Temporal changes in glucose metabolism reflect polarization in resident and monocyte-derived macrophages after myocardial infarction

Abstract

Introduction: Metabolic reprogramming from glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle and oxidative phosphorylation may mediate macrophage polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype. We hypothesized that changes in cardiac macrophage glucose metabolism would reflect polarization status after myocardial infarction (MI), ranging from the early inflammatory phase to the later wound healing phase.

Methods: MI was induced by permanent ligation of the left coronary artery in adult male C57BL/6J mice for 1 (D1), 3 (D3), or 7 (D7) days. Infarct macrophages were subjected to metabolic flux analysis or gene expression analysis. Monocyte versus resident cardiac macrophage metabolism was assessed using mice lacking the Ccr2 gene (CCR2 KO).

Results: By flow cytometry and RT-PCR, D1 macrophages exhibited an M1 phenotype while D7 macrophages exhibited an M2 phenotype. Macrophage glycolysis (extracellular acidification rate) was increased at D1 and D3, returning to basal levels at D7. Glucose oxidation (oxygen consumption rate) was decreased at D3, returning to basal levels at D7. At D1, glycolytic genes were elevated (Gapdh, Ldha, Pkm2), while TCA cycle genes were elevated at D3 (Idh1 and Idh2) and D7 (Pdha1, Idh1/2, Sdha/b). Surprisingly, Slc2a1 and Hk1/2 were increased at D7, as well as pentose phosphate pathway (PPP) genes (G6pdx, G6pd2, Pgd, Rpia, Taldo1), indicating increased PPP activity. Macrophages from CCR2 KO mice showed decreased glycolysis and increased glucose oxidation at D3, and decreases in Ldha and Pkm2 expression. Administration of dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, robustly decreased pyruvate dehydrogenase phosphorylation in the non-infarcted remote zone, but did not affect macrophage phenotype or metabolism in the infarct zone.

Discussion: Our results indicate that changes in glucose metabolism and the PPP underlie macrophage polarization following MI, and that metabolic reprogramming is a key feature of monocyte-derived but not resident macrophages.

Keywords: glycolysis; heart failure; immunometabolism; inflammation; macrophage.

Figure 1

Flow cytometry assessment of cardiac immune cells after MI. (A) Representative flow cytometry plots of cardiac immune cells at day 0, day 1, and day 7 after MI. After gating for total live cells, myeloid cells were determined by CD45-APC and CD11b-PE-Cy7 expression. This population was then separated into neutrophils and macrophages based on Ly6G-PE, and Ly6G- macrophages were divided into CD11b + Ly6G-Ly6C^high monocytes and CD11b + Ly6G-Ly6C^low macrophages. (B) Quantification of sorted cells. *p < 0.05, n = 3–4 per group.

Figure 2

Changes in glucose metabolism in cardiac macrophages after MI. (A) Representative Seahorse plots for ECAR (top) and OCR (bottom) for glycolysis stress test. Dashed lines indicate time point at which each stimulation began. (B) Quantification of glycolysis, ECAR/OCR upon glucose exposure, glucose oxidation, and ECAR/OCR upon oligomycin exposure. ECAR—extracellular acidification rate, OCR—oxygen consumption rate. *p < 0.05, n = 6–8 per group.

Figure 3

Changes in macrophage gene expression at different time points after MI. (A) M1/M2 markers. (B) Genes relates to glycolysis. (C) Genes related to the PPP pathway. (D) Genes related to the TCA cycle. PPP—pentose phosphate pathway. TCA—tricarboxylic acid cycle. *p < 0.05, n = 5–6 per group.

Figure 4

Flow cytometry assessment of cardiac immune cells and inflammatory markers after MI in CCR2 KO mice. (A) Representative flow plots for detecting myeloid (CD45 + CD11b+), macrophages (CD11b + Ly6G-) and neutrophils (CD11b + Ly6G+), and monocytes (Ly6C^high) at day 1 after MI; and quantification of immune cell subtypes. (B) Gene expression in the LV infarct tissue at day 1 after MI. *p < 0.05, n = 3–4 per group.

Figure 5

Changes in glucose metabolism in cardiac macrophages after MI in CCR2 KO mice. (A) Left—representative Seahorse plots for ECAR and OCR in cardiac macrophages at day 3 after MI, and quantification of glycolysis, ECAR/OCR, and glucose oxidation. ECAR—extracellular acidification rate; OCR—oxygen consumption rate. (B) Inflammatory genes (Ccr2 and Il1b), glycolysis (Gapdh, Ldha, Pkm2) and TCA cycle (Sdha and Sdhb, Idh1 and Idh2) were measured. *p < 0.05, n = 3–6 per group.

Figure 6

Effects of dichloroacetate (DCA) on cardiac function after MI. (A) DCA significantly decreased inhibitory phosphorylation of PDH (S293), indicating PDH activation. (B) DCA attenuated anterior wall thinning at day 3 post-MI, but did not affect posterior wall thickness, end-diastolic diameter or volume, or ejection fraction. *p < 0.05, n = 9–10 per group.

Figure 7

Effects of DCA on post-MI inflammation and macrophage phenotype. (A) DCA did not affect immune cell infiltration into the heart as assessed by flow cytometry. (B) DCA decreased Il1b and Tnf mRNA in the LVI tissue. In LV infarct macrophages, DCA decreased Il18, and increased Ccl2. (C) DCA did not affect macrophage glycolysis or OXPHOS. *p < 0.05. n = 6–7 per group.