Toll-interacting protein contributes to mortality following myocardial infarction through promoting inflammation and apoptosis

Abstract

Background and purpose: Toll-interacting protein (Tollip) is an endogenous inhibitor of toll-like receptors, a superfamily that plays a pivotal role in various pathological conditions, including myocardial infarction (MI). However, the exact role of Tollip in MI remains unknown.

Experimental approach: MI models were established in Tollip knockout (KO) mice, mice with cardiac-specific overexpression of human Tollip gene and in their Tollip(+/+) and non-transgenic controls respectively. The effects of Tollip on MI were evaluated by mortality, infarct size and cardiac function. Hypoxia-induced cardiomyocyte damage was investigated in vitro to confirm the role of Tollip in heart damage.

Key results: Tollip expression was dramatically up-regulated in human ischaemic hearts and infarcted mice hearts. MI-induced mortality, infarct size and cardiac dysfunction were decreased in Tollip-KO mice compared with Tollip(+/+) controls. Ischaemic hearts from Tollip-KO mice exhibited decreased inflammatory cell infiltration and reduced NF-κB activation. Tollip depletion also alleviated myocardial apoptosis by down-regulating pro-apoptotic protein levels and up-regulating anti-apoptotic protein expressions in infarct border zone. Conversely, MI effects were exacerbated in mice with cardiac-specific Tollip overexpression. This aggravated MI injury by Tollip in vivo was confirmed with in vitro assays. Inhibition of Akt signalling was associated with the detrimental effects of Tollip on MI injury; activation of Akt largely reversed the deleterious effects of Tollip on MI-induced cardiomyocyte death.

Conclusions and implications: Tollip promotes inflammatory and apoptotic responses after MI, leading to increased mortality and aggravated cardiac dysfunction. These findings suggest that Tollip may serve as a novel therapeutic target for the treatment of MI.

Figure 1

Up-regulation of Tollip in hearts subjected to MI. (A) Representative Western blotting of Tollip in the heart samples from normal donors (n = 6) and patients with IHD (n = 7). (B) Western blotting results for Tollip in the infarcted border zone of C57BL/6J mice at the indicated time points post-MI (n = 5). Left, representative Western blotting. Right, quantitative results. *P < 0.05 versus normal donors or sham-operated hearts.

Figure 2

Tollip increases MI-induced mortality, infarct size and cardiac dysfunction. (A) Kaplan–Meier survival curves for Tollip^+/+ and Tollip KO mice after MI (*P < 0.05 vs. Tollip^+/+/MI group). (B) Representative images of haematoxylin and eosin staining in heart cross-sections of Tollip^+/+ and Tollip^−/− mice at 3 days and 4 weeks after MI surgery (n = 6 mice per experimental group). (C) Measurement of infarct size in the heart tissue of mice from Tollip^+/+ and Tollip^−/− groups. *P < 0.05 versus Tollip^+/+/MI group at 3 days; #P < 0.05 versus Tollip^+/+/MI group at 4 weeks. (D) Echocardiographic and haemodynamic assessment of cardiac function in Tollip^+/+ and Tollip^−/− mice after MI (n = 8 mice per experimental group). *P < 0.05 versus Tollip^+/+/sham operated group; #P < 0.05 versus Tollip^+/+/MI group. (E) Comparison of mortality between NTG mice and Tollip-TG mice at 3 days and 4 weeks post-MI injury (*P < 0.05 vs. NTG/MI group). (F,G) Representative images and evaluation of infarct size in NTG and Tollip-TG mice at 3 days and 4 weeks after MI surgery (n = 6 mice per experimental group). *P < 0.05 versus NTG/MI group at 3 days; #P < 0.05 versus NTG/MI group at 4 weeks. (H) The parameters of cardiac function in NTG and Tollip-TG mice after 4 weeks of MI surgery (n = 8 for each group). *P < 0.05 versus NTG/sham operated group; #P < 0.05 versus NTG/MI group.

Figure 3

Tollip increases inflammatory and immune cell infiltration and NF-κB activation after MI. (A,B) Representative immunofluorescence staining images of macrophage (Mac-1), neutrophil (LY6G) and T cell (CD3) accumulation in the infarct border zone of Tollip^+/+ and Tollip-KO mice (A) and in NTG and Tollip-TG mice (B) at 3 days and 4 weeks after MI. (C,D) Representative Western blotting and quantitative results showing the phosphorylation levels of p65 and IκBα in the infarct border zone of hearts from (C) Tollip^+/+ and Tollip-KO mice and from (D) NTG and Tollip-TG mice at 4 weeks after MI (n = 13 mice per experimental group). Left, representative Western blotting. Right, quantitative results. *P < 0.05 versus Tollip^+/+/sham or NTG/sham-operated group; #P < 0.05 versus Tollip^+/+/MI or NTG/MI group.

Figure 4

Tollip promotes apoptosis and regulates the expression of apoptosis-related genes in response to MI. (A–C) Representative TUNEL staining images (A) and quantitative results (B,C) TUNEL-positive cells in heart sections from (B) Tollip^+/+ and Tollip^−/− mice and from (C) NTG and Tollip-TG mice at 3 days and 4 weeks after MI (n = 6 mice per experimental group, *P < 0.05 vs. Tollip^+/+/MI or NTG/MI group at 3 days; #P < 0.05 vs. Tollip^+/+/MI or NTG/MI group at 4 weeks). (D,E) Representative Western blotting and quantitative analysis showing the protein levels of Bax, Bcl-2 and cleaved caspase-3 in (D) Tollip^+/+ and Tollip-KO mice and in (E) NTG and Tollip-TG mice at 4 weeks after MI induction (n = 13 mice per experimental group). Left, representative Western blotting. Right, quantitative results. *P < 0.05 versus Tollip^+/+/sham or NTG/sham-operated group; #P < 0.05 versus Tollip^+/+/MI or NTG/MI group.

Figure 5

Tollip enhances hypoxia-induced cardiomyocyte death and regulates the expression of apoptosis-related genes in vitro. (A,C) Representative photomicrographs of (A) AdshTollip- or (C) AdTollip-infected primary cardiomyocytes double labelled with Hoechst 33258 for nuclei (blue) and PI for dead cells (red) after 24 h of hypoxia stimulation (n = 5). (B,D) Determination of cell viability and cell toxicity by cell counting kit-8 (CCK-8) and LDH release assay in NRCMs with (B) Tollip knockdown or (D) overexpression after exposure to hypoxia for 24 h (n = 5). *P < 0.05 versus AdshRNA/normoxia or AdGFP/normoxia; #P < 0.05 versus AdshRNA/hypoxia or AdGFP/hypoxia. (E,F) Western blotting results for the protein levels of Bax, Bcl-2 and cleaved caspase-3 in NRCMs infected with (E) AdshTollip or (F) AdTollip after hypoxia treatment for 24 h (n = 5 samples). Top, representative Western blotting. Bottom, quantitative results. *P < 0.05 versus AdshRNA/normoxia or AdGFP/normoxia; #P < 0.05 versus AdshRNA/hypoxia or AdGFP/hypoxia.

Figure 6

Tollip exacerbates cardiac injury post-MI by inactivating the Akt signalling pathway. (A,B) Representative Western blotting showing phosphorylated and total MEK1/2, ERK1/2, JNK1/2 and p38 protein levels from (A) Tollip^+/+ and Tollip^−/− mice and from (B) NTG and Tollip-TG mice at 4 weeks after MI (n = 13 mice per experimental group). (C– E) The (C) representative Western blotting and (D and E) quantitative results showing the levels of phosphorylated and total Akt, mTOR, S6, FOXO1 and GSK3β protein from heart tissue of mice in the indicated groups at 4 weeks after MI (n = 13 mice per experimental group). *P < 0.05 versus Tollip^+/+/MI or NTG/MI group. (F–H) Representative Western blotting results showing the expression levels of proteins in Akt signalling in NRCMs infected with AdshTollip or AdTollip after hypoxia treatment for 24 h (n = 6 samples). *P < 0.05 versus AdshRNA/hypoxia or AdGFP/hypoxia. (I) The cell viability and LDH release of cardiomyocytes infected with AdshRNA, AdshTollip, AddnAkt or AdshTollip + AddnAkt after exposure to hypoxia for 24 h (n = 6 samples). (J) The cell viability and LDH release of cardiomyocytes in the indicated groups after treatment of hypoxia for 24 h (n = 6 samples). ns, no significant difference.

Figure 7

Akt inhibition was responsible for the detrimental role of Tollip on MI-induced cardiac damage. (A) Representative Western blotting showing phosphorylated and total Akt protein levels from Tollip^+/+ and Tollip^−/− mice treated with Akt inhibitor MK-2206 at 1 week after MI (n = 8 mice per experimental group). (B) Representative images of haematoxylin and eosin staining in heart cross-sections of Tollip^+/+ and Tollip^−/− mice treated with Akt inhibitor MK-2206 at 1 week after MI surgery (n = 6 mice per experimental group). (C) Measurement of infarct size in the heart tissue of mice from Tollip^+/+ and Tollip^−/− groups treated with Akt inhibitor MK-2206. (D) Echocardiographic and haemodynamic assessment of cardiac function in Tollip^+/+ and Tollip^−/− mice treated with Akt inhibitor MK-2206 after MI (n = 6 mice per experimental group). ns, no significant difference.