Glycogen synthase kinase-3β inhibition alleviates activation of the NLRP3 inflammasome in myocardial infarction

https://doi.org/10.1016/j.yjmcc.2020.09.009Get rights and content

Highlights

  • GSK-3β inhibition improved myocardial dysfunction and remodeling after myocardial infarction (MI).

  • GSK-3β inhibition decreased NLRP3 inflammasome activation after MI.

  • GSK-3β inhibition reduced NLRP3 inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes.

  • GSK-3β promoted NLRP3 inflammasome activation by interacting with ASC phosphorylation.

Abstract

Inflammasome-promoted sterile inflammation following cardiac damage is critically implicated in heart dysfunction after myocardial infarction (MI). Glycogen synthase kinase-3 (GSK-3β) is a prominent mediator of the inflammatory response, and high GSK-3 activity is associated with various heart diseases. We investigated the regulatory mechanisms of GSK-3β in activation of the nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome in a rat model with successful induction of MI on days 2–28. An in vitro investigation was performed using newborn rat/human cardiomyocytes and fibroblast cultures under typical inflammasome stimulation and hypoxia treatment. GSK-3β inhibition markedly improved myocardial dysfunction and prevented remodeling, with parallel reduction in the parameters of NLRP3 inflammasome activation after MI. GSK-3β inhibition reduced NLRP3 inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes. GSK-3β's interaction with activating signal cointegrator (ASC) as well as GSK-3β inhibition reduced ASC phosphorylation and oligomerization at the tissues and cellular levels. Taken together, these data show that GSK-3β directly mediates NLRP3 inflammasome activation, causing cardiac dysfunction in MI.

Introduction

Myocardial infarction (MI) is the leading cause of death worldwide [1]. Although modern healthcare has reduced the rate of acute infarction-related mortality, the prevalence of heart failure continues to increase. Therefore, novel therapeutic strategies to repair infarcted hearts are urgently needed.

MI is closely related to sterile inflammation, which is a critical condition for tissue healing and may lead to excessive heart damage and maladaptive ventricular remodeling [2]. Growing evidence indicates that the inflammasome, a large multiprotein complex in the cytosol that may induce caspase-1 activation, plays a key role in sterile inflammation. We have an understanding of many inflammasome functions [3], of which the nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome has been extensively studied. It has also been shown that the NLRP3 inflammasome plays an indispensable role in the development and progression of aseptic inflammation [4].

When NLRP3 is activated, it binds to the activating signal cointegrator (ASC) adaptor molecule and aggregates with pro-caspase-1, which are components of the NLRP3 inflammasome [5]. The NLRP3 inflammasome converts pro-caspase-1 to caspase-1, which catalyzes the conversion of pro-IL-1β to its mature product IL-1β. IL-1β secretion from cells causes inflammation and tissue damage [6]. IL-1β plays an important role in the inflammatory response following MI by regulating immune cell recruitment, cytokine production, and extracellular matrix turnover [7]. Recent research has shown that early IL-1β elevation impairs cardiac function, reduces left ventricular ejection fraction (LVEF), and induces hypertrophy [[8], [9], [10]]. In preclinical and clinical studies, inhibition of IL-1 signaling after MI has been shown to improve left ventricular function and reduce the incidence of heart failure [[11], [12], [13]].

Glycogen synthase-3β (GSK-3β) is a multifunctional serine/threonine kinase that was initially described as a key enzyme involved in glycogen metabolism, but is now known to regulate various cell functions including cellular structure, growth, motility, metabolism, and survival [14]. Studies on various signal transduction pathways, including Wnt/wingless, nuclear factor-kappa B, insulin, and apoptotic signaling, have shown that cell survival is largely dependent on GSK-3β [15]. The role of GSK-3β in myocyte biology and disease has been studied [[16], [17], [18]]. GSK-3β also contributes to cardiac hypertrophy [19] and heart failure [20]. Furthermore, if GSK-3β activity is inhibited, myocardial ischemia-reperfusion and doxorubicin-induced heart damage are improved and restored to some extent [21].

Recent reports have shown that GSK-3β plays a key role in controlling the inflammatory immune response [22,23]. Moreover, GSK-3β inhibitors decrease IL-1β expression [22]. Although GSK-3β is involved in activation of the NLRP3 inflammasome in various diseases [[24], [25], [26]], it is unclear whether GSK-3β regulates activation of the NLRP3 inflammasome in MI.

Accordingly, we evaluated the mechanisms involved in GSK-3β-mediated activation of the NLRP3 inflammasome after MI. GSK-3β may be a novel therapeutic target for cardiovascular disease.

Section snippets

Animals

Male Sprague-Dawley rats (220–240 g; Experimental Animal Center of Zhengzhou University, Zhengzhou, Henan, China) were kept under standard conditions. Animal experiments were approved by the Animal Experiments Committee of Zhengzhou University and the Experimental Animal Center of Zhengzhou University (Permit No. SYXK [YU] 2011–0001) and conformed to the Guidelines for the Care and Use of Laboratory Animals (NIH Publication, 2011). All of the studies followed editorial guidelines for

SB216763 inhibits GSK-3β activation in ischemic hearts

Rats were treated with SB216763 at 1 h before MI surgery and once daily thereafter. To evaluate GSK-3β activation, we first observed the mRNA expression of GSK-3β in the LV at various time points. In the ischemic zone, MI induced an increase in GSK-3β mRNA expression on day 2 post-MI (Fig. 1A), with maximum expression observed on day 7 post-MI (Fig. 1B) and a slow decrease thereafter (day 28) (Fig. 1C). In the border zone, MI increased GSK-3β mRNA expression on day 7 post-MI; this increase was

Discussion

GSK-3β is an important therapeutic target in a variety of pathologies [37]. Several studies indicate that the inhibition of GSK-3β before ischemia or reperfusion has marked cardioprotective effects [38]. Our study found that the inhibition of GSK-3β conferred cardioprotection by attenuating activation of the NLRP3 inflammasome in MI-induced rats. Moreover, GSK-3β affects the activation of NLRP3 inflammasomes by affecting the phosphorylation of ASC, and GSK-3β inhibition reduced cytoplasmic

Conclusions

Our results identified a novel mechanism through which GSK-3β regulates myocardial fibrotic remodeling in the infarcted heart of rats. GSK-3β may exert these effects via the direct regulation of NLRP3 inflammasome activation. Clinically, pharmacological inhibitors targeting GSK-3β show potential for the treatment of MI.

The following is the supplementary data related to this article.

. GSK-3β inhibition reduced the expression of NLRP3 inflammasome components in hypoxia-induced RCFs.

RCFs derived

Disclosures

None.

Declaration of Competing Interest

None.

Acknowledgments

The National Natural Science Foundation of China (No. 81670311) supported this work.

References (73)

  • D.A. Chistiakov et al.

    The role of cardiac fibroblasts in post-myocardial heart tissue repair

    Exp. Mol. Pathol.

    (2016)
  • T. Prochnicki et al.

    Inflammasomes on the crossroads of innate immune recognition and metabolic control

    Cell Metab.

    (2017)
  • J.M. Lambert et al.

    Macrophage roles following myocardial infarction

    Int. J. Cardiol.

    (2008)
  • W. Chen et al.

    Fibroblasts in post-infarction inflammation and cardiac repair

    Biochim. Biophys. Acta

    (2013)
  • O. Dewald et al.

    Of mice and dogs: species-specific differences in the inflammatory response following myocardial infarction

    Am. J. Pathol.

    (2004)
  • L. Liu et al.

    Stroke and stroke care in China: huge burden, significant workload, and a national priority

    Stroke.

    (2011)
  • N.G. Frangogiannis et al.

    The inflammatory response in myocardial infarction

    Cardiovasc. Res.

    (2002)
  • E. Latz et al.

    Activation and regulation of the inflammasomes

    Nat. Rev. Immunol.

    (2013)
  • K.V. Swanson et al.

    The NLRP3 inflammasome: molecular activation and regulation to therapeutics

    Nat. Rev. Immunol.

    (2019)
  • M.S.J. Mangan et al.

    Targeting the NLRP3 inflammasome in inflammatory diseases

    Nat. Rev. Drug Discov.

    (2018)
  • M. Takahashi

    NLRP3 inflammasome as a novel player in myocardial infarction

    Int. Heart J.

    (2014)
  • A. Abbate et al.

    Anakinra, a recombinant human interleukin-1 receptor antagonist, inhibits apoptosis in experimental acute myocardial infarction

    Circulation.

    (2008)
  • S. Orn et al.

    Increased interleukin-1beta levels are associated with left ventricular hypertrophy and remodelling following acute ST segment elevation myocardial infarction treated by primary percutaneous coronary intervention

    J. Intern. Med.

    (2012)
  • A. Abbate et al.

    Interleukin-1 blockade with anakinra to prevent adverse cardiac remodeling after acute myocardial infarction (Virginia Commonwealth University Anakinra Remodeling trial [VCU-ART] pilot study)

    Am. J. Cardiol.

    (2010)
  • A. Abbate et al.

    Interleukin-1beta modulation using a genetically engineered antibody prevents adverse cardiac remodelling following acute myocardial infarction in the mouse

    Eur. J. Heart Fail.

    (2010)
  • H.B. Sager et al.

    Targeting interleukin-1beta reduces leukocyte production after acute myocardial infarction

    Circulation.

    (2015)
  • P. Cohen et al.

    GSK3 inhibitors: development and therapeutic potential

    Nat. Rev. Drug Discov.

    (2004)
  • S. Haq et al.

    Glycogen synthase kinase-3beta is a negative regulator of cardiomyocyte hypertrophy

    J. Cell Biol.

    (2000)
  • K.C. Woulfe et al.

    Glycogen synthase kinase-3beta regulates post-myocardial infarction remodeling and stress-induced cardiomyocyte proliferation in vivo

    Circ. Res.

    (2010)
  • T. Matsuda et al.

    Distinct roles of GSK-3alpha and GSK-3beta phosphorylation in the heart under pressure overload

    Proc. Natl. Acad. Sci. U. S. A.

    (2008)
  • S. Hirotani et al.

    Inhibition of glycogen synthase kinase 3beta during heart failure is protective

    Circ. Res.

    (2007)
  • T. Miura et al.

    Mitochondria and GSK-3beta in cardioprotection against ischemia/reperfusion injury

    Cardiovasc. Drugs Ther.

    (2010)
  • K. Rehani et al.

    Toll-like receptor-mediated production of IL-1Ra is negatively regulated by GSK3 via the MAPK ERK1/2

    J. Immunol.

    (2009)
  • J. Zhao et al.

    Lupus nephritis: glycogen synthase kinase 3beta promotion of renal damage through activation of the NLRP3 inflammasome in lupus-prone mice

    Arthritis Rheum.

    (2015)
  • T. Xiong et al.

    GSK-3beta/mTORC1 couples synaptogenesis and axonal repair to reduce hypoxia ischemia-mediated brain injury in neonatal rats

    J. Neuropathol. Exp. Neurol.

    (2018)
  • M.J. Curtis et al.

    Experimental design and analysis and their reporting: new guidance for publication in BJP

    Br. J. Pharmacol.

    (2015)
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