2.1 Animals

All the rats were purchased from Slaccas Company (Shanghai, China) and kept in the animal facility at Tongji University. All of the procedures were approved by Institutional Animal Care and Use Committee at Tongji University (Approval No: TJLAC-016-022). All of animal experiments were performed in accordance with the National Institutes of Health guide for the care and use of Laboratory animals.

2.2 Endotoxemia model

All experiments were operated on male Sprague Dawley rats that weighed between 220 g and 250 g. Rats were randomly divided into four groups: sham group, endotoxin group, negative group and positive group. Endotoxemia was induced by intraperitoneal (ip) injection with 4 mg/kg LPS (Sigma). Negative controls or positive groups were pre-treated with 100 mg/kg NaCl (Sigma) or with 100 mg/kg LiCl (Sigma), respectively, 3 days before or after LPS injection. 6 hours after stimulation with LPS, cardiac functions were evaluated and samples from each group were collected. All rats were killed under deep anaesthesia with isoflurane (Gene&I).

2.3 Cell culture and treatment

Neonatal Sprague Dawley rats were purchased from Slaccas Company (Shanghai, China). Primary newborn rat's cardiac myocytes (CMs) were isolated and cultured as previously described.22 Briefly, cells were digested with 0.1% type II collagenase (Invitrogen) and 0.1% trypsin (Gibco) mixture with gently shaking. Cells were resuspended with DMEM including 10% FBS (Gibco) and cultured at 37°C with 5% CO₂ for 1 hour. Non-adherent cells (myocytes) were collected and cultured in fresh medium (Gibco) harbouring 0.1 mM BrdU (sigma) for 48 hours. To induce sepsis, CMs were treated with LPS at different concentrations (0.1, 0.2, 0.5, 1.0 and 2.0 μg/mL) and time-points (3, 6, 12, 24 and 48 hours). The GSK-3β inhibitors LiCl (sigma) and CHIR-99021 (Selleck) were added into CMs during culture for stimulation.

2.4 Western blot

CMs were lysed directly in the culture plate on ice with RIPA lysis buffer containing protease inhibitor cocktail (Roche), and tissues were also grinded with the same condition. Then, the lysates were centrifuged to remove any debris, and protein concentrations were measured using the BCA protein assay kit (TaKaRa). Protein samples were resolved using SDS-PAGE gel and then transferred to 0.45 μm Immobilon-PVDF membranes (Millipore). Primary antibodies used included cleaved-caspase3 (Abcam, ab49822), NF-κB (Abcam, ab16502), Bcl-2 (Abcam, ab59348), GSK-3β (Abcam, ab2602), p-GSK-3β (Abcam, ab75745), FOXO3A (Abcam, ab17026), β-catenin (Abcam, ab32572), ERK (Cell Signaling Technology, CST4695), Bim (Cell Signaling Technology, CST2933T), p-ERK (Cell Signaling Technology, CST4370), AMPK (Cell Signaling Technology, CST2532) and p-AMPK (Cell Signaling Technology, CST2531). GAPDH (Proteintech, 60004-1-Ig) was applied as a loading control. The membranes were then incubated with secondary antibodies and imaged with ImageQuant LAS 4000 or Amersham Imager 600.

2.5 Quantitative real-time PCR

The total RNA was isolated from tissues or cells with TRIzol reagent (Invitrogen), and cDNA was synthesized using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa) followed by a gene expression assay applying TB Green™ Premix Ex Taq™ (TaKaRa) on a Bio-Rad CFX Connect™ real-time system. The related primers were listed in Table S1.

2.6 Immunofluorescence staining

Cells were seeded on glass slides in 48-well plate. After treatment, CMs were fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature (RT) and then permeabilized with 0.1% triton X-100 before blocked with 10% goat serum for 1 hour. Primary antibodies (1:200) were incubated at 4°C overnight, following by fluorescent secondary antibodies (1:250) for 1 hour and Hoechst for 12 minutes at RT. Images were taken using Leica confocal microscope after blocking with aqueous mounting medium.

2.7 TUNEL assay

CMs apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) assay. After fixed and permeated, each sample was treated with 80 μL of TUNEL reaction mixture for 60 minutes at 37°C in dark, and then, the samples were incubated with Hoechst for 12 minutes at RT, following by rinsing with PBS three times, and finally imaged using Leica confocal microscope.

2.8 siRNAs for β-catenin and FOXO3A

Cells were seeded at 2 × 10⁶ in 6-well plate or at 4 × 10⁴ in 24-well plate. After growing for 48 hours, siRNA was transfected using Lipofectamine 2000 (Invitrogen) with a terminal concentration of 50 nM. 4-6 hours later, cells were treated by changing fresh medium and cultured for another 24-48 hours before conducting next experiments. The siRNA sequences were as following: si-FOXO3A# CUCUAUAACGUAUGCAAAU, si-β-catenin#1 GCUGACCAAACUGCUAAAU and si-β-catenin#2 GCACCAUGCAGAAUACAAA. All of the siRNAs and control siRNA were synthesized by GenePharma as previously reported.23, 24

2.9 Separation of nuclear and cytoplasmic protein

Nuclear and cytoplasmic proteins were separated using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific) according to the manufacturer's protocol. Proteinase inhibitors were added to each buffer immediately before use. Cells were harvested by scraping and rinsing with ice-cold PBS, and then, the cells were lysed in ice-cold CER I buffer for 15 minutes, following by adding ice-cold CER II buffer and incubating on ice for 1 minute. Cell suspensions were centrifuged at 16 000 g for 5 minutes, and the supernatant containing the cytoplasmic proteins was collected for next experiments. The precipitation was resuspended with ice-cold NER buffer and incubated on ice for 40 minutes. The samples were centrifuged at 16 000 g for 10 minutes, and the nuclear protein was collected and stored at −80°C for further use.

2.10 Echocardiography

After 6 hours of the treatment with LPS or saline through ip injection, rats were anaesthetized with isoflurane (3.0% induction in room air, followed with 0.5% maintenance in room air) and subjected to echocardiography using Vevo770 (Visual Sonics Inc) as previously described.22 The M-mode images of left ventricular (LV) dimensions were obtained. The left ventricular EF (%) and FS (%) were measured, respectively. Echocardiography data were recorded and analysed individually.

2.11 Wheat germ agglutinin staining

Cardiomyocyte size was evaluated using wheat germ agglutinin staining. The rat heart was fixed in 4% paraformaldehyde, and then, the frozen tissues were sectioned into 20 μm slides, rinsed with PBS and stained for cardiomyocyte membrane with FITC-conjugated wheat germ agglutinin (Sigma). Finally, the heart cross section was imaged with Leica confocal microscope.

2.12 Statistical analysis

ANOVA test was used to compare among three or more groups, followed by Bonferroni's post hoc test. Student's t test was applied to compare two groups, and the error bar represented the standard error of mean (SEM). A value of P < .05 was considered significant. All data were analysed using Prism 5.0 (GraphPad Software, Inc).

3.1 LPS induces activation of GSK-3β in rat cardiomyocytes

Uncontrolled inflammation and apoptosis are two main features of endotoxin-induced cardiac dysfunction.10, 25 Here, we examined the apoptosis rate of CMs exposed to LPS at different concentrations and incubation time. Our results showed a concentration-dependent increase for the expression of the pro-apoptosis proteins, cleaved-caspase3 and Bim after treatment of the CMs with LPS for 24 hours. However, the expression of anti-apoptosis gene Bcl-2 was significantly decreased (Figure 1A). Furthermore, the expression of cleaved-caspase3, Bim and Bcl-2 protein was elevated in presence of LPS (Figure 1B). We then investigated the inflammatory response in CMs under different concentrations of LPS. The results displayed that LPS significantly increased the release of pro-inflammatory cytokines IL-6, IL-1β and TNF-α. Meanwhile, LPS treatment also promoted the mRNA expression of the chemotactic cytokine, iNOs (Figure 1C).

GSK-3β can either positively or negatively affect a variety of transcription factors that are critical in regulating pro- and anti-inflammatory cytokine production as well as cell survival.26 Therefore, we initially determined whether LPS could regulate the expression of GSK-3β. To this end, CMs were treated for 12 hours by different concentrations of LPS (0.1, 0.2, 0.5, 1.0 and 2.0 μg/mL). Protein expression of GSK-3β was up-regulated as a concentration-dependent manner rather than a stimulating-time manner (Figure 1D,E). Interestingly, phosphorylation of GSK-3β at Y216 showed a peak in the presence of 500 ng/mL LPS for 12 hours (Figure 1D-F), which might be related to the potential role GSK-3β at the early stage of inflammation injury.

3.2 GSK-3β inhibition attenuates LPS-induced cardiac inflammation injury

CHIR-99021 and LiCl were widely used as GSK-3β inhibitors in a variety of cell types.10 We applied CHIR-99021 and LiCl to CMs to assess their potential inhibition on GSK-3β. β-catenin was used as an indicator of GSK-3β activity because β-catenin could be phosphorylated and degraded by GSK-3β. Our results showed that CHIR-99021 and LiCl could significantly increase the β-catenin level (Figure 2A,B), which demonstrated their ability to inhibit GSK-3β activity in CMs.

To investigate whether GSK-3β played a critical role in LPS-induced CMs inflammation injury, we treated the CMs with LPS in the presence and absence of CHIR-99021 and LiCl. We found that the GSK-3β inhibition decreased the LPS-induced activation of pro-inflammatory cytokines IL-6, IL-1β, TNF-α and iNOs (Figure 2C). Moreover, during the LPS-induced inflammatory response, CHIR-99021 and LiCl could significantly reduce the TUNEL-positive cell numbers in CMs (Figure 2D,E). It has been showed that Bcl-2, as an indicator of anti-apoptosis, can suppress cell death through inhibiting Bim. As expected, the inhibition of GSK-3β could increase the expression of Bcl-2 but decrease the expression of Bim (Figure 2F), suggesting that GSK-3β may aggravate cardiac dysfunction and promote apoptosis in LPS-stimulated rats.

3.3 GSK-3β inhibition improves cardiac function in vivo

To evaluate the in vivo role of GSK-3β, LPS (4 mg/kg) was intraperitoneally administrated into rats to induce septic myocardial dysfunction as previously described,12 and LiCl was also applied to inhibit the activity of GSK-3β for three consecutive days at different concentrations (50, 100 and 200 mg/kg) before or after LPS treatment (Figure 3A). The up-regulation of β-catenin in heart suggested an inhibition role on GSK-3β (Figure 3B). Lipopolysaccharide significantly impaired heart function, including decrement ejection and shortening fraction, while GSK-3β inhibition could improve the EF by 13.85% and FS by 12.22% in the rats (Figure 3C; Figure S1). Myocardium damage assay demonstrated that GSK-3β inhibition not only attenuated the LPS-induced injury in hearts (Figure 3D) but, meanwhile, could partially alleviate the change in the cell size in LPS-treated CMs (Figure 3E). However, no obvious effect on cardiac dimensions was observed in the condition (Figure S2).

TUNEL staining confirmed that GSK-3β inhibition also reduced apoptotic cells in heart challenged with LPS (Figure 3F). Consistent with experiments in vitro, LPS could increase the expression level of GSK-3β, FOXO3A and Bim, which could be reversed when exposed to LiCl (Figure 3G,H). In addition, the GSK-3β inhibition suppressed the LPS-induced activation for pro-inflammatory cytokines IL-6, IL-1β, TNF-α and iNOs in vivo (Figure 3I). Although left ventricular function was reduced, no obvious cardiac fibrosis was detected in LPS-treated rats by Masson's trichrome staining assay even the corresponding genes expression signatures revealed a decrease in fibrosis markers (Col1a, Col3a, Fibronectin and α-SMA), which could be partly reversed by GSK-3β inhibition (Figure S3). These results suggest that the block of GSK-3β could significantly improve cardiac function and attenuate apoptosis in LPS-treated rats.

3.4 Inhibition of β-catenin aggravates the LPS-induced cell apoptosis

β-catenin is the major downstream transcription factors and proteolyzed by the destruction complex containing GSK-3β.10 To determine the role of β-catenin on the GSK-3β regulation in LPS-induced apoptosis of CMs, the specific siRNAs were transfected into CMs to inhibit β-catenin expression (Figure 4A,B). We treated CMs with LPS, two siRNAs that targeted β-catenin, and Wnt/β-catenin inhibitors, XAV939 and ICG-001. The knock-down of β-catenin really attenuated the Bcl-2 and increased Bim expression after LPS exposure (Figure 4D; Figure S4). Meanwhile, TUNEL-positive cells were also significantly increased under the condition (Figure 4C; Figure S4). Then, we exposed CMs to β-catenin siRNAs as treated with GSK-3β inhibitor, CHIR-99021. The results shown that the effect of GSK-3β inhibition on sepsis was prevented by the knock-down of β-catenin (Figure 4E,F), indicating that β-catenin exerts an important role in GSK-3β related sepsis.

3.5 GSK-3β inhibition decreases FOXO3A expression mediated by β-catenin

We next examined whether the activation of LPS-induced inflammation injury by GSK-3β was involved in the regulation of FOXO3A signalling. Lipopolysaccharide treatment with different concentrations (0.1, 0.2, 0.5, 1.0 and 2.0 μg/mL) and time-points (0, 3, 6, 12, 24 and 48 hours) could significantly increase the FOXO3A expression (Figure 5A,B; Figure S5), in which FOXO3A expression level reached a peak after 12-hour exposure with 500 ng/mL in CMs. To prove our hypothesis that FOXO3A might be one of the targets of GSK-3β in sepsis-induced cardiac dysfunction, we used CHIR-99021 and LiCl to inhibit GSK-3β. As indicated in Figure 5C; Figure S3, expression of both FOXO3A and its target gene LC3 were decreased along with inhibition of GSK-3β (Figure S5). Moreover, the inhibition of FOXO3A could be rescued by knock-down of β-catenin through transfecting siRNA (Figure 5C). Then, we further examined whether FOXO3A was responsible for the protection of inflammation injury in the CMs induced by the GSK-3β inhibition. We treated CMs with the GSK-3β inhibitors (CHIR-99021 and LiCl) in the presence and absence of the FOXO3A activator TIC10 and exposed them to LPS. We found that TIC10 really increased the FOXO3A level (Figure 5D), with an up-regulation of inflammatory cytokines IL-1β, TNF-α and iNOs as well (Figure 5F). In addition, TUNEL staining and Western blot analysis for Bcl-2/Bim showed that TIC10 significantly enhanced the apoptosis of the CMs (Figure 5E,G), indicating that the FOXO3A as one of the target of the GSK-3β could regulate endotoxemia cardiac dysfunction which might attribute to its pro-apoptotic effect on the CMs.

3.6 FOXO3A knock-down attenuates LPS-induced cardiac inflammation injury

To confirm the FOXO3A function in the LPS-induced sepsis in CMs, knock-down assay showed the expression level of FOXO3A was significantly decreased in the CMs transfected with the siRNA targeting FOXO3A for 48 hours (Figure 6A,B), in which the LPS-induced up-regulation of IL-6, IL-1β, TNF-α and iNOs was suppressed (Figure 6C). Meanwhile, FOXO3A knock-down also attenuated the activation of NF-κB (Figure S6). These results suggest that the LPS-induced FOXO3A could regulate inflammation process in CMs. Moreover, the down-regulation of FOXO3A could decrease the LPS-induced apoptotic index (Figure 6D), accompanying with an increase in Bcl-2 and a decrease in Bim. All of the data indicated that FOXO3A was associated with the endotoxin-induced myocardial apoptosis (Figure 6E,F) and played a crucial role in the inflammation injury of the heart.

3.7 GSK-3β inhibition negatively involved in the activations of ERK/NF-κB signalling and positively regulated AMPK pathway

IκBα is degraded upon phosphorylation which results in the consequent release and nuclear translocation of NF-κB.27 When cells were stimulated with LPS, the degradation of IκBα induced an increase in NF-κB level in the nuclear fraction (Figure 7A-C). However, GSK-3β inhibitors LiCl or CHIR-99021 increased the IκBα level and reduced nuclear NF-κB only under LPS condition (Figure 7A-C). The results suggest that NF-κB is involved in the effect of GSK-3β on regulating inflammation injury in heart.

In addition, ERK and AMPK signalling are two important pathways activated by LPS in many cell types. ERK contributes to the induction of inflammation and apoptosis, whereas AMPK exerts reverse effects in the processes through cross-talking with NF-κB.28 As shown in Figure 7D-I, ERK and AMPK signalling were activated by the exposure of CMs to LPS for 15 or 30 minutes. GSK-3β inhibition suppressed the LPS-induced ERK1/2 phosphorylation and enhanced the LPS-induced AMPK phosphorylation with different extent, suggesting a crucial role of GSK-3β in inflammation injury.