31P NMR studies of the intact perfused rat heart: a novel analytical approach for determining functional-metabolic correlates, temporal relationships, and intracellular actions of cardiotoxic chemicals nondestructively in an intact organ model.

Intact hearts isolated from adult male, Sprague-Dawley rats were perfused under standardized conditions in an apparatus designed for use in a high-resolution nuclear magnetic resonance (NMR) spectrometer system. Myocardial phosphate metabolite concentrations (ATP, PCr, Pi, and phosphomonoesters) and intracellular pH were determined sequentially at timed intervals coincident with the functional assessments of the intact heart by phosphorus-31 (31P) NMR spectroscopic methods. Myocardial functional and metabolic parameters were unaffected by sustained control perfusion (2 hr). The negative inotropic actions of cadmium were associated with significant changes in the chemical environment of inorganic phosphate (Pi) within the cells. This initial cellular response to cadmium, which correlated with the onset and magnitude of the contractile disturbances, appeared to represent the formation of an acidic, intracellular Pi pool (pH, 6.0). This pH compartment reached a steady state during the period in which maximal changes in contractile function were manifested, and before cellular ATP and PCr concentrations were altered. These findings are consistent with the interpretation that the functional deficits caused by cadmium originated primarily from changes in the chemical environment experienced by intracellular metabolites, rather than changes in the amounts of cellular high energy substrates. In contrast, the time-dependent negative inotropic effects of arsenate were proportional to the loss of cellular ATP stores. Intracellular pH was not affected in these hearts. A distinctive metabolic finding associated with the cardiotoxicity of arsenate was the time-dependent accumulation of previously undetected phosphate metabolites in the arsenate-treated hearts. Efforts to chemically identify these metabolites proved inconclusive; however, existing evidence suggests the possibility that these phosphorus-containing compounds may be arsenophosphate derivatives of naturally occurring cellular metabolites. The present findings provide experimental evidence demonstrating that toxicologic assessments in an intact organ model are feasible using whole organ 31P NMR spectroscopic methods and that meaningful, new insights regarding the biochemical mechanisms responsible for the cardiotoxic actions of xenobiotic agents can be obtained by this analytical approach.

Cardiotoxicity of lead at various perfusate calcium concentrations: functional and metabolic responses of the perfused rat heart.

Equilibrated rat hearts were perfused for 60 min with a standard crystalloid buffer containing either 0.9, 1.8, 3.5, or 5.0 mM Ca with or without added lead (0.3 and 30 microM). Contractile tension (T), rate of tension development (dT/dt), electrocardiographic (EKG), His bundle electrographic (HBE) indices, heart rate (HR), preejection period (PEP), and coronary flow rate (CFR) were recorded as a function of perfusion time. Endpoint analyses of myocardial phosphatic metabolites were performed on heart perchloric acid extracts by standard phosphorus-31 nuclear magnetic resonance (P-31 NMR) spectroscopic techniques. The contractile activity and glycerol 3-phosphorylcholine content of the myocardium were found to vary directly as a function of the perfusate calcium concentration; however, except for a prolonged atrioventricular-His bundle conduction time detected in hearts treated with 0.9 mM Ca, the variable perfusate calcium concentrations were devoid of any other significant physiologic and metabolic effects. In contrast, perfusion of control equilibrated hearts with 30 microM lead significantly attenuated the positive, and exacerbated the negative, inotropic responses to elevated, and low perfusate calcium concentrations, respectively. Moreover, a secondary, time-dependent decline in myocardial contractile strength was also observed in response to this lead concentration, which was progressively more pronounced with each increment in the perfusate calcium concentration. The preejection period of ventricular systole was also prolonged in response to 30 microM lead; however, this effect was less pronounced at higher perfusate calcium concentrations. Hearts perfused with 30 microM lead were also characterized by significant prolongation in atrioventricular node and His bundle conduction time, reduced coronary flow rate, and decreased heart rate, irrespective of the perfusate calcium concentration. Hearts treated with 0.3 microM lead exhibited functional properties that were diminished, but still comparable to control hearts. Analysis of myocardial phosphatic metabolite amounts following 60 min of perfusion revealed a significant lead-induced reduction in the energy status of the heart. The combination of 5.0 mM calcium with either 0.3 or 30 microM lead resulted in significant disturbances in phosphoglyceride, glycolytic, and high-energy phosphate pathways. These findings suggest that the cardiotoxic actions of lead are linked to complex mechanisms that are partially related to an interference with calcium-dependent cellular processes.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium-dependent effects of cadmium on energy metabolism and function of perfused rat heart.

Postequilibrated isolated rat hearts were perfused for 60 min with a standard supporting electrolyte buffer containing one of the following calcium concentrations: 0.9, 1.8, 3.5, or 5.0 mM, either with or without added cadmium. Doses of cadmium which proved to be minimally (0.03 microM Cd)--and maximally (3.0 microM Cd)--effective at 0.9 mM Ca were studied at all other calcium concentrations. A dose-dependent positive inotropy that persisted throughout the 60-min perfusion period was induced by the graded increases in the perfusate calcium concentration throughout the range from 0.9 to 5.0 mM. Atrioventricular node conductivity was prolonged significantly in hearts perfused with 0.9 mM Ca as compared to hearts perfused with higher calcium concentrations. Increasing the perfusate calcium concentration caused a dose-dependent increase in heart glycerol 3-phosphorylcholine (GPC) content. The other measured phosphatic metabolites of the heart were not altered significantly by varying the perfusate calcium level. In contrast, cadmium (3.0 microM Cd) induced extensive functional and metabolic aberrations which varied in magnitude as an inverse function of the perfusate calcium concentration. Contractile tension, rate of tension development (dT/dt), heart rate, coronary flow rate, and atrioventricular node conductivity were decreased significantly in response to cadmium perfusion. Moreover, these hearts characteristically had significantly elevated low energy phosphate (inosine monophosphate and inorganic phosphate) and decreased high energy phosphate (ATP, PCr) levels relative to their respective calcium controls. Furthermore, various phosphorylated intermediates of glycolysis (glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate), as well as glycerol 3-phosphate, and uridine diphosphoglucose accumulated significantly in hearts perfused with cadmium at certain calcium concentrations below 5.0 mM. The calcium-activated increase in heart GPC was inhibited completely by 3 microM cadmium. At the minimally effective dose of cadmium (0.03 microM), demonstrable changes were apparent only at the lowest perfusate calcium concentration examined (0.9 mM). These findings are consistent with the hypothesis that cadmium interferes with calcium-activated and calcium-mediated physiologic and biochemical processes of the mammalian heart. The primary mechanistic basis for the action of cadmium appears to be linked to a competition with calcium for membrane and possibly intracellular binding and activation sites.