By reducing the extracellular Ca2+ level, FBP would make it easier for cells to maintain low intracellular levels and prevent such damage. In the experiments shown in Fig. 3, the calcium-chelating agent EGTA reduced the death rate by 60–70%, similar to the effects of FBP. Thus, chelation of calcium is protective in our experimental system, even though the final medium used to wash and incubate the myocytes is nominally calcium-free. It is difficult to measure the levels of calcium in this medium. However, the results indicated that the medium likely contained micromolar levels of calcium, and that most of this probably came from the albumin. Thus, the idea of a chelating effect of extracellular FBP is reasonable. Using a dissociation constant of Mogroside-III would bind approximately 60% of the calcium in the medium. We found that there was considerable variation in the quality of myocytes prepared using different lots of albumin having the same product number. Possibly this was the result of differences in calcium content of the different batches. Evidence for effects of FBP on calcium homeostasis is shown in Fig. 4. For freshly-prepared myocytes, cytosolic calcium was an average of 33% lower in FBP-treated cells compared to control cells. For freshly-prepared cells there is expected to be little effect of FBP on the cellular ATP levels; in Ref. we observed only a 16% higher ATP level for cells treated with 5 mM FBP compared to control cells after 2 hours of hypothermic incubation. Thus, the differences for the fresh myocytes in Fig. 4 are likely direct effects of calcium chelation rather than due to increased availability of ATP for Ca2+ pumping. Our earlier experiments found that ATP levels were about 30% higher at 6 hours and 50% higher at 24 hours in FBP-treated cells. Therefore the larger reduction in calcium with 24 hours of FBP treatment may be due to both chelation of Ca2+ by FBP and the cumulative effects of maintaining higher ATP levels in the cells, which should allow them to reduce the calcium levels through Ca2+-ATPase activity. In addition to producing extracellular effects, it is possible that FBP could be taken up by the cells and chelate intracellular Ca2+. We previously showed that label from radiolabeled FBP at 5 mM could be taken up by myocytes, both at room temperature and at 3uC. However, it is likely that much of this label was converted to other metabolites, and it seems unlikely that intracellular FBP would rise to levels high enough to provide a significant chelating effect. Additional support for a chelating effect of extracellular FBP,Mogroside-IIA2 rather than an effect via glycolytic ATP production, comes from a comparison of the effects of BDM and FBP. These compounds at 5 mM had similar effects on the hypothermic survival of myocytes. However, in myocytes incubated for 24 hours, BDM produced a much smaller reduction in free calcium than did FBP. Nevertheless, the data in Fig. 3 indicate that FBP may have protective effects beyond those due to calcium chelation. For both 0.3 and 1.0 mM EGTA, it appeared that the combination of FBP and EGTA produced greater reductions in the death rate than EGTA alone, although the differences were not statistically significant at the p,0.05 level by paired ttests. Because of the much greater affinity of EGTA for Ca2+ compared to the affinity of FBP, 5 mM FBP would not lower the Ca2+ level significantly in the presence of these levels of EGTA. We previously showed that FBP could be taken up by cardiac myocytes at 21uC and 3uC. It is possible that FBP taken up during or after the transition to hypothermia could be used to provide glycolytic ATP even at the reduced temperature. Since energyconsuming processes would also be slowed by the hypothermia, this ATP could provide significant protection against cell death. As described above, we previously demonstrated that FBP helped maintain higher levels of ATP during hypothermic incubation.