Metabolomic profiling of the heart during acute ischemic preconditioning reveals a role for SIRT1 in rapid cardioprotective metabolic adaptation

Abstract

Ischemic preconditioning (IPC) protects tissues such as the heart from prolonged ischemia-reperfusion (IR) injury. We previously showed that the lysine deacetylase SIRT1 is required for acute IPC, and has numerous metabolic targets. While it is known that metabolism is altered during IPC, the underlying metabolic regulatory mechanisms are unknown, including the relative importance of SIRT1. Thus, we sought to test the hypothesis that some of the metabolic adaptations that occur in IPC may require SIRT1 as a regulatory mediator. Using both ex-vivo-perfused and in-vivo mouse hearts, LC-MS/MS based metabolomics and (13)C-labeled substrate tracing, we found that acute IPC altered several metabolic pathways including: (i) stimulation of glycolysis, (ii) increased synthesis of glycogen and several amino acids, (iii) increased reduced glutathione levels, (iv) elevation in the oncometabolite 2-hydroxyglutarate, and (v) inhibition of fatty-acid dependent respiration. The majority (83%) of metabolic alterations induced by IPC were ablated when SIRT1 was acutely inhibited with splitomicin, and a principal component analysis revealed that metabolic changes in response to IPC were fundamentally different in nature when SIRT1 was inhibited. Furthermore, the protective benefit of IPC was abrogated by eliminating glucose from perfusion media while sustaining normal cardiac function by burning fat, thus indicating that glucose dependency is required for acute IPC. Together, these data suggest that SIRT1 signaling is required for rapid cardioprotective metabolic adaptation in acute IPC.

Keywords: Fatty acids; Glucose; Ischemia; Preconditioning; Reperfusion; Sirtuin.

Figure 1. In-vivo vs. ex-vivo metabolomics

Whole hearts were rapidly sampled for metabolomic analysis as described in the methods, with starting material comprising either in-vivo hearts, or isolated hearts perfused with Krebs Henseleit buffer containing 5 mM glucose and 100 μM palmitate-BSA. Graph shows log-transformed steady-state metabolite levels, with each point representing a single metabolite. Data are means of 7 (in-vivo) or 8 (ex-vivo) independent experiments. The highlighted red point is di-P-glycerate, a contaminant from blood (see text). All data excluding this point were fitted with a linear regression shown on the graph (r² for all data including di-P-glycerate was 0.745).

Figure 2. Steady-state metabolomics of IPC

Schematics of perfusion protocols for steady-state metabolomics studies are presented in Fig. S1A. Metabolites are binned by pathway. Levels of each metabolite in the IPC condition are normalized to the same metabolite in the control condition. Ctrl = 1 (horizontal dotted line). Data are means ± SEM, n=8, *p<0.05 vs. Ctrl (paired t-test). Original data are in Table S1.

Figure 3. Fractional carbon saturation (F-SAT) of glycolytic intermediates originating from U¹³C-labeled substrates; effect of IPC ±SIRT1 inhibition

Following control perfusion or IPC, ± splitomicin (Sp), hearts were perfused for 5 min. with U¹³C-labeled glucose or palmitate (see methods and Fig. S1A), with the corresponding non-labeled partner substrates (glucose or palmitate) also present. Graphs show F-SAT, i.e., the fraction of a metabolite that became occupied by ¹³C from the labeled substrate within 5 min. (A): Schematic presentation of glycolysis and associated pathways. (B): F-SAT of metabolites originating from U¹³C-glucose (blue) or U¹³C-palmitate (red), after control perfusion (pale colors) or IPC (dark colors). (C): F-SAT of metabolites originating from U¹³C-glucose (green) or U¹³C-palmitate (brown) after control perfusion (pale colors) or IPC (dark colors), all in the presence of the SIRT1 inhibitor splitomicin (Sp). All data are means ± SEM, n=4–6, *p<0.05 between Ctrl. and IPC groups (ANOVA). ND = not detectable. Full data set in Table S2.

Figure 4. Fractional carbon saturation (F-SAT) of TCA cycle intermediates originating from U¹³C-labeled substrates; effect of IPC ±SIRT1 inhibition

Following control perfusion or IPC, ± splitomicin (Sp), hearts were perfused for 5 min. with U¹³C-labeled glucose or palmitate (see methods), with the corresponding non-labeled partner substrates (glucose or palmitate) also present. Graphs show F-SAT, i.e., the fraction of a metabolite that became occupied by ¹³C from the labeled substrate within 5 min. (A): Schematic presentation of TCA cycle and related pathways. ETFQOR = electron transfer flavoprotein quinone oxido-reductase of FAO. (B): F-SAT of metabolites originating from U¹³C-glucose (blue) or U¹³C-palmitate (red), after control perfusion (pale colors) or IPC (dark colors). (C): F-SAT of metabolites originating from U¹³C-glucose (green) or U¹³C-palmitate (brown) under control perfusion (pale colors) or IPC (dark colors), all in the presence of the SIRT1 inhibitor splitomicin (Sp). All data are means ± SEM, n=6, *p<0.05 between Ctrl. and IPC groups (ANOVA). ND = not detectable. Full data set in Table S2.

Figure 5. Simulated cardiomyocyte IR injury (sIR) and simulated IPC (sIPC) in seahorse XF

(A): Schematic showing treatment and measurement protocol for adult mouse cardiomyocytes. OCR = time point at which oxygen consumption rate was measured. (B): Cell death (LDH release) following sIR with or without prior sIPC. Data are means ± SEM, n=4, *p<0.05 vs. sIR (paired t-test). (C): Oxygen consumption rate (OCR) in cardiomyocytes at baseline and after sIPC. Cells were incubated with either glucose, palmitate or both substrates. Data are means ± SEM, n=6, *p<0.05 vs. glucose (ANOVA), #p<0.05 vs. corresponding baseline (ANOVA).

Figure 6. Steady state metabolomics of IPC with SIRT1 inhibition

Hearts were subjected to control perfusion or IPC, in the presence of 10 μM splitomicin (Sp). The subset of metabolites that were significantly altered in IPC alone (Fig. 2) are shown as circles, using the same color scheme as Fig. 2 (expressed relative to control, dotted line = 1). N.B. symbols to indicate significance in this IPC alone group (circles) are removed for clarity, since all are significant. The effect of IPC on these metabolites in the presence of splitomicin is shown in corresponding triangles, relative to control perfusion with splitomicin alone (no IPC, dotted line = 1). Data are means ± SEM, n=8. *p<0.05 (paired t-test) for IPC with splitomicin, vs. control perfusion with splitomicin (i.e. triangles).

Figure 7. IR and IPC in the absence of glucose

Isolated hearts were perfused with buffer supplemented with 100 μM palmitate-BSA alone (no glucose), and were subjected to IR injury as described in the methods. Optionally, hearts were also subjected to IPC prior to IR injury. Schematics of perfusion protocols for ischemia reperfusion injury and ischemic preconditioning are presented in Fig. S1B. (A): Cardiac function (rate pressure product) in IR and IPC+IR hearts. Data are means ± SEM. (B): Myocardial infarct, plotted as individual points (left) and means ± SEM (right). Images inset to the graph show representative TTC-stained cardiac cross-section slices (white = infarct, red = live myocardium). Data are means ± SEM, n=7. No significant differences were observed between IR and IPC groups.