It should be noted that Hassinen et al. reported metabolic effects of FBP in perfused hearts that were not produced by EGTA and thus did not appear to be due to calcium chelation. Effects on basal levels were not reported. While the authors suggested that preservation of ATP was responsible for the FBP effect, they also suggested that Ca2+ chelation could be involved. Cavallini et al. showed that FBP inhibited the thrombin-induced increase in cytosolic Ca2+ in platelets, though it did not appear that there was any effect on the resting Ca2+ level. The authors proposed effects on ‘‘the transmission of signal at the level of the receptor-G-protein-phospholipase C system.’’ Tamaki et al. reported that FBP inhibited the increase in cytosolic Ca2+ in response to phorbol ester treatment of Kupffer cells. The authors proposed that the effects came about via both chelation of extracellular Ca2+ and by provision of glycolytic ATP, allowing greater Ca2+-ATPase activity. Two of the studies that looked at intracellular Ca2+ concerned neurons, and found increases in cytosolic Ca2+ in response to FBP treatment, in contrast to our observations. However, in Ref., FBP, while it increased basal Ca2+ levels, prevented the increase in Ca2+ in response to hypoxia. In another study, using synaptosomes rather than intact cells, Zeng et al. showed that FBP reduced Ginsenoside-Rb2 level during ischemic conditions. They proposed metabolic effects of FBP and did not address the possible role of chelation. Thus, our studies add to the weight of evidence concerning a role for Ca2+ in protective effects of FBP, and provide the first experimental evidence related to an effect of FBP on Pseudoginsenoside-F11 homeostasis in heart preservation. Possible further experiments that would help confirm our hypothesis would include determining the effect of FBP on survival under conditions in which extracellular Ca2+ is fixed, as well as measurements of intracellular Ca2+ for EGTA-treated myocytes. Determining the combined effect of EGTA and FBP on intracellular Ca2+ would help establish whether or not FBP acts by multiple mechanisms. These include effects on calcium fluxes at low concentrations and inhibition of myosin ATPase at somewhat higher concentrations. Beneficial effects of BDM in the preparation of cardiac myocytes have been characterized, and the compound is used in the procedure we employed. Several studies have found benefits in preservation of the intact heart. However, at high concentrations, BDM can also have deleterious effects, possibly through action as a phosphatase. However, the effect was much lower than that of FBP. Possibly this is because the effect of BDM on calcium is indirect; by inhibiting myosin ATPase and preserving ATP, it enables the cells to maintain calcium pumping activity better than untreated cells. Because they have different modes of action, the combination of FBP and BDM may have benefits beyond either alone, as indicated by the furthest right hatched bar in Fig. 5. Pyruvate is employed during the procedure we used for the preparation of cardiac myocytes. The general metabolic benefits of pyruvate in the heart have been reviewed by Mallet. Most studies of pyruvate’s effects on the heart have examined periods of reperfusion after ischemia, rather than effects during cold storage. We found that pyruvate produced, at best, relatively small decreases in the death rate of myocytes incubated at 3uC. When combined with 5 mM FBP, effects were no greater than those of FBP alone. While we hypothesized that there might be sufficient residual oxygen in the ischemic cell suspensions to support pyruvate oxidation, the results suggest that either such metabolism is relatively small, or it provides little survival advantage to the myocytes. This is consistent with our previous finding that dichloroacetate, which stimulates pyruvate dehydrogenase, produced no beneficial effects under these conditions.