Acetoacetate, when present as the only fuel for respiration in rat hearts, causes an impairment in contractile function that is reversible with the addition of substrates that can contribute to anaplerosis. To determine the importance of pyruvate carboxylation via NADP(+)-dependent malic enzyme on metabolism and function in hearts oxidizing acetoacetate, isolated working rat hearts were perfused with [1-14C]pyruvate and acetoacetate. While the cardiac power output after 60 min of perfusion in hearts utilizing acetoacetate alone had fallen to 44% of the initial value, the addition of pyruvate resulted in a stable performance with no fall in the work output. When hydroxymalonate, an inhibitor of NADP(+)-dependent malic enzyme and malate dehydrogenase, was added to the two substrates, function at 60 min was similar to the value for hearts oxidizing acetoacetate alone. Measurements of the specific activities of malate, aspartate, and citrate confirm inhibition of both pyruvate carboxylation and malate oxidation. The findings are consistent with a mechanism in which the enrichment of malate by pyruvate improves function by increasing the production of reducing equivalents by the malate dehydrogenase and the isocitrate dehydrogenase reactions increase flux through the span of the tricarboxylic acid cycle from malate to 2-oxoglutarate. The present study demonstrates the physiological importance of anaplerotic pathways in maintaining contractile function in the heart.

Prior evidence has suggested that propionyl-L-carnitine improves function in ischemic hearts by providing carnitine for dissipation of acyl-CoA derivatives and propionate for enrichment of the citric acid cycle. Because contractile failure in hearts oxidizing ketone bodies is due to sequestration of free coenzyme A, which can be reversed by the addition of anaplerotic substrates that enrich the citric acid cycle, experiments were performed to determine whether the addition of propionyl-L-carnitine (2 mM) can improve performance in working rat hearts utilizing acetoacetate (7.5 mM). Whereas the addition of propionyl-L-carnitine to acetoacetate resulted in a sustained improvement in the work output of the heart, the addition of propionate (2 mM) or L-carnitine (2 mM) alone to acetoacetate had negligible effects on contractile function. Propionyl-L-carnitine increased the uptake of acetoacetate by 130%, whereas beta-hydroxybutyrate release was minimal and unchanged compared with other groups. These observations show that rates of acetoacetate oxidation are increased commensurate with increased contractile function. Tissue metabolite data indicate that the utilization of propionyl-L-carnitine did not lead to accumulation of citric acid cycle intermediates in the span from citrate to 2-oxoglutarate but to an increase in the tissue content of malate. The results show that addition of propionyl-L-carnitine in hearts oxidizing acetoacetate results in improved mechanical performance that is comparable to the mechanical performance of hearts perfused with glucose as the only substrate. This improvement is most likely conferred by anaplerosis, as suggested by enhanced rates of acetoacetate utilization and citric acid flux.