Cardiomyocyte Pyroptosis Research Articles

Geniposide protects against sepsis-induced myocardial dysfunction through AMPKα-dependent pathway

Uncontrolled inflammatory response and subsequent cardiomyocytes loss (apoptosis and pyroptosis) are closely involved in sepsis-induced myocardial dysfunction. Our previous study has found that geniposide (GE) can protect the murine hearts against obesity-induced inflammation. However, the effect of GE on sepsis-related cardiac dysfunction is still unknown. Mice were exposed to lipopolysaccharide (LPS) to generate sepsis-induced myocardial dysfunction. And 50 mg/kg GE was used to treat mice for consecutive 7 days. Our results showed that GE treatment significantly improved survival rate and cardiac function, and suppressed myocardial inflammatory response, as well as myocardial loss in LPS-treated mice. Those effects of GE were largely abolished in NOD-like receptor protein 3 (NLRP3)-deficient mice. Further detection revealed that the inhibition of NLRP3 inflammasome activation depended on the reduction of p47phox by GE. GE treatment restored the phosphorylation and activity of AMP-activated protein kinase α (AMPKα) in the hearts of sepsis mice, and knockout of AMPKα abolished the protection of GE against reactive oxygen species (ROS) accumulation, NLRP3 inflammasome activation and cardiomyocytes loss in sepsis mice. In conclusion, our findings revealed that GE activated AMPKα to suppress myocardial ROS accumulation, thus blocking NLRP3 inflammasome-mediated cardiomyocyte apoptosis and pyroptosis and improving cardiac function in mice with sepsis.

Macrophage-Derived Exosomal Mir-155 Regulating Cardiomyocyte Pyroptosis and Hypertrophy in Uremic Cardiomyopathy

miR-155 was synthesized and loaded into exosomes in increased infiltration of macrophages in a uremic heart.The released exosomal fusion with the plasma membrane leads to the release of miR-155 into the cytosoland translational repression of forkhead transcription factors of the O class (FoxO3a) in cardiomyocytes. Finally, macrophage-derived miR-155-containing exosomes promoted cardiomyocyte pyroptosis and uremic cardiomyopathy changes (cardiac hypertrophy and fibrosis) by directly targeting FoxO3a in uremic mice.

Bnip3 mediates doxorubicin-induced cardiomyocyte pyroptosis via caspase-3/GSDME

AimsThis study was aimed to investigate the role of GSDME-mediated pyroptosis in cardiac injury induced by Doxorubicin (DOX), and to evaluate the role of BH3-only protein Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (Bnip3) in regulation of DOX-induced pyroptosis. Main methodsHL-1 cardiomyocytes and C57BL/6J mice were treated by DOX to establish DOX-induced cardiotoxicity in vitro and in vivo models, respectively. Cell transfection was applied to regulate the expression of caspase-3, GSDME and Bnip3. Western blot was used for measuring expression of protein level. LDH-cytotoxicity assay was used to detect the LDH release. The Flow cytometry analysis was used to detect the cell death. Echocardiography was used to determine the cardiac function. HE staining was used for observing pathological feature of heart tissues. Key findingsOur results showed that GSDME-mediated pyroptosis was involved in DOX-induced cardiotoxicity in vivo. We showed that HL-1 cardiomyocytes exposed to DOX exhibited morphological features of pyroptosis in vitro. We also showed that DOX induced activation of caspase-3 and eventually triggered GSDME-dependent pyroptosis, which was reduced by the silence or inhibitor of caspase-3. We further showed that knockdown of GSDME inhibited DOX-induced cardiomyocyte pyroptosis in vitro. Finally, DOX increased the expression of Bnip3, whereas silencing of Bnip3 blunted cardiomyocyte pyroptosis induced by DOX, which was regulated through caspase-3 activation and GSDME cleavage. SignificanceOur findings revealed a novel pathway that cardiomyocyte pyroptosis is regulated through Bnip3-caspase-3-GSDME pathway following DOX treatment, suggesting that Bnip3-dependent pyroptosis may offer a novel therapeutic strategy to reduce cardiotoxicity induced by DOX.

Exendin-4 Protects against Hyperglycemia-Induced Cardiomyocyte Pyroptosis via the AMPK-TXNIP Pathway.

Diabetic cardiomyopathy is a common cardiac condition in patients with diabetes mellitus, which results in cardiac hypertrophy and subsequent heart failure. Chronic inflammation in the diabetic heart results in loss of cardiomyocytes and subsequentially cardiac dysfunction. Accumulated evidence implicated pyroptosis as a vital contributor to the hyperglycemia-induced cardiac inflammatory response. Exendin-4, a GLP analog, promotes survival of cardiomyocytes in cardiovascular diseases, including diabetic cardiomyopathy. However, the role of Exendin-4 in cardiac pyroptosis remains to be elucidated. Our study revealed that Exendin-4 treatment protected against heart remolding and dysfunction and attenuated cardiac inflammation in high-fat diet-fed rats. The activity of caspase-1 and production of pyroptotic cytokines were significantly inhibited by Exendin-4 treatment in the diabetic heart and in high glucose-treated cardiomyocytes as well. In an effort to understand the signaling mechanisms underlying the antipyroptotic property of Exendin-4, we found that blockade of AMPK, an oxidative stress sensor, activity diminished the antipyroptotic property of Exendin-4. Phosphorylation of AMPK resulted in degeneration of TXNIP that promoted the activation of the NLRP3 inflammasome. Exendin-4 treatment decreased the protein level of TXNIP. Moreover, RNA silencing of TXNIP mimicked the antipyroptotic actions of Exendin-4. These findings promoted us to propose a new signaling pathway mediating cardioprotective effect of Exendin-4 under hyperglycemic conditions: Exendin-4 → ROS↓ → pAMPK↑ → TXNIP↓ → caspase-1↓ → IL-1β and IL-18↓ → pyroptosis↓. In general, our study identified Exendin-4 as a pyroptotic inhibitor protecting against hyperglycemia-induced cardiomyocyte pyroptosis via the AMPK-TXNIP pathway.

LncRNA KLF3-AS1 in human mesenchymal stem cell-derived exosomes ameliorates pyroptosis of cardiomyocytes and myocardial infarction through miR-138-5p/Sirt1 axis

AimMyocardial infarction (MI) is a severe disease with increased mortality and disability rates, posing heavy economic burden for society. Exosomes were uncovered to mediate intercellular communication after MI. This study aims to explore the effect and mechanism of lncRNA KLF3-AS1 in exosomes secreted by human mesenchymal stem cells (hMSCs) on pyroptosis of cardiomyocytes and MI.MethodsExosomes from hMSCs were isolated and identified. Exosomes from hMSCs with transfection of KLF3-AS1 for overexpression were injected into MI rat model or incubated with hypoxia cardiomyocytes. Effect of KLF3-AS1 on MI area, cell viability, apoptosis, and pyroptosis was determined. The relationship among miR-138-5p, KLF3-AS1, and Sirt1 was verified by dual-luciferase reporter assay. Normal cardiomyocytes were transfected with miR-138-5p inhibitor or sh-Sirt1 to clarify whether alteration of miR-138-5p or sh-Sirt1 can regulate the effect of KLF3-AS1 on cardiomyocytes.ResultsExosomes from hMSCs were successfully extracted. Transfection of KLF3-AS1 exosome in rats and incubation with KLF3-AS1 exosome in hypoxia cardiomyocytes both verified that overexpression of KLF3-AS1 in exosomes leads to reduced MI area, decreased cell apoptosis and pyroptosis, and attenuated MI progression. KLF3-AS1 can sponge miR-138-5p to regulate Sirt1 expression. miR-138-5p inhibitor transfection and KLF3-AS1 exosome incubation contribute to attenuated pyroptosis and MI both in vivo and in vitro, while transfection of sh-Sirt1 could reverse the protective effect of exosomal KLF3-AS1 on hypoxia cardiomyocytes.ConclusionLncRNA KLF3-AS1 in exosomes secreted from hMSCs by acting as a ceRNA to sponge miR-138-5p can regulate Sirt1 so as to inhibit cell pyroptosis and attenuate MI progression.

Kanglexin, a novel anthraquinone compound, protects against myocardial ischemic injury in mice by suppressing NLRP3 and pyroptosis.

Pyroptosis is a form of inflammatory cell death that could be driven by the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation following myocardial infarction (MI). Emerging evidence suggests the therapeutic potential for ameliorating MI-induced myocardial damages by targeting NLRP3 and pyroptosis. In this study, we investigated the myocardial protection effect of a novel anthraquinone compound (4,5-dihydroxy-7-methyl-9,10-anthraquinone-2-ethyl succinate) named Kanglexin (KLX) in vivo and in vitro. Male C57BL/6 mice were pre-treated either with KLX (20, 40 mg· kg−1per day, intragastric gavage) or vehicle for 7 consecutive days prior to ligation of coronary artery to induce permanent MI. KLX administration dose-dependently reduced myocardial infarct size and lactate dehydrogenase release and improved cardiac function as compared to vehicle-treated mice 24 h after MI. We found that MI triggered NLRP3 inflammasome activation leading to conversion of interleukin-1β (IL-1β) and IL-18 into their active mature forms in the heart, which could expand the infarct size and drive cardiac dysfunction. We also showed that MI induced pyroptosis, as evidenced by increased DNA fragmentation, mitochondrial swelling, and cell membrane rupture, as well as increased levels of pyroptosis-related proteins, including gasdermin D, N-terminal GSDMD, and cleaved caspase-1. All these detrimental alterations were prevented by KLX. In hypoxia- or lipopolysaccharide (LPS)-treated neonatal mouse ventricular cardiomyocytes, we showed that KLX (10 μM) decreased the elevated levels of terminal deoxynucleotidyl transferase dUTP nick end labeling- and propidium iodide-positive cells, and pyroptosis-related proteins. We conclude that KLX prevents MI-induced cardiac damages and cardiac dysfunction at least partly through attenuating NLRP3 and subsequent cardiomyocyte pyroptosis, and it is worthy of more rigorous investigations for its potential for alleviating ischemic heart disease.

Doxorubicin induces cardiomyocyte pyroptosis via the TINCR-mediated posttranscriptional stabilization of NLR family pyrin domain containing 3

AimsDoxorubicin (DOX), a widely used powerful chemotherapeutic component for cancer treatment, can give rise to severe cardiotoxicity that limits its clinical use. Pyroptosis is characterized by proinflammation and has been defined as a new type of programmed cell death in recent years. However, whether the DOX-induced cardiotoxicity is related to pyroptosis, and if so, which genes are involved in this process is largely unknown. In this study, we sought to identify the effect of DOX on cardiomyocyte pyroptosis and further reveal the underlying regulatory mechanism. Methods and resultsIn vitro and in vivo experiments showed that DOX treatment induced cardiomyocyte pyroptosis as evidenced by increased cell death and upregulated expression levels of NLR family pyrin domain containing 3 (NLRP3), caspase-3, IL-1β, IL-18 and GMDSD-N. Inhibition of NLRP3 rescued the DOX-induced pyroptosis. qRT-PCR showed that TINCR lncRNA was upregulated by DOX treatment and knockdown of TINCR reversed the DOX-induced pyroptosis both in vitro and in vivo. Mechanistic investigations revealed that TINCR increased NLRP3 level via recruiting IGF2BP1 to enhance NLRP3 mRNA. And the effect of TINCR on cardiomyocyte pyroptosis was attenuated by the inhibition of NLRP3 or IGF2BP1. Finally, TINCR was not involved in DOX-induced pyroptosis in cancer cells. ConclusionTINCR mediates the DOX-induced cardiotoxicity and pyroptosis in an IGF2BP1-dependent manner. Therefore, TINCR may serve as a promising therapeutic target to overcome the cardiotoxicity of chemotherapy for cancer therapy.

A Novel Circular RNA Mediates Pyroptosis of Diabetic Cardiomyopathy by Functioning as a Competing Endogenous RNA

Diabetic cardiomyopathy (DCM) is a vital cause of fatalities in diabetic patients. The programmed death of cardiomyocytes and inflammation critically contribute to cardiac hypertrophy and fibrosis in DCM. Furthermore, circular RNA (circRNA) is a key regulator of various diseases. However, the role of circRNAs in DCM remains to be elucidated. Our previous study found that pyroptosis was markedly activated in the cardiomyocytes subjected to high-glucose conditions, and miR-214-3p regulated the expression of caspase-1. The aim of this study was to elucidate whether circRNA is involved in DCM pyroptosis via the miR-214-3p/caspase-1 pathway. Herein, we identified that hsa_circ_0076631, named caspase-1-associated circRNA (CACR), was increased both in high-glucose-treated cardiomyocytes and in the serum of diabetic patients. CACR also sponged an endogenous miR-214-3p to sequester and inhibit its expression. CACR knockdown in cardiomyocytes counteracted high-glucose-induced caspase-1 activation. Conversely, miR-214-3p knockdown partially abolished the beneficial effects of CACR silencing on pyroptosis in cardiomyocytes. Therefore, this study elucidated that CACR might be a novel therapeutic target via the CACR/miR-214-3p/caspase-1 pathway in DCM.

Calpain silencing alleviates myocardial ischemia-reperfusion injury through the NLRP3/ASC/Caspase-1 axis in mice

AimsPrior to reperfusion, Calpains remain inactive due to the acidic pH and elevated ionic strength in the ischemic myocardium; but Calpain is activated during myocardial reperfusion. The underlying mechanism of Calpain activation in the ischemia-reperfusion (I/R) is yet to be determined. Therefore, the present study aims to investigate the mechanism of Calpain in I/R-induced mice. Main methodsIn order to detect the function of Calpain and the NLRP3/ASC/Caspase-1 axis in cardiomyocyte pyroptosis, endoplasmic reticulum (ER) stress and myocardial function, the cardiomyocytes were treated with hypoxia-reoxygenation (H/R), and NLRP3 were silenced, Calpain was overexpressed and Caspase-1 inhibitors were used to determine cardiomyocyte pyroptosis. The results obtained from the cell experiments were then verified with an animal experiment in I/R mice. Key findingsThere was an overexpression in Calpain, ASC, NLRP3, GRP78 and C/EBP homologous protein (CHOP) in cardiomyocytes following H/R. A significant increase was witnessed in lactic acid dehydrogenase (LDH) activity, cardiomyocyte pyroptosis rate, Calpain activity, reactive oxygen species (ROS) concentration, as well as activation of ER stress in cardiomyocytes after H/R. However, opposing results were observed in H/R cardiomyocytes that received siRNA Calpain, siRNA NLRP3 or Caspase-1 inhibitor treatment. Overall, the results obtained from the animal experiment were consistent with the results from the cell experiment. SignificanceThe silencing of Calpain suppresses the activation of the NLRP3/ASC/Caspase-1 axis, thus inhibiting ER stress in mice and improving myocardial dysfunction induced by I/R, providing a novel therapeutic pathway for I/R.

STING-IRF3 contributes to lipopolysaccharide-induced cardiac dysfunction, inflammation, apoptosis and pyroptosis by activating NLRP3

Mountainous evidence suggests that inflammation, cardiomyocyte apoptosis and pyroptosis are involved in the development of sepsis and sepsis-induced cardiomyopathy (SIC). Stimulator of interferon genes (STING) is an indispensable molecule that could regulate inflammation and immune response in multiple diseases. However, the role of STING in cardiovascular disease, especially SIC remains unclear. This study was designed to investigate the potential molecular mechanisms of STING in lipopolysaccharide (LPS)-induced cardiac injury using STING global knockout mice. In wild type mice and cardiomyocytes, LPS stimulation triggered the perinuclear translocation of STING, which further bound to Type-I interferons (IFN) regulatory factor 3 (IRF3) and phosphorylated IRF3. Phosphorylated (P-) IRF3 subsequently translocated into nucleus and increased the expression of NOD-like receptor protein 3 (NLRP3). Knockout of STING in mice significantly improved survival rate and cardiac function, apart from suppressing myocardial and serum inflammatory cytokines, apoptosis, as well as cardiomyocyte pyroptosis. In vitro experiments revealed that NLRP3 overexpression by adenovirus could offset protective effects of STING knockdown in LPS-induced cardiomyocytes. Additionally, LPS stimulation also promoted the production of intracellular reactive oxygen (ROS), which further induced the NLRP3 translocation to the cytoplasm from the nucleus. Dissociative TXNIP could directly interact with cytoplasmic NLRP3 and form inflammasome, eventually triggering cardiomyocyte injury. Collectively, our findings disclose that STING deficiency could alleviate LPS-induced SIC in mice. Hence, targeting STING in cardiomyocytes may be a promising therapeutic strategy for preventing SIC.

Emodin alleviates myocardial ischemia/reperfusion injury by inhibiting gasdermin D-mediated pyroptosis in cardiomyocytes.

BackgroundEmodin has recently been reported to have a powerful antiinflammatory effect, protecting the myocardium against ischemia/reperfusion (I/R) injury. Pyroptosis is a proinflammatory programmed cell death that is related to many diseases. The present study investigated the effect of emodin on pyroptosis in cardiomyocytes.Materials and methodsSprague Dawley rats were randomly divided into sham, I/R, and I/R+Emodin groups. I/R model was subjected to 30 minutes’ ligation of left anterior descending coronary artery, followed by 2 hours of reperfusion. Cardiomyocytes were exposed to hypoxic conditions for 1 hour and normoxic conditions for 2 hours. The level of the pyroptosis was detected by Western blot, real-time PCR analysis, and ELISA.ResultsThe level of gasdermin D-N domains was upregulated in cardiomyocytes during I/R or hypoxia/reoxygenation (H/R) treatment. Moreover, emodin increased the rate of cell survival in vitro and decreased the myocardial infarct size in vivo via suppressing the levels of I/R-induced pyroptosis. Additionally, the expression of TLR4, MyD88, phospho-IκBα, phospho-NF-κB, and the NLRP3 inflammasome was significantly upregulated in cardiomyocytes subjected to H/R treatment, while emodin suppressed the expression of these proteins.ConclusionThis study confirms that emodin treatment was able to alleviate myocardial I/R injury and inhibit pyroptosis in vivo and in vitro. The inhibitory effect of emodin on pyroptosis was mediated by suppressing the TLR4/MyD88/NF-κB/NLRP3 inflammasome pathway. Therefore, emodin may provide an alternative treatment for myocardial I/R injury.

Lipopolysaccharide (LPS) Aggravates High Glucose- and Hypoxia/Reoxygenation-Induced Injury through Activating ROS-Dependent NLRP3 Inflammasome-Mediated Pyroptosis in H9C2 Cardiomyocytes.

Diabetes aggravates myocardial ischemia-reperfusion (I/R) injury because of the combination effects of changes in glucose and lipid energy metabolism, oxidative stress, and systemic inflammatory response. Studies have indicated that myocardial I/R may coincide and interact with sepsis and inflammation. However, the role of LPS in hypoxia/reoxygenation (H/R) injury in cardiomyocytes under high glucose conditions is still unclear. Our objective was to examine whether lipopolysaccharide (LPS) could aggravate high glucose- (HG-) and hypoxia/reoxygenation- (H/R-) induced injury by upregulating ROS production to activate NLRP3 inflammasome-mediated pyroptosis in H9C2 cardiomyocytes. H9C2 cardiomyocytes were exposed to HG (30 mM) condition with or without LPS, along with caspase-1 inhibitor (Ac-YVAD-CMK), inflammasome inhibitor (BAY11-7082), ROS scavenger N-acetylcysteine (NAC), or not for 24 h, then subjected to 4 h of hypoxia followed by 2 h of reoxygenation (H/R). The cell viability, lactate dehydrogenase (LDH) release, caspase-1 activity, and intracellular ROS production were detected by using assay kits. The incidence of pyroptosis was detected by calcein-AM/propidium iodide (PI) double staining kit. The concentrations of IL-1β and IL-18 in the supernatants were assessed by ELISA. The mRNA levels of NLRP3, ASC, and caspase-1 were detected by qRT-PCR. The protein levels of NF-κB p65, NLRP3, ASC, cleaved caspase-1 (p10), IL-1β, and IL-18 were detected by western blot. The results indicated that pretreatment LPS with 1 μg/ml not 0.1 μg/ml could efficiently aggravate HG and H/R injury by activating NLRP3 inflammasome to mediate pyroptosis in H9C2 cells, as evidenced by increased LDH release and decreased cell viability in the cells, and increased expression of NLRP3, ASC, cleaved caspase-1 (p10), IL-1β, and IL-18. Meanwhile, Ac-YVAD-CMK, BAY11-7082, or NAC attenuated HG- and H/R-induced H9C2 cell injury with LPS stimulated by reversing the activation of NLRP3 inflammasome-mediated pyroptosis. In conclusion, LPS could increase the sensitivity of H9C2 cells to HG and H/R and aggravated HG- and H/R-induced H9C2 cell injury by promoting ROS production to induce NLRP3 inflammasome-mediated pyroptosis.

Mesenchymal Stem Cells-Derived Exosomal microRNA-182-5p Downregulates Gasdermin D and Ameliorates Myocardial Ischemia/Reperfusion Injury

Objective: Exosomes contain abundant microRNAs (miRNAs/miRs) and play an intermediary role in cell-to-cell communications. Recently, miR-182-5p was found to have cardioprotective effects against ischemia/reperfusion (I/R) injury. However, whether exosomes delivering miR-182-5p functions post I/R injury remains unknown. The present study aimed at exploring whether exosomal miR-182-5p would protect against myocardial I/R injury-induced pyroptosis. Methods: I/R mouse model was developed by left anterior descending coronary artery occlusion. Myocardial cell exposed to hypoxia/reoxygenation (H/R) was also used as in vitro I/R model. Binding relationship between miR-182-5p and gasdermin D (GSDMD) was evaluated by dual-luciferase reporter gene assay. MiR-182-5p and GSDMD loss- and gain-of-function experiments were conducted to explore the effects of miR-182-5p via mesenchymal stem cells (MSCs)-derived exosomes on I/R- or H/R- induced pyroptosis, cell viability, pyroptosis-related proteins and inflammatory cytokines, reactive oxygen species (ROS) production, and lactate dehydrogenase (LDH) release in vitro and in vivo. Results: miR-182-5p expression was decreased while GSDMD increased in I/R-injured myocardial tissues. In addition, miR-182-5p targeted and reduced GSDMD expression. Overexpression of GSDMD facilitated H/R-induced myocardial cell pyroptosis and inhibited viability, accompanied by increased LDH release, ROS production, and levels of pyroptosis markers. Importantly, transfer of miR-182-5p-loaded exosomes derived from MSCs downregulated GSDMD in H/R-exposed cardiomyocytes. Moreover, miR-182-5p-loaded exosome injection significantly improved cardiac function and decreased myocardial infarction and cell damage in I/R mice. Conclusions: Collectively, results from the present study demonstrated that transfer of miR-182-5p-loaded exosomes derived from MSCs downregulated GSDMD and alleviated I/R-induced pyroptosis in cardiomyocytes. Funding Statement: This study was supported by the National Natural Science Foundation of China (81600232), the Key Projects of Education Department of Sichuan Province (17ZA0183), Municipal-School Cooperative Scientific Research Project of Nanchong City (18SXHZ0458; NSMC20170210), and Doctoral Research Initiation Foundation of North Sichuan Medical College (CBY15-QD12). Declaration of Interests: The authors declare that they have no conflicts of interest. Ethics Approval Statement: All animal experiments were in compliance with the Guide for the Care and Use of Laboratory Animal (8th Edition, National Institute of Health, 2011) and approved by Animal Care Committee, North Sichuan Medical College First Affiliated Hospital.

GW29-e1490 Macrophage-Derived miR-155-Containing Exosomes Promote Cardiomyocyte Pyroptosis by Directly Targeting Foxo3a in Uremic Cardiomyopathy

GW29-e1490 Macrophage-Derived miR-155-Containing Exosomes Promote Cardiomyocyte Pyroptosis by Directly Targeting Foxo3a in Uremic Cardiomyopathy

GW29-e0741 The clock gene, brain and muscle Arnt-like 1, regulates NLRP3 inflammasome in diabetic cardiomyocyte pyroptosis

GW29-e0741 The clock gene, brain and muscle Arnt-like 1, regulates NLRP3 inflammasome in diabetic cardiomyocyte pyroptosis

Trimetazidine Attenuates Cardiac Dysfunction in Endotoxemia and Sepsis by Promoting Neutrophil Migration.

Aims: Cardiac dysfunction can be a fatal complication during severe sepsis. The migration of neutrophils is significantly impaired during severe sepsis. We sought to determine the role of trimetazidine (TMZ) in regulation of neutrophil migration to the heart in a mouse model of sepsis and endotoxemia, and to identify the mechanism whereby TMZ confers a survival advantage.Methods and Results: C57/BL6 mice were (1) injected with LPS followed by 24-h TMZ administration, or (2) treated with TMZ (20 mg/kg/day) for 1 week post cecal ligation and puncture (CLP) operation. Echocardiography and Millar system detection showed that TMZ alleviated cardiac dysfunction and histological staining showed the failure of neutrophils migration to heart in both LPS- and CLP-induced mice. Bone marrow transplantation revealed that TMZ-pretreated bone marrow cells improved LPS- and CLP-induced myocardial dysfunction and enhanced neutrophil recruitment in heart. In CXCL2-mediated chemotaxis assays, TMZ increased neutrophils migration via AMPK/Nrf2-dependent up-regulation of CXCR2 and inhibition of GRK2. Furthermore, using luciferase reporter gene and chromatin immunoprecipitation assays, we found that TMZ promoted the binding of the Nrf2 and CXCR2 promoter regions directly. Application of CXCR2 inhibitor completely reversed the protective effects of TMZ in vivo. Co-culture of neutrophils and cardiomyocytes further validated that TMZ decreased LPS-induced cardiomyocyte pyroptosis by targeting neutrophils.Conclusion: Our findings suggested TMZ as a potential therapeutic agent for septic or endotoxemia associated cardiac dysfunction in mice.STUDY HIGHLIGHTS What is the current knowledge on the topic?Migration of neutrophils is significantly impaired during severe sepsis, but the underlying mechanisms remain unknown.What question did this study address?The effects of TMZ on cardiac dysfunction via neutrophils migration.What this study adds to our knowledgeTMZ attenuated LPS-induced cardiomyocyte pyroptosis and cardiac dysfunction by promoting neutrophils recruitment to the heart tissues via CXCR2.How this might change clinical pharmacology or translational scienceOur findings suggested TMZ as a potential therapeutic agent for septic cardiac dysfunction.

NF-κB-Gasdermin D (GSDMD) Axis Couples Oxidative Stress and NACHT, LRR and PYD Domains-Containing Protein 3 (NLRP3) Inflammasome-Mediated Cardiomyocyte Pyroptosis Following Myocardial Infarction.

BackgroundPyroptosis and oxidative stress play pivotal roles in cardiomyocyte loss after myocardial infarction. NF-κB is associated with oxidative stress and gasdermin D (GSDMD), the effector molecule of pyroptosis. However, the exact relationship between oxidative stress and cardiomyocyte pyroptosis remains unknown.Material/MethodsWe measured inflammasome-mediated cardiomyocyte pyroptosis in vivo via membrane pore formation, lactate dehydrogenase (LDH) release, and expression of caspase-1, cleaved caspase-1, NACHT, LRR and PYD domains-containing protein 3 (NLRP3), and apoptosis-associated speck-like protein containing a CARD (ASC). Furthermore, we induced pyroptosis in vitro by oxygen-glucose deprivation (OGD) in H9C2 cells. NLRP3 inflammasome-mediated pyroptosis was confirmed by LDH assay kit and Western blot. Oxidative stress was evaluated by reactive oxygen species (ROS) and superoxide dismutase (SOD) activity. We suppressed oxidative stress with N-acetyl-cysteine (NAC) and measured subsequent changes to the NF-κB-GSDMD axis and pyroptosis by LDH assay kit and Western blot. Then, we inhibited NF-κB activation with pyrrolidine dithiocarbamate (PDTC) and measured changes to GSDMD activity and pyroptosis by qRT-PCR, Western blot, and LDH assay kit.ResultsSuppression of oxidative stress by NAC reduced NF-κB and GSDMD activation and increased pyroptosis, characterized by LDH release and NLRP3 inflammasome activation in H9C2 cells under OGD. Moreover, inhibition of NF-κB activation reduced GSDMD transcription and activation and NLRP3 inflammasome-mediated pyroptosis of H9C2 cells under OGD.ConclusionsWe demonstrated that the NF-κB-GSDMD axis functioned as a bridge between oxidative stress and NLRP3 inflammasome-mediated cardiomyocyte pyroptosis. Our findings provide important insight into the mechanism of myocardial infarction-related ventricular remodeling.

Abstract 372: MicroRNA-9 Inhibits Hyperglycemia-induced Cardiac Pyroptosis by Targeting ELAVL1

Diabetic cardiomyopathy is a common complication in patients with diabetes and is associated with underlying chronic inflammation and cardiac cell death, subsequently leading to left ventricular dysfunction and heart failure. ELAV-like protein 1 (ELAVL1, mRNA stabilizing protein) and NLRP3 activation (inflammasome complex protein)-mediated IL-1beta synthesis play a critical role in the progression of heart failure. However, ELAVL1 regulation of pyroptosis (caspase-1-mediated programmed apoptosis) and associated IL-1beta release in cardiomyocytes, especially under diabetic condition, remains elusive. Human diabetic, non-diabetic heart tissues, human ventricular cardiomyocytes and rat cardiomyoblasts exposed to high glucose (HG) were used for our studies. Our data demonstrates that human ventricular cardiomyocytes exposed to high glucose condition showed significant increase in ELAVL1 and NLRP3 expression with a concomitant increase in caspase-1 and IL-1 beta expression. Furthermore, human cardiac tissue from diabetic patients showed increased ELAVL1, caspase-1 and NLRP3 expression as compared to non-diabetic hearts. Intriguingly, ELAVL1 knockdown abrogates TNF-α induced canonical pyroptosis via NLRP3, caspase-1 and IL-1beta suppression. Interestingly, miRNA-9 expression significantly reduces in high glucose treated cardiomyocytes and in human diabetic hearts. Bioinformatics analysis and target validation assays showed that miR-9 directly targets ELAVL1. Inhibition of miR-9 up regulates ELAVL1 expression and activates caspase-1. Alternatively, miR-9 mimics attenuate hyperglycemia-induced ELAVL1 and inhibit cardiomyocyte pyroptosis. To our knowledge, this is the first report to demonstrate the role of miR-9/ELAVL1 in hyperglycemia-induced cardiac pyroptosis. Taken together our study highlights the potential therapeutic implications in preventing cardiomyocyte cell loss in human diabetic failing heart.

MicroRNA-9 inhibits hyperglycemia-induced pyroptosis in human ventricular cardiomyocytes by targeting ELAVL1.

Diabetic cardiomyopathy is a common complication in patients with diabetes and is associated with underlying chronic inflammation and cardiac cell death, subsequently leading to heart failure (HF). ELAV-like protein 1 (ELAVL1) plays a critical role in the progression of inflammation and HF. However the role of ELAVL-1 in inflammation induced cardiac cell death (pyroptosis) under hyperglycemic condition remains elusive. Our data demonstrates that ELAVL1 expression augmented with a concomitant increase in caspase-1 and IL-1 beta expression in human hearts and human ventricular cardiomyocytes under hyperglycemic condition. Furthermore, ELAVL1 knockdown abrogates TNF-α induced canonical pyroptosis via NLRP3, caspase-1 and IL-1beta suppression. Bioinformatics analysis and target validation assays showed that miR-9 directly targets ELAVL1. Interestingly, miRNA-9 expression significantly reduced in high glucose treated cardiomyocytes and in human diabetic hearts. Inhibition of miR-9 upregulates ELAVL1 expression and activates caspase-1. Alternatively, treatment with miR-9 mimics attenuates hyperglycemia-induced ELAVL1 and inhibits cardiomyocyte pyroptosis. Taken together our study highlights the potential therapeutic implications of targeting miR-9/ELAVL1 in preventing cardiomyocyte cell loss during HF in diabetics.

MicroRNA-30d regulates cardiomyocyte pyroptosis by directly targeting foxo3a in diabetic cardiomyopathy.

Diabetic cardiomyopathy is a common cardiac condition in patients with diabetes mellitus, which can result in cardiac hypertrophy and subsequent heart failure, associated with pyroptosis, the pro-inflammatory programmed cell death. MicroRNAs (miRNAs), small endogenous non-coding RNAs, have been shown to be involved in diabetic cardiomyopathy. However, whether miRNAs regulate pyroptosis in diabetic cardiomyopathy remains unknown. Our study revealed that mir-30d expression was substantially increased in streptozotocin (STZ)-induced diabetic rats and in high-glucose-treated cardiomyocytes as well. Upregulation of mir-30d promoted cardiomyocyte pyroptosis in diabetic cardiomyopathy; conversely, knockdown of mir-30d attenuated it. In an effort to understand the signaling mechanisms underlying the pro-pyroptotic property of mir-30d, we found that forced expression of mir-30d upregulated caspase-1 and pro-inflammatory cytokines IL-1β and IL-18. Moreover, mir-30d directly repressed foxo3a expression and its downstream protein, apoptosis repressor with caspase recruitment domain (ARC). Furthermore, silencing ARC by siRNA mimicked the action of mir-30d: upregulating caspase-1 and inducing pyroptosis. These findings promoted us to propose a new signaling pathway leading to cardiomyocyte pyroptosis under hyperglycemic conditions: mir-30d↑→foxo3a↓→ ARC↓→caspase-1↑→IL-1β, IL-18↑→pyroptosis↑. Therefore, mir-30d may be a promising therapeutic target for the management of diabetic cardiomyopathy.