Among these additives, fructose-1,6-bisphosphate, 2,3-butanedione monoxime, pyruvate, adenosine, ribose, and adenine have all been reported to have some effects. Of special interest is FBP, which has been reported to be useful in protecting a variety of tissues during ischemia and hypoxia. These include heart, liver, kidney, brain, smooth muscle, lung, and intestine. New studies concerning benefits of FBP appear every year. Our group has characterized effects of FBP in preserving heart function during hypothermic storage, and has demonstrated uptake of FBP by cardiac myocytes, even at 3uC. We also showed that in an experimental model for hypothermic heart preservation, isolated cardiac myocytes maintained in ischemic suspension at 3uC, FBP greatly reduced the death rate and helped preserve cellular ATP. In other papers concerning use of FBP with the heart,Cycloastragenol the compound has been included in the preservation solution in a study of continuous perfusion during cold storage, and in clinical trials of coronary artery bypass graft surgery. Several hypotheses have been proposed for the mechanism by which FBP protects tissues. One possibility is that FBP enters cells and is used in glycolysis, providing ATP without the necessity of the two prior ATP-consuming phosphorylation steps. Another is that FBP exerts its effects via chelation of calcium ions. Other proposals include allosteric activation of phosphofructokinase and stimulation of the pentose phosphate pathway. However, none of the proposals have been definitely established. Hassinen et al. determined a value of about 3 mM for the dissociation constant of the Ca2+-FBP complex. Thus, millimolar levels of FBP,Calycosin as used in our previous experiments, could reduce extracellular levels of Ca2+, which in turn would allow the myocytes to maintain their intracellular Ca2+ at lower levels and reduce the amount of ATP consumed by Ca2+ transport. The work described here focused on several additives that have been reported to be effective in myocardial protection. We paid special attention to the calcium chelation hypothesis for FBP effects, again using isolated cardiac myocytes as an experimental system. Our results indicate that chelation of extracellular calcium is an important potential mechanism by which FBP protectscells. We also tested whether 2,3-butanedione monoxime and pyruvate, both of which have shown protective effects with intact heart and with cardiac myocytes, would be beneficial in our experimental system. BDM was strongly protective, while pyruvate had little effect. Finally, we tested the hypothesis that adenine and ribose, either individually or in combination, could enhance the survival of the myocytes due to their ability to serve as precursors for adenine nucleotides. The results did not support this hypothesis. We observed that 5 mM FBP could protect myocytes from death during ischemic, hypothermic incubation even if added after one day or two days. This suggested that a mechanism other than metabolism of FBP might be involved in its protective effects, with chelation of extracellular calcium by FBP being a possibility. Although the myocytes are referred to as “calcium tolerant” because they survived increasing amounts of calcium during the isolation procedure, the final preparation nevertheless shows reduced viability during hypothermic incubation with added calcium. Likely this is because increasing extracellular calcium will lead to a rise in intracellular calcium. The latter can be damaging via several mechanisms, including expenditure of ATP for Ca2+ATPases, as the cells attempt to lower their cytosolic calcium levels; activation of contractile activity, also reducing ATP; and activation of Ca2+dependent proteinases.