Enhanced Glycolytic Metabolism Contributes to Cardiac Dysfunction in Polymicrobial Sepsis

Abstract

Background: Cardiac dysfunction is present in >40% of sepsis patients and is associated with mortality rates of up to 70%. Recent evidence suggests that glycolytic metabolism plays a critical role in host defense and inflammation. Activation of Toll-like receptors on immune cells can enhance glycolytic metabolism. This study investigated whether modulation of glycolysis by inhibition of hexokinase will be beneficial to septic cardiomyopathy.

Methods: Male C57B6/J mice were treated with a hexokinase inhibitor (2-deoxy-d-glucose [2-DG], 0.25-2 g/kg, n = 6-8) before cecal ligation and puncture (CLP) induced sepsis. Untreated septic mice served as control. Sham surgically operated mice treated with or without the 2-DG inhibitor served as sham controls. Cardiac function was assessed 6 hours after CLP sepsis by echocardiography. Serum was harvested for measurement of inflammatory cytokines and lactate.

Results: Sepsis-induced cardiac dysfunction was significantly attenuated by administration of 2-DG. Ejection fraction and fractional shortening in 2-DG-treated septic mice were significantly (P < .05) greater than in untreated CLP mice. 2-DG administration also significantly improved survival outcome, reduced kidney and liver injury, attenuated sepsis-increased serum levels of tumor necrosis factor α and interleukin 1β as well as lactate, and enhanced the expression of Sirt1 and Sirt3 in the myocardium, which play an important role in mitochondrial function and metabolism. In addition, 2-DG administration suppresses sepsis-increased expression of apoptotic inducers Bak and Bax as well as JNK phosphorylation in the myocardium.

Conclusions: Glycolytic metabolism plays an important role in mediating sepsis-induced septic cardiomyopathy. The mechanisms may involve regulation of inflammatory response and apoptotic signaling.

Keywords: 2-deoxy-D-glucose; cardiomyopathy; glycolysis; inflammatory responses.; sepsis.

Figure 1.

Inhibition of hexokinase attenuates sepsis-induced cardiac dysfunction. Mice were treated with a single dose of 2-deoxy-d-glucose (2-DG) (2 g/kg) 3 hours prior to cecal ligation and puncture (CLP). Cardiac function was examined by echocardiography 6 hours after CLP. A, Ejection fraction (EF%). B, Fractional shortening (FS%). C and D, 2-DG administration at 0.5 g/kg body weight also significantly attenuated CLP sepsis-induced cardiac dysfunction. E and F, 2-DG treatment at 0.25 and 1 g/kg also significantly improves cardiac function. n = 6–8/group. *P < .05, **P < .01, and ***P < .001 compared with indicated groups.

Figure 2.

Administration of 2-deoxy-d-glucose (2-DG) attenuates sepsis-induced organ injury and improves survival outcome. Mice were treated with 2-DG (0.25 g/kg body weight) 3 hours before induction of cecal ligation and puncture (CLP) sepsis. Serum levels of aspartate aminotransferase (AST; A), creatinine (B), and creatine kinase (C) were measured by commercially available enzyme-linked immunosorbent assay kits. D–F, 2-DG administration improves survival outcome. Mice were treated with 2-DG (2 g/kg body weight [D] or 0.25 g/kg body weight [E]) 3 hours prior to CLP and were then re-treated with 2-DG (500 mg/kg) 24 hours after CLP. D and E, Acute CLP sepsis. F, Chronic CLP septic mice treated with 2-DG at 0.5 kg/kg body weight 3 hours prior to CLP. The animals were monitored for lethality every 2 hours for up to 250 hours following CLP. n = 10–18/group. *P < .05, **P < .01, and ***P < .001 compared with indicated groups.

Figure 3.

Administration of 2-deoxy-d-glucose (2-DG) attenuates sepsis-induced accumulation of immune cells and prevents intercellular cell adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression in the myocardium. A, Eight hours after cecal ligation and puncture (CLP)–induced sepsis, hearts were harvested and sectioned for immunostaining of immune cells with specific antibody NIMP-R1411b, which can recognize Ly-6G and Ly-6C neutrophils, monocytes, and macrophages. 2-DG prevents sepsis-induced increases in the expression of ICAM-1 (B) and VCAM-1 (C). n = 4–6 per group. 2-DG treatment attenuated lipopolysaccharide (LPS)–induced ICAM-1 (D) and VCAM-1 (E) expression in endothelial cells. Human umbilical vein endothelial cells were treated with LPS (1 µg/mL) in the presence or absence of 2-DG. The cells were harvested 12 hours after treatment. Cellular proteins were prepared for immunoblotting. n = 8 replicates in each group. *P < .05, **P < .01, and ***P < .001 compared with indicated groups.

Figure 4.

Administration of 2-deoxy-d-glucose (2-DG) attenuates sepsis-induced lactate and inflammatory cytokine production. Serum was harvested 8 hours after cecal ligation and puncture (CLP) sepsis. The levels of lactate (A), tumor necrosis factor (TNF-α; B), interleukin 1β (IL-1β; C), and interleukin 6 (IL-6; D) were analyzed with enzyme-linked immunosorbent assay. Administration of 2-DG at 0.25 or 1 g/kg body weight also significantly reduced serum lactate (E), TNF-α (F), and IL-1β (G). 2-DG (0.25 g/kg) attenuated sepsis-induced increases in the nuclear hypoxia-inducible factor (HIF-1α) levels (H). CLP sepsis did not significantly alter cytosolic HIF-1α levels in the myocardium (I). n = 4–6/group. *P < .05, **P < .01, and ***P < .001 compared with indicated groups.

Figure 5.

Administration of 2-deoxy-d-glucose (2-DG) attenuates sepsis-induced myocardial apoptosis. A, Hearts were harvested 8 hours after cecal ligation and puncture (CLP) sepsis and sectioned for TUNEL assay. B, Myocardial caspase-3/7 activity was analyzed by enzyme-linked immunosorbent assay. 2-DG treatment prevents sepsis-induced Bak (C) and Bax (D) expression in the myocardium. 2-DG treatment prevents lipopolysaccharide (LPS)–induced Bak (E) and Bax (F) expression in endothelial cells. n = 8 replicates/group. 2-DG treatment prevents sepsis-induced JNK phosphorylation in the myocardium (G; n = 4–6/group) and LPS-increased JNK phosphorylation in the endothelial cells (H). Human umbilical vein endothelial cells were treated with lipopolysaccharide (1 µg/mL) in the presence or absence of 2-DG. The cells were harvested 12 hours after treatment. Cellular proteins were prepared for immunoblotting. n = 8 replicates/group. *P < .05, **P < .01, and ***P < .001 compared with indicated groups.

Figure 6.

Administration of 2-deoxy-d-glucose (2-DG) attenuated sepsis-induced decreases in the levels of nicotinamide adenine dinucleotide (NAD⁺, oxidized form), Sirt1, and Sirt3 in the myocardium. Hearts were harvested 8 hours after cecal ligation and puncture (CLP) for preparation of cellular proteins. NAD⁺ (A) and nicotinamide adenine dinucleotide (NADH, reduced form) (B) levels were examined by commercially available kits. C, Ratio of NAD⁺/NADH. D and E, 2-DG treatment increased the levels of Sirt1 (D) and Sirt3 (E) in the myocardium. F and G, 2-DG treatment prevents MKK3 phosphorylation and expression in the myocardium. n = 4–6/group. *P < .05, **P < .01, and ***P < .001 compared with indicated groups.

Figure 7.

Two-deoxy-d-glucose (2-DG) suppresses HK2 activity, thus reducing lactate production. Abbreviations: GLUT, glucose transport protein; HK2, hexokinase 2; PFK1, phosphofrutokinase 1; PKM2, pyruvate kinase isozymes M1/2; TCA, tricarboxylic acid.