The Toll-like receptor 9 ligand, CpG oligodeoxynucleotide, attenuates cardiac dysfunction in polymicrobial sepsis, involving activation of both phosphoinositide 3 kinase/Akt and extracellular-signal-related kinase signaling

Abstract

Background: Toll-like receptors (TLRs) play a role in the pathophysiology of sepsis and multiple organ failure. This study examined the effect of CpG oligodeoxynucleotide (CpG-ODN), the TLR9 ligand, on polymicrobial sepsis-induced cardiac dysfunction.

Methods: Male C57BL/6 mice were treated with CpG-ODN, control CpG-ODN (control-ODN), or inhibitory CpG-ODN (iCpG-ODN) 1 hour prior to cecal ligation and puncture (CLP)-induced sepsis. Mice that underwent sham surgery served as sham controls. Cardiac function was examined by echocardiography before and 6 hours after CLP.

Results: Cardiac function was significantly decreased 6 hours after CLP. CpG-ODN prevented CLP-induced cardiac dysfunction, as evidenced by maintenance of the ejection fraction and fractional shortening. Control-ODN or iCpG-ODN did not alter CLP-induced cardiac dysfunction. CpG-ODN significantly attenuated CLP-induced myocardial apoptosis and increased myocardial Akt and extracellular-signal-related kinase (ERK) phosphorylation levels following CLP. In vitro experiments demonstrated that CpG-ODN promotes an association between TLR9 and Ras, resulting in Akt and ERK phosphorylation. Inhibition of phosphoinositide 3-kinase (PI3K) by Ly294002 or inhibition of ERK by U0126 in vivo abolished CpG-ODN attenuation of CLP-induced cardiac dysfunction.

Conclusions: CpG-ODN prevents CLP-induced cardiac dysfunction, in part through activation of PI3K/Akt and ERK signaling. Modulation of TLR9 could be an effective approach for treatment of cardiovascular dysfunction in patients with sepsis or septic shock.

Figures

Figure 1.

CpG oligodeoxynucleotide (CpG-ODN) attenuated cecal ligation and puncture (CLP)–induced myocardial apoptosis. Mice were treated with CpG-ODN, control CpG-ODN (control-ODN), and inhibitory CpG-ODN (iCpG-ODN) by intraperitoneal injection 1 hour prior to CLP (n = 6/group). Sham surgery served as a sham control (n = 6/group). Hearts were harvested, and cellular proteins were prepared. A, Myocardial apoptosis was examined by the TUNEL assay. Red arrows indicate cardiac myocyte apoptosis. Quantitative data are shown at right. B, Caspase-3 and -8 activities in the myocardium were measured by enzyme-linked immunosorbent assay kits. C, Fas and FasL levels in the myocardium were measured by Western blotting. Representative blots are shown above in graphs. *P < .05. Abbreviation: RLU, relative light units.

Figure 2.

Cecal ligation and puncture (CLP) decreased the levels of Akt and glycogen synthase kinase G β (GSK3β) phosphorylation was attenuated by CpG oligodeoxynucleotide (CpG-ODN). Mice were treated with CpG-ODN, control CpG-ODN (control-ODN), and inhibitory CpG-ODN (iCpG-ODN) by intraperitoneal injection 1 hour prior to CLP (n = 6/group). Sham surgery served as a sham control (n = 6/group). Hearts were harvested, and cellular proteins were prepared. The levels of phosphorylation of Akt (p-Akt; A) and GSK-3β (p-GSK3β; B) were examined by Western blotting. Representative blots are shown above the graph. Abbreviations: S, Sham; C, CLP; C-ODN, Control-ODN; CpG, CpG-ODN; iCpG, inhibitory CpG-ODN. *P < .05.

Figure 3.

CpG oligodeoxynucleotide (CpG-ODN) increased the levels of extracellular-signal-related kinase (ERK) phosphorylation in the myocardium following cecal ligation and puncture (CLP). Mice were treated with CpG-ODN, control CpG-ODN (control-ODN), and inhibitory CpG-ODN (iCpG-ODN) by intraperitoneal injection 1 hour prior to CLP (n = 6/group). Sham surgery served as a sham control (n = 6/group). Hearts were harvested, and cellular proteins were prepared. The levels of phosphorylated ERK (p-ERK) were examined by Western blotting. Representative blots are shown above in the graph. *P < .05.

Figure 4.

CpG oligodeoxynucleotide (CpG-ODN) increased both Akt and extracellular-signal-related kinase (ERK) phosphorylation and induced an association between Ras and Toll-like receptor 9 (TLR9) in H9C2 cells. H9C2 cells were treated with CpG-ODN or control CpG-ODN (control-ODN) at indicated times. Untreated cells served as a control. Cells were harvested, and cellular proteins were isolated for analysis of phosphorylated Akt (p-Akt; A) and phosphorylated ERK (B; p-ERK) by Western blotting. Immunoprecipitation (IP) was performed with a specific anti-Ras antibody, followed by immunoblotting (IB) with a specific anti-TLR9 antibody (C). Representative blots are shown above in the graph. There were 4 replicates for each time point. *P < .05.

Figure 5.

Phosphoinositide 3-kinase (PI3K) or extracellular-signal-related kinase (ERK) inhibition abrogated CpG oligodeoxynucleotide (CpG-ODN)–induced attenuation of cardiac dysfunction in polymicrobial sepsis. Mice were treated with the PI3K-specific inhibitor Ly294002 (LY) or the ERK-specific inhibitor U0126 (U0) 15 minutes prior to CpG-ODN administration. Cardiac function was measured by echocardiography before and 6 hours after cecal ligation and puncture. Hearts were harvested, and cellular proteins were isolated for analysis of phosphorylation of Akt (p-Akt; A) and phosphorylation of ERK (p-ERK; B) by Western blotting. Representative blots are shown above in the graph. There were 6 mice in each group. *P < .05. Abbreviations: LY+CpG, CpG-ODN+LY294002; Unt, untreated; U + CpG: CpG-ODN+U0126.