Bound inorganic phosphate and early contractile failure in global ischaemia

Inorganic phosphate (P_i) accumulates extremely rapidly in ischaemic heart muscle and intracellular binding of this metabolite may account for the precipitous loss of function seen at the onset of severe ischaemia. We have used³¹P-NMR spectroscopy to measure the free cytosolic [P_i] and chemical assay techniques to measure total tissue P_i at 0, 1, 2, 3, 5, and 12 min of complete global ischaemia in the isolated isovolumic rat heart. At zero time, the P_i assayed chemically was 30.77±5.52 mol/g dry wt (mean±SD, n=7) whilst P_i assayed by NMR was 3.39±1.21 mol/g dry wt (n=15). Thus, 27.38 mol/g dry wt of P_i was bound at a cytosolic [P_i] of 0.82 mM. After 12 min of ischaemia, 49.88 mol/g dry wt of P_i was bound at a cytosolic [P_i] of 4.11 mM. When all data were fitted, using a non-linear, least squares fit (p<0.05), to the binding isotherm: Bound P_i=B_max. [P_i]/(K_d+[P_i]), the apparent binding parameters K_d and B_max were estimated to be 1.1±0.6 mM and 64.0±10.2 mol/g dry wt respectively. During the first minute of global ischaemia when the rate-pressure product had decreased by 79% of its pre-ischaemic value, bound P_i had increased by 58% and free cytosolic [P_i] by 162%. When functional and metabolite changes were expressed as a fraction of the total change which occurred during the 12-min ischaemic period, bound P_i had the profile most similar to the rate-pressure product. Both the amount of bound P_i and free cytosolic [P_i] correlated with loss of contractile function as the ischaemic period progressed. The results snow that during ischaemia, P_i is bound progressively as free cytosolic [P_i] is increased as the result of high energy phosphate hydrolysis. While these results are consistent with the possibility that P_i binding may contribute to ischaemic contractile failure, no molecular explanation for the possible effect of bound P_i on contraction has been propsed.