Elevation of serum sphingosine-1-phosphate attenuates impaired cardiac function in experimental sepsis

Abstract

Serum levels of the lipid mediator sphingosine-1-phosphate (S1P) are reduced in septic patients and are inversely associated with disease severity. We show that serum S1P is reduced in human sepsis and in murine models of sepsis. We then investigated whether pharmacological or genetic approaches that alter serum S1P may attenuate cardiac dysfunction and whether S1P signaling might serve as a novel theragnostic tool in sepsis. Mice were challenged with lipopolysaccharide and peptidoglycan (LPS/PepG). LPS/PepG resulted in an impaired systolic contractility and reduced serum S1P. Administration of the immunomodulator FTY720 increased serum S1P, improved impaired systolic contractility and activated the phosphoinositide 3-kinase (PI3K)-pathway in the heart. Cardioprotective effects of FTY720 were abolished following administration of a S1P receptor 2 (S1P2) antagonist or a PI3K inhibitor. Sphingosine kinase-2 deficient mice had higher endogenous S1P levels and the LPS/PepG-induced impaired systolic contractility was attenuated in comparison with wild-type mice. Cardioprotective effects of FTY720 were confirmed in polymicrobial sepsis. We show here for the first time that the impaired left ventricular systolic contractility in experimental sepsis is attenuated by FTY720. Mechanistically, our results indicate that activation of S1P2 by increased serum S1P and the subsequent activation of the PI3K-Akt survival pathway significantly contributes to the observed cardioprotective effect of FTY720.

Figure 1. Decreased S1P serum concentrations in patients with sepsis.

(a,b) S1P serum levels were assessed by LC/MS/MS in control patients (undergoing cardiac surgery prior to operation (T1 n = 11) and early postoperative (T2 n = 11) and in patients with severe sepsis/septic shock at day 1 (D1 n = 19) and day 3 (D3 n = 17) after diagnosis. (b) Septic patients were divided in 28-d survivors (D1 n = 9; D3 n = 9) and 28-d non-survivors (D1 n = 10; D3 n = 8). Data are expressed as box and whisker min to max for n number of observations. + = mean value. *P < 0.05 sepsis vs. control T1 ^#P < 0.05 sepsis vs. control T2 (Kruskall-Wallis test with Dunn´s multiple comparisons test).

Figure 2. Effect of LPS/PepG on S1P serum levels and on cardiac function in C57BL/6J wild type mice.

At 18 h after LPS/PepG or vehicle (sham) administration to 2-month-old male C57BL/6J wild type mice (a) S1P serum levels were assessed by LC/MS/MS, or (b) the percentage ejection fraction was assessed by echocardiography. The following groups were studied: sham + vehicle (n = 8); LPS/PepG + vehicle (n = 8). Data are expressed as means ± SEM, ***P = 0.005 vs. sham + vehicle, ****P = 0.0001 vs. sham + vehicle (unpaired t-test).

Figure 3. Effect of LPS/PepG co-administration and treatment with FTY720 on cardiac function and S1P serum levels in C57BL/6J wild type mice.

(a) Representative M-mode echocardiograms and legend of all groups studied. (b) Percentage ejection fraction, (c) fractional shortening (d), fractional area change were assessed via echocardiography and (e) S1P serum levels were assessed by LC/MS/MS 18 h subsequent to vehicle administration (sham) or LPS/PepG co-administration in 2-month-old male C57BL/6J wild type mice. At 1 h after LPS/PepG challenge mice were treated either with FTY720 (0.1 mg/kg) or vehicle (10% DMSO). The following groups were studied: C57BL/6J sham + vehicle (n = 8); C57BL/6J sham + FTY720 (n = 3); C57BL/6J LPS/PepG + vehicle (n = 14); C57BL/6J LPS/PepG + FTY720 (n = 15). Data are expressed as means ± SEM for n number of observations. *P < 0.05 vs. C57BL/6J LPS/PepG + vehicle (Kruskall-Wallis test with Dunn´s multiple comparisons test).

Figure 4. Effect of LPS/PepG co-administration and treatment with FTY720 on cardiac function and S1P serum levels in sphingosine kinase-2 deficient mice.

(a) Representative M-mode echocardiograms and legend of all groups studied. (b) Percentage ejection fraction, (c) fractional shortening (d), fractional area change were assessed via echocardiography and (e) S1P serum levels were assessed by LC/MS/MS 18 h subsequent to vehicle administration (sham) or LPS/PepG co-administration in 2-month-old male SPHK-2^−/− mice. At 1 h after LPS/PepG challenge mice were treated either with FTY720 (0.1 mg/kg) or vehicle (10% DMSO). The following groups were studied: SPHK-2^−/− sham + vehicle (n = 6); SPHK-2^−/− sham + FTY720 (n = 6), SPHK-2^−/− LPS/PepG + vehicle (n = 16), SPHK-2^−/− LPS/PepG + FTY720 (n = 14). Data are expressed as means ± SEM for n number of observations. *P < 0.05 vs. SPHK-2^−/− LPS/PepG + vehicle (Kruskall-Wallis test with Dunn´s multiple comparisons test).

Figure 5. Effect of LPS/PepG co-administration and treatment with FTY720 on Akt, eNOS and ERK1/2 phosphorylation in heart tissue of C57BL/6J wild type mice.

At 1 h after administration of LPS/PepG or vehicle (sham), mice were treated with FTY720 (0.1 mg/kg) or vehicle (10% DMSO). Signaling events in heart tissue were assessed at 18 h. Each immunoblot is from a single experiment and is representative of three separate experiments. All values were corrected for the corresponding β-actin band. Densitometric analysis of the bands is expressed as relative optical density (OD) of (a) phosphorylated Akt (pSer⁴⁷³) corrected for the corresponding total Akt content (Σ Akt) and normalized using the related sham band; (b) phosphorylated eNOS (pSer¹¹⁷⁷) corrected for the corresponding total eNOS content (Σ eNOS) and normalized using the related sham band and; (c) phosphorylated ERK1 (pThr²⁰²/pTyr²⁰⁴) and ERK2 (pThr¹⁸⁵/pTyr¹⁸⁷) corrected for the corresponding total ERK1 or ERK2 content (Σ ERK1 or Σ ERK2) and normalized using the related sham band. Data are expressed as mean ± SEM for n number of observations. *P < 0.05 vs. LPS/PepG + vehicle (Kruskall-Wallis test with Dunn´s multiple comparisons test).

Figure 6. Role of S1P₁ and S1P₂ on observed LPS/PepG- and FTY720-mediated effects on cardiac function in mice.

(a) S1P receptors S1P_1-3, and the activation of ERK and PI3K/Akt/eNOS through G_i coupled signaling pathways. S1P activates S1P receptors. The S1P mimetic FTY720 acts in its phosphorylated isoform as an unselective agonist on S1P₁ and S1P₃ and a selective functional antagonist on S1P₁. S1P₁ exclusively couples to G_i, while S1P₂ and S1P₃ couple to G_i, G_12/13 and G_q. Coupling of S1P receptors to G_i leads to activation of the Ras/ERK pathway and the PI3K/Akt/eNOS pathway. SEW2871: S1P₁ agonist. JTE 013: S1P₂ antagonist. LY294002: PI3K inhibitor. S1P: sphingosine-1-phosphate. FTY720-P: phosphorylated FTY720. (b) Percentage ejection fraction was assessed via echocardiography 18 h subsequent to LPS/PepG co-administration in 2-month-old male C57BL/6J mice. At 1 h after LPS/PepG challenge mice were treated either with vehicle (10% DMSO) (n = 14), FTY720 (0.1 mg/kg) (n = 15) or the selective S1P₁ agonist SEW2871 (1 mg/kg i.v.) (n = 3). Or mice received (45 min after LPS/PepG and 15 min prior to FTY720) the selective PI3K inhibitor LY294002 (0.3 mg/kg i.v.) (n = 6) or the selective S1P₂antagonist JTE 013 (1 mg/kg i.v.) (n = 6). One animal, which received JTE 013, died and was not included in the statistics. Data are expressed as means ± SEM for n number of observations. *P < 0.05 LPS/PepG + FTY720 vs. LPS/PepG + vehicle; ^#P < 0.05 vs. LPS/PepG + FTY720 (Kruskall-Wallis test with Dunn´s multiple comparisons test). (c) Summary of the experimental setup for acquisition of data provided in panel b.

Figure 7. Effect of cecal ligation and puncture and treatment with FTY720 on cardiac function in C57BL/6J wild type mice.

(a) Representative M-mode echocardiograms and legend of all groups studied. (b) Percentage ejection fraction, (c) fractional shortening (d), fractional area change were assessed via echocardiography 24 h subsequent to CLP or sham-operation in 8-month-old male C57BL/6J wild type mice. The following groups were studied: sham + vehicle (n = 8); sham + FTY720 (n = 3), CLP + vehicle (n = 8), CLP + FTY720 (n = 10). Data are expressed as means ± SEM for n number of observations. *P < 0.05 vs. CLP + vehicle (Man Whitney test, two-tailed).

Figure 8. Effect of cecal ligation and puncture (CLP) and treatment with FTY720 on Akt, eNOS and ERK1/2 phosphorylation in murine heart tissue.

At 1 h after CLP or sham operation, mice were treated with either FTY720 (0.1 mg/kg) or vehicle. Signaling events in heart tissue were assessed at 24 h. Each immunoblot is from a single experiment and is representative of three separate experiments. All values were corrected for the corresponding β-actin band. Densitometric analysis of the bands is expressed as relative optical density (OD) of (a) phosphorylated Akt (pSer⁴⁷³) corrected for the corresponding total Akt content (Σ Akt) and normalized using the related sham band; (b) phosphorylated eNOS (pSer¹¹⁷⁷) corrected for the corresponding total eNOS content (Σ eNOS) and normalized using the related sham band and; (c) phosphorylated ERK1 (pThr²⁰²/pTyr²⁰⁴) and ERK2 (pThr¹⁸⁵/pTyr¹⁸⁷) corrected for the corresponding total ERK1 or ERK2 content (ΣERK1 or Σ ERK2) and normalized using the related sham band. Data are expressed as mean ± SEM for n number of observations. *P < 0.05 vs. CLP + vehicle (Kruskall-Wallis test with Dunn´s multiple comparisons test).