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 13C-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 principle 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.

Potential mechanisms linking SIRT activity and hypoxic 2-hydroxyglutarate generation: no role for direct enzyme (de)acetylation

“…As shown in Figures 4B/C, Sirt3 −/− hearts exhibited the expected hyper-acetylation of cardiac mitochondrial proteins (vs. wild type, WT), but surprisingly this was accompanied by a 3.5-fold elevation in 2-HG. Such a result contrasts with the situation in both IPC [14] and cellular anoxia (Figure 1A), wherein deacetylation is associated with elevated 2-HG. Together these data support a disconnect between acetylation and 2-HG generation.…”

“…We previously demonstrated a role for SIRT1 in the elevation of 2-HG observed in cardiac ischemic preconditioning (IPC) [14]. In addition, 2-HG is elevated in cells exposed to hypoxia [3,4].…”

“…Finally, to uncover the basis for Sp sensitivity in the hypoxic increase in 2-HG ([14] and Figure 1A), we measured the effects of Sp on ischemia-induced acidification of primary mouse cardiomyocytes. As shown in Figures 4G and 4H, Sp elicited a slight alkalinization alone and blunted the acidification caused by ischemia.…”

“…Previously we demonstrated that an elevation in 2-HG levels observed in cardiac ischemic preconditioning (IPC) was associated with lysine deacetylation [14,15], and we and others showed that IPC-induced deacetylation occurs on LDH-A, IDH2 and MDH2 [15–17]. Intriguingly, both lysine deacetylation and the elevation in 2-HG seen in IPC were inhibited by the SIRT1 inhibitor splitomicin (Sp) [14,15]. As such, we hypothesized herein that the 2-HG generating capacity of these dehydrogenases may be regulated by lysine acetylation status.…”

Metabolomics reveals critical adrenergic regulatory checkpoints in glycolysis and pentose–phosphate pathways in embryonic heart

“…Samples were prepared as previously described (73). Briefly, metabolites were serially extracted from tissue powder of each heart in 80% methanol (1 then 0.5 mL) and extracts were dried under nitrogen gas and stored at -80º C. Samples were suspended in 120 μL of 50% methanol and, for amino acid detection, were derivatized with 2% (v/v) triethylamine and benzyl chloroformate.…”