Elsevier

Cellular Signalling

Volume 86, October 2021, 110084
Cellular Signalling

Deficiency of tenascin-C attenuated cardiac injury by inactivating TLR4/NLRP3/caspase-1 pathway after myocardial infarction

https://doi.org/10.1016/j.cellsig.2021.110084Get rights and content

Highlights

  • TN-C deficiency attenuates post-infarction myocardial injury by inactivating both caspase-1 and TLR4/NF-kB/NLRP3 pathway.

Abstract

Inflammation and pyroptosis play a deleterious role in cardiac dysfunction after myocardial infarction (MI). NLRP3/caspase-1 is a well-established axis in pyroptosis and inflammation. In this study, we examined the effects of TN-C on pyroptosis through NLRP3 is unclear. We constructed 18 TN-C-knockout and 38 WT male mice model and divided into WT sham (n = 16), WT MI (n = 22), TNKO sham (n = 6), TNKO MI (n = 12). Elisa, immunostaining, TTC, qPCR, CCK8, flow cytometry, and western blot, echocardiographic, TUNEL staining technologies were applied. Here, we found a positive correlation between TN-C and NLRP3 in heart tissue via the GEPIA database (r = 0.52, p < 0.05). The findings indicate that TN-C was elevated and peaked on the fifth day after MI. TN-C deficiency alleviated cardiac dysfunction (LVEF, FS, LVIDd, and LVIDs) and cardiomyocyte death. Though the intracellular levels of pyroptosis-related cytokine caspase-1, cleaved caspase-1, NLRP3, IL-18, IL-1β were upregulated both in MI and H2O2 stimulation, knockout of TN-C resisted such injury and alleviated cardiac pyroptosis, which further decreased IL-6, TNF-α, MCP-1 expression. TN-C knockdown inhibited TLR4 expression, reduces the release of downstream factors by inactivating the TLR4/NF-kB pathway, while protects the cardiomyocytes. And TLR4 inhibitor TAK-242 significantly reduced NLRP3 expression levels after MI. We demonstrated for the first time a direct link between MI-induced TN-C upregulation and caspase-1-dependent cardiomyocyte pyroptosis, a process mediated, at least in part, by TLR4/NF-kB/NLRP3 and IL-18, IL-1β signaling pathways. These findings provide new insights into the role of TN-C in post-MI cardiomyocytes' pyroptosis and inflammation.

Graphical abstract

The role of TN-C was demonstrated in cardiomyocyte pyroptosis after MI by applying TN-C KO mice. Our findings confirmed that TN-C deficiency attenuates post-infarction cardiomyocyte pyroptosis, resulting in the reduction of the inflammatory response and improvement of cardiac function in vivo and in vitro. Studies on the mechanism revealed that TN-C mediated cardiomyocyte pyroptosis was dependent upon caspase-1 activation and partially mediated through TLR4/NF-κB/NLRP3 pathway.

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Introduction

MI is a continuous process from atherosclerosis to heart failure, and the inflammatory immune response is involved in the whole process from initial to end-stage. Cardiac injury and minimal regenerative capacity after myocardial infarction (MI) are the key factors affecting myocardial repair and prognosis [1]. Different types of cell death, including autophagy, apoptosis, and necrosis, occur after MI [2]. Pyroptosis, a new type of programmed inflammatory cell death, is characterized by rapid onset and activation of the release of pro-inflammatory immune cellular substances [3].

Previous research has established that pyroptosis may be a potential mechanism for myocardial cell loss [1]. A large number of fibrous scar formations occur after MI because of cardiomyocyte inflammation and pyroptosis [4]. NOD-like receptor protein3 (NLRP3) inflammasome is a multi-protein complex, activate of NLRP3/caspase-1 axis increases the release of IL-1β and IL-18, and inducing cell pyroptosis [5], [6]. NLRP3 inflammasome and caspase-1 have been shown to play an important role in MI [7], [8].

Tenascin-C (TN-C) is an extracellular matrix glycoprotein with four distinct domains: epidermal growth factors–like repeats, fibronectin type III–like repeats, and a fibrinogen-like globe. Though TN-C is rarely detected in adults, it is up-regulated after injury and regulates cell signaling via ligand or integrin to such as TLR4 or αvβ6 signaling [9], [10]. TN-C also acts as a member of the inflammatory immune response, plays an important role in the development and progression of MI. Whether TN-C plays a role in modulating pyroptosis after MI remains unknown.

In this study, we found TN-C was upregulated after MI in mice. TN-C deficiency alleviated myocardial death and inflammation via TLR4/NF-κB/NLRP3/caspase-1. Furthermore, TN-C deficiency inhibited the NLRP3 activation directly, as a result of reducing inflammasome expression and improved cardiac function after MI in vivo and in vitro.

Section snippets

Animals MI model

All animal experiments were approved by the Institutional Animal Care and Use Committee of Dalian Medical University (approval no. L2014029). TN-C-deficient mice (C57BL/6N-TgH (Tnc)) were purchased from RIKEN BioResource Center (Koyadai, Tsukuba, Japan). All animals were bred in specific pathogen-free conditions under staff supervision.

MI model

38 adult male WT and 18 TN-C KO mice at 6–8 weeks old were starved for 12 h before surgery. Animals were anesthetized with a mask inhaling isoflurane gas at a

TN-C is highly expressed after MI and peaks on the fifth day

There are several studies suggest that TN-C is expressed at a low level in adult hearts, but other studies have presented different results for the TN-C expression peak [13], [14]. Thus, the present study determined TN-C expression in a mouse heart. TN-C is almost not expressed in normal heart tissues of wild-type (WT) mice, but is significantly highly expressed when MI occurs in WT mice (Fig. 1A). And, TN-C was detected at the junction of intact heart tissue and necrotic area (Fig. 1B). We

Discussion

In this study, the role of TN-C was first demonstrated in cardiomyocyte pyroptosis after MI by applying TN-C KO mice. Our findings confirmed that TN-C deficiency attenuates post-infarction cardiomyocyte pyroptosis, resulting in a reduction of the inflammatory response and improvement of cardiac function. Studies on the mechanism revealed that TN-C-mediated cardiomyocyte pyroptosis was dependent upon caspase-1 activation and partially mediated through TLR4/NF-kB/NLRP3 pathway.

The increasing

Authorship contribution

Huang RC and Xu MY designed study. Ye ZS, Zhao Xin and Xu MY conducted experiments. Data collection and analysis were performed by Gong XH and Guo HZ. The first draft of the manuscript was written by Xu MY. All authors read and approved the final manuscript.

Sources of funding

This work was funded by the National Natural Science Foundation of China (grant nos. 81770340, 82070468, 82000402 and 81670324).

Ethics statement

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Dalian Medical University (approval no. L2014029). The animal study was complied with the ARRIVE guidelines and carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments.

Declaration of Competing Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

We would like to thank the support and assistance of the Biochemistry Laboratory of Dalian Medical University.

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