Figure 4. ¹³C-enrichment of metabolites in ¹³C-labeled substrate-perfused cMPC1^−/− hearts.

(a-d) Metabolic flux analysis of Langendorff hearts from 8-week-old mice perfused with 10mM [U-¹³C₆]-glucose and unlabeled substrates (0.4mM palmitate, 0.5mM lactate, 0.1mM β-HB and 0.5mM glutamine). (a-b) Total molar percentage enrichment (MPE) of all ¹³C-labeled isotopomers of tissue pyruvate, lactate, alanine, serine and citric acid cycle (CAC) intermediates were determined by GC-MS (Control, 6; cMPC1^−/−, 9). (c-f) The flux ratio of pyruvate dehydrogenase/citrate synthase (PDC/CS), pyruvate carboxylation/citrate synthase (PC/CS), PC/PDC and percentage of ¹³C-labeled citrate recycling into CAC were calculated from tissue ¹³C-MPE of isotopomers of citrate, OAA moiety of citrate (OAA^CIT), pyruvate and succinate (Control, 6; cMPC1^−/−, 9). (g) Fractional enrichment (FE) analysis of ¹³C-glutamate performed on neutralized acid extracts of Langendorff hearts perfused with 10mM [1,6-¹³C₂]-glucose, 0.5mM [3-¹³C]-pyruvate and 1mM [3-¹³C]-lactate, in addition to 0.4mM unlabeled palmitate (Control, 7; cMPC1^−/−, 8). (h) Ratio of anaplerosis to citrate synthase flux (y) was determined in neutralized acid extracts of Langendorff hearts perfused with 0.4mM U-¹³C-palmitate, in addition to unlabeled substrates (10mM glucose, 0.5mM pyruvate and 1mM lactate) (Control, 9; cMPC1^−/−, 4). (i) Langendorff perfusion was performed with 0.5mM [2,4-¹³C₂]-β-HB (β-hydroxybutyrate) and unlabeled substrates (10mM glucose, 0.4mM palmitate, 0.5mM pyruvate and 1mM lactate) (Control, 3; cMPC1^−/−, 5). Neutralized acid extracts were analyzed by NMR to determine fractional ¹³C-enrichment of acetyl-CoA entering the CAC from oxidation of ¹³C-β-HB, (Fc_{β-HB→Acetyl-CoA}). Data are presented as mean ± SEM and P values were determined by two-tailed unpaired Student’s t-test.