Advantages of perfluorochemical perfusion in the isolated working rabbit heart preparation using ^(31)P-NMR

Abstract

Quantitative ^(31)P-NMR and enzymatic analysis of high-energy phosphates were used to characterize an isolated perfused working rabbit heart preparation. In this model, the left side of the heart works against a physiological after-load. Two perfusates, Krebs-Henseleit saline and the perfluorocarbon emulsion FC-43 (perfluorotributylamine), were evaluated in their ability to maintain cardiac function and high-energy phosphate metabolites over a period of 2–3 h. Adenine nucleotides ATP, ADP, phosphocreatine and inorganic phosphate (P_i) were measured by ^(31)P-NMR while monitoring cardiac output and coronary flow. Intracellular pH was determined using the chemical shift of P_i. At the end of each experiment, hearts were freeze clamped and enzymatically assayed for adenine nucleotides, phosphocreatine and P_i. In every experiment, hearts perfused with FC-43 emulsion maintained the same rate of cardiac output as hearts perfused with Krebs-Henseleit saline, but with half the coronary flow rate: FC-43, 22 ± 2.5 (n = 5), Krebs-Henseleit saline 42 ± 2.7 (n = 6) ml/min, P < 0.001. Hearts perfused with FC-43 emulsion showed higher [phosphocreatine] and [ATP] measured by 31P-NMR. For [phosphocreatine]: FC-43 3.2 ± 0.7 (n = 5), Krebs-Henseleit saline 1.7 ± 0.2 (n = 6) μmol/g wet wt., P < 0.01. For [ATP]: FC-43 1.8 ± 0.7 (n = 5), Krebs-Henseleit saline 0.9 ± 0.2 (n = 6) μmol/g wet wt., P < 0.02. [phosphocreatine] and [ATP] determined by ^(31)P-NMR values were identical within experimental error to those values obtained by enzymatic analysis. Comparing [P_i] determined by both methods, 36% of P_i in FC-43-perfused hearts, and only 24% of P_i in Krebs-Henseleit saline-perfused hearts were visible by NMR, indicating that a large proportion of P_i is bound in the intact functioning heart. Similar results were obtained for [ADP]. Using the combined techniques of ^(31)P-NMR and enzymatic assay, we have shown in this model of the isolated working rabbit heart preparation, that FC-43 emulsion maintains significantly better function and high-energy phosphate levels than Krebs-Henseleit saline.