiments. Filled bars in these figures are for treatments without FBP, while hatched bars are for treatments including 5 mM FBP. Results are means of 4 determinations; error bars indicate S.E.M. In this figure, myocytes were incubated with the indicated concentrations of EGTA. Statistically significant by paired t-tests: **, p<0.01 vs. control; ***, p<0.005 vs. control; +, p<0.05 vs. FBP alone.

Figure 4. Effects of FBP and BDM on intracellular calcium.

Aliquots of myocytes were given no additions, 5 mM FBP, or 5 mM BDM, and analyzed for intracellular Ca2+ either immediately or after 24 hours of incubation at 3°C. Individual results are plotted, with means of the groups indicated by horizontal lines. The first two sets of data are for five experiments with fresh myocytes without (▪) or with (▴) FBP. The second two sets of data are for four experiments with myocytes incubated for one day without (▾) or with (♦) FBP. The last two sets of data are for three experiments with myocytes incubated for one day without (•) or with (□) BDM.

Figure 5. Effects of 2,3-butanedione monoxime on preservation.

Myocytes were incubated with the indicated concentrations of BDM with or without 5 mM FBP. Results are means of 3 determinations. Statistically significant by paired t-tests: ***, p<0.005 vs. control; ****, p<0.002 vs. control; +, p<0.05 vs. FBP alone.

Figure 6. Effects of BDM addition after one or two days of hypothermic incubation.

Cardiac myocytes were incubated at 3°C for the time indicated on the x-axis, with additions of 5 mM 2,3-butanedione monoxime prior to the beginning of the incubation (triangles) or after one (squares) or two (diamonds) days; controls (circles) had no addition. The percentage of rod-shaped cells at each time point is shown. Results are means from two experiments.