Cardiomyocyte Pyroptosis Research Articles

Amentoflavone mitigates doxorubicin-induced cardiotoxicity by suppressing cardiomyocyte pyroptosis and inflammation through inhibition of the STING/NLRP3 signalling pathway

BackgroundDoxorubicin (DOX) is a potent anticancer chemotherapeutic agent whose clinical application is substantially constrained by its cardiotoxicity. The pathophysiology of DOX-induced cardiotoxicity manifests as cardiomyocyte pyroptosis and inflammation. Amentoflavone (AMF) is a naturally occurring biflavone possessing anti-pyroptotic and anti-inflammatory properties. However, the mechanism through which AMF alleviates DOX-induced cardiotoxicity remains undetermined. PurposeThis study aimed at investigating the role of AMF in alleviating DOX-induced cardiotoxicity. Study design and methodsTo assess the in vivo effect of AMF, DOX was intraperitoneally administered into a mouse model to induce cardiotoxicity. To elucidate the underlying mechanisms, the activities of STING/NLRP3 were quantified using the NLRP3 agonist nigericin and the STING agonist amidobenzimidazole (ABZI). Primary cardiomyocytes isolated from neonatal Sprague-Dawley rats were treated with saline (vehicle) or DOX with or without AMF and/or ABZI. The echocardiogram, haemodynamics, cardiac injury markers, heart/body weight ratio, and pathological alterations were monitored; the STING/NLRP3 pathway-associated proteins were detected by western blot and cardiomyocyte pyroptosis was analysed by immunofluorescence staining of cleaved N-terminal GSDMD and scanning electron microscopy. Furthermore, we evaluated the potential of AMF in compromising the anticancer effects of DOX in human breast cancer cell lines. ResultsAMF substantially alleviated cardiac dysfunction and reduced heart/body weight ratio and myocardial damage in mice models of DOX-induced cardiotoxicity. AMF effectively suppressed DOX-mediated upregulation of IL-1β, IL-18, TNF-α, and pyroptosis-related proteins, including NLRP3, cleaved caspase-1, and cleaved N-terminal GSDMD. The levels of apoptosis-related proteins, namely Bax, cleaved caspase-3, and BCL-2 were not affected. In addition, AMF inhibited STING phosphorylation in DOX-affected hearts. Intriguingly, the administration of nigericin or ABZI dampened the cardioprotective effects of AMF. The in vitro anti-pyroptotic effect of AMF was demonstrated in attenuating the DOX-induced reduction in cardiomyocyte cell viability, upregulation of cleaved N-terminal GSDMD, and pyroptotic morphology alteration at the microstructural level. AMF exhibited a synergistic effect with DOX to reduce the viability of human breast cancer cells. ConclusionAMF alleviates DOX-induced cardiotoxicity by suppressing cardiomyocyte pyroptosis and inflammation via inhibition of the STING/NLRP3 signalling pathway, thereby validating its efficacy as a cardioprotective agent.

Dibutyl phthalate causes heart damage by disrupting Ca2+ transfer from endoplasmic reticulum to mitochondria and triggering subsequent pyroptosis

Dibutyl phthalate (DBP) is a typical plasticizer and is widely used in industrial manufacturing. DBP has been reported to be cardiotoxic, manifested by oxidative stress and inflammatory damage. However, the potential mechanism of heart damage caused by DBP remains unclear. By in vivo and in vitro experiments, first, this study demonstrated that DBP induced endoplasmic reticulum (ER) stress, mitochondrial damage, and pyroptosis in cardiomyocytes; second, it was confirmed that the ER stress increased mitochondrial-associated ER membrane (MAM), which led to mitochondrial damage by abnormalizing Ca2+ transfer within MAMs; finally, it was confirmed that mitochondrial reactive oxygen species (mtROS) production was increased after mitochondrial damage, which activated NLRP3 inflammasome and pyroptosis in cardiomyocytes. In summary, ER stress is the initiation of DBP cardiotoxicity, which leads to mitochondrial damage by disrupting Ca2+ transfer from ER to mitochondria. Subsequently, released mtROS promotes the activation of NLRP3 inflammasome and pyroptosis, eventually leading to heart damage.

Dihydroartemisinin alleviates mitochondrial damage and improves cardiomyocyte pyroptosis and ferroptosis through Sirt1-mediated PGC-1α

This study assesses the effect of dihydroartemisinin on pyroptosis and ferroptosis. Rat H9C2 cardiomyocytes were intervened with 35 mmol/L high glucose through assigned blank control, dihydro artemisinin, and dihydroartemisinin+Sirt1 groups. Confocal microscopy was used to observe the ROS levels, while proliferation ability was detected by CCK-8 method, and apoptosis was assessed by flow cytometry, and migration ability by Transwell transfer method. Moreover, analysis of pyroptosis-related factors expression and content of lipid peroxide were done using laser confocal microscopy. The average fluorescence intensity of dihydro artemisinin group and dihydroartemisinin+SIRT 1 group decreased significantly (P <0.05), among which the dihydroartemisinin+SIRT 1 group had lowest average fluorescence intensity (P <0.05). SIRT 1 level in the dihydroartemisinin and dihydroartemisinin+SIRT 1 groups was higher than blank control (P <0.05), with highest level in the dihydroartemisinin+SIRT 1 group (P <0.05). Cell proliferation in the dihydroartemisinin and dihydroartemisinin+SIRT 1 group was reduced (P <0.05), with lowest proliferation in combination group (P < 0.05). Cell migration in the dihydroartemisinin and dihydroartemisinin+SIRT 1 groups was reduced (P <0.05), with lowest number of migratory cells in the dihydroartemisinin+SIRT 1 group (P <0.05). Cell apoptosis in the dihydroartemisinin and dihydroartemisinin+SIRT 1 groups was increased (P <0.05), with lowest apoptosis in the dihydroartemisinin+SIRT 1 group (P <0.05). There was upregulation of SIRT 1 and PGC-1α mRNA expression in the dihydroartemisinin and dihydroartemisinin+SIRT 1 groups was elevated (P <0.05). The expression of NLRP3, GSDMD, and Caspase-1 were all decreased (P <0.05), while that of GPX4 was increased (P <0.05). Dihydroartemisinin inhibits the function of H9C2 cardiomyocytes, pyroptosis and ferroptosis, playing a positive role in ameliorating Diabetic cardiomyopathy (DCM).

TRPA1 deficiency aggravates dilated cardiomyopathy by promoting S100A8 expression to induce M1 macrophage polarization in rats.

Transient receptor potential ankyrin 1 (TRPA1) plays an important role in different cardiovascular diseases. However, the role of TRPA1 in dilated cardiomyopathy (DCM) remains unclear. Here, we aimed to investigate the role of TRPA1 in DCM induced by doxorubicin (DOX) and explore its possible mechanisms. GEO data were used to explore the expression of TRPA1 in DCM patients. DOX (2.5 mg/kg/week, 6 weeks, i.p.) was used to induce DCM. Bone marrow-derived macrophages (BMDMs) and neonatal rat cardiomyocytes (NRCMs) were isolated to explore the role of TRPA1 in macrophage polarization, cardiomyocyte apoptosis, and pyroptosis. In addition, DCM rats were treated with the TRPA1 activator, cinnamaldehyde to explore the possibility of clinical translation. TRPA1 expression was increased in left ventricular (LV) tissue in DCM patients and rats. TRPA1 deficiency aggravated the cardiac dysfunction, cardiac injury, and LV remodeling in DCM rats. In addition, TRPA1 deficiency promoted the M1 macrophage polarization, oxidative stress, cardiac apoptosis, and pyroptosis induced by DOX. RNA-seq results showed that TRPA1 knockout promoted the expression of S100A8, an inflammatory molecule that belongs to the family of Ca2+ -binding S100 proteins, in DCM rats. Furthermore, S100A8 inhibition attenuated M1 macrophage polarization in BMDMs isolated from TRPA1 deficiency rats. Recombinant S100A8 promoted the apoptosis, pyroptosis, and oxidative stress in primary cardiomyocytes stimulated with DOX. Finally, TRPA1 activation via cinnamaldehyde alleviated the cardiac dysfunction and reduced S100A8 expression in DCM rats. Taken together, these results suggested that TRPA1 deficiency aggravates DCM by promoting S100A8 expression to induce M1 macrophage polarization and cardiac apoptosis.

Ubiquitin-specific protease 11 Aggravates Ischemia-reperfusion-induced Cardiomyocyte Pyroptosis and Injury by Promoting TRAF3 Deubiquitination

In myocardial ischemia-reperfusion injury, myocardial damage is aggravated when blood perfusion is restored in myocardial infarction. Ubiquitin-specific protease 11 (USP11), a deubiquitinating enzyme, could remove the ubiquitination of substrate proteins and regulate protein stability, thereby affecting multiple pathological processes. This study aimed to investigate the potential function of USP11 in myocardial ischemia-reperfusion injury and its underlying mechanisms. In vivo and in vitro experimental study. The ischemia-reperfusion rat model in vivo was evolved, wherein the left anterior descending coronary artery was ligated for 30 min, followed by ligature release for 120 min. Meanwhile, H9C2 cells were brought to hypoxia for 6 h and then reoxygenated for 18 h to establish a cell hypoxia-reoxygenation (H/R) injury in vitro. Then, the loss-of-function experiments of USP11 were performed. Triphenyltetrazolium chloride and hematoxylin and eosin staining were performed to observe myocardial injury. The MTT assay was utilized to detect H9C2 cell viability. Pyroptosis was analyzed by TUNEL staining and flow cytometry. Pyroptosis-related protein expression and TRAF3 were analyzed via Western blot. The content of inflammatory factors was examined by enzyme-linked immunoassay. Co-immunoprecipitation and ubiquitination assays were performed to analyze for USP11 interacting with TRAF3. USP11 was upregulated in the ischemic heart tissue. Ischemia-reperfusion and H/R injuries increased USP11 expression. USP11 loss-of-function assays showed that USP11 knockdown alleviated ischemia-reperfusion- and H/R-induced myocardial cell damage, pyroptosis, pro-inflammatory factor secretion, and IKKβ/NF-κB pathway activation. In H9C2 cells, USP11 stabilized TRAF3 by deubiquitination. Furthermore, rescue experiments revealed that TRAF3 overexpression reversed the protection of silencing USP11 on H/R-induced H9C2 cell injury. This study confirmed that USP11 knockdown ameliorated myocardial ischemia-reperfusion injury by downregulating TRAF3, suggesting that USP11 silencing can be a novel target of myocardial infarction.

The lncRNA HOTAIR attenuates pyroptosis of diabetic cardiomyocytes by recruiting FUS to regulate SIRT3 expression.

Diabetic cardiomyopathy (DCM) is a serious cardiovascular complication of diabetes that severely affects the quality of life of diabetic patients. Long noncoding RNAs (lncRNAs) play important roles in the pathogenesis of DCM. However, the role of the lncRNA homeobox transcript antisense RNA (HOTAIR) in the progression of DCM remains unclear. The present study aimed to investigate the role of HOTAIR in high glucose (HG)-induced pyroptosis in cardiomyocytes. The expression of the lncRNA HOTAIR, FUS, and SIRT3 in H9C2 cardiomyocytes was detected by RT-qPCR. Western blotting was used to evaluate the expression of FUS and SIRT3 as well as that of pyroptosis- and inflammation-related proteins. RT-qPCR and ELISA were used to determine the expression and secretion of IL-1β and IL-18. RNA pulldown and RIP experiments were used to validate the binding relationship among HOTAIR, FUS, and SIRT3. Flow cytometry was performed to detect pyroptosis. HG induced pyroptosis and elevated the expression of proteins associated with pyroptosis and inflammation (NLRP3, GSDMD-N, cleaved caspase-1, IL-1β, and IL-18) in cardiomyocytes. HOTAIR and SIRT3 levels were decreased in HG-exposed H9C2 cells. Additionally, overexpression of HOTAIR inhibited the HG-induced pyroptosis and inflammatory response in cardiomyocytes. HOTAIR upregulated SIRT3 expression in H9C2 cells by targeting FUS. Moreover, SIRT3 upregulation suppressed HG-mediated pyroptosis of cardiomyocytes. Notably, SIRT3 depletion reversed the inhibitory effect of HOTAIR on HG-triggered pyroptosis in cardiomyocytes. Our research indicates that HOTAIR alleviates pyroptosis in diabetic cardiomyocytes through the FUS/SIRT3 axis, providing a potential marker for the diagnosis and treatment of DCM.

GSDMD-mediated pyroptosis promotes cardiac remodeling in pressure overload

ABSTRACT Background Gasdermin D (GSDMD) forms membrane pores to execute pyroptosis. But the mechanism of how cardiomyocyte pyroptosis induces cardiac remodeling in pressure overload remains unclear. We investigated the role of GSDMD-mediated pyroptosis in the pathogenesis of cardiac remodeling in pressure overload. Methods Wild-type (WT) and cardiomyocyte-specific GSDMD-deficient (GSDMD-CKO) mice were subjected to transverse aortic constriction (TAC) to induce pressure overload. Four weeks after surgery, left ventricular structure and function were evaluated by echocardiographic, invasive hemodynamic and histological analysis. Pertinent signaling pathways related to pyroptosis, hypertrophy and fibrosis were investigated by histochemistry, RT-PCR and western blotting. The serum levels of GSDMD and IL-18 collected from healthy volunteers or hypertensive patients were measured by ELISA. Results We found TAC induced cardiomyocyte pyroptosis and release of pro-inflammatory cytokines IL-18. The serum GSDMD level was significantly higher in hypertensive patients than in healthy volunteers, and induced more dramatic release of mature IL-18. GSDMD deletion remarkably mitigated TAC-induced cardiomyocyte pyroptosis. Furthermore, GSDMD deficiency in cardiomyocytes significantly reduced myocardial hypertrophy and fibrosis. The deterioration of cardiac remodeling by GSDMD-mediated pyroptosis was associated with activating JNK and p38 signaling pathways, but not ERK or Akt signaling pathway. Conclusion In conclusion, our results demonstrate that GSDMD serves as a key executioner of pyroptosis in cardiac remodeling induced by pressure overload. GSDMD-mediated pyroptosis activates JNK and p38 signaling pathways, and this may provide a new therapeutic target for cardiac remodeling induced by pressure overload.

CircRNA DICAR as a novel endogenous regulator for diabetic cardiomyopathy and diabetic pyroptosis of cardiomyocytes

In this study, we identified that a conserved circular RNA (circRNA) DICAR, which was downregulated in diabetic mouse hearts. DICAR had an inhibitory effect on diabetic cardiomyopathy (DCM), as the spontaneous cardiac dysfunction, cardiac cell hypertrophy, and cardiac fibrosis occurred in DICAR deficiency (DICAR+/−) mice, whereas the DCM was alleviated in DICAR-overexpressed DICARTg mice. At the cellular level, we found that overexpression of DICAR inhibited, but knockdown of DICAR enhanced the diabetic cardiomyocyte pyroptosis. At the molecular level, we identified that DICAR-VCP-Med12 degradation could be the underlying molecular mechanism in DICAR-mediated effects. The synthesized DICAR junction part (DICAR-JP) exhibited a similar effect to the entire DICAR. In addition, the expression of DICAR in circulating blood cells and plasma from diabetic patients was lower than that from health controls, which was consistent with the decreased DICAR expression in diabetic hearts. DICAR and the synthesized DICAR-JP may be drug candidates for DCM.

Resveratrol pretreatment alleviates NLRP3 inflammasome-mediated cardiomyocyte pyroptosis by targeting TLR4/MyD88/NF-κB signaling cascade in coronary microembolization-induced myocardial damage

Percutaneous coronary intervention and acute coronary syndrome are both closely tied to the frequently occurring complication of coronary microembolization (CME). Resveratrol (RES) has been shown to have a substantial cardioprotective influence in a variety of cardiac diseases, though its function and potential mechanistic involvement in CME are still unclear. The forty Sprague-Dawley rats were divided into four groups randomly: CME, CME + RES (25 mg/kg), CME + RES (50 mg/kg), and sham (10 rats per group). The CME model was developed. Echocardiography, levels of myocardial injury markers in the serum, and histopathology of the myocardium were used to assess the function of the cardiac muscle. For the detection of the signaling of TLR4/MyD88/NF-κB along with the expression of pyroptosis-related molecules, ELISA, qRT-PCR, immunofluorescence, and Western blotting were used, among other techniques. The findings revealed that myocardial injury and pyroptosis occurred in the myocardium following CME, with a decreased function of cardiac, increased levels of serum myocardial injury markers, increased area of microinfarct, as well as a rise in the expression levels of pyroptosis-related molecules. In addition to this, pretreatment with resveratrol reduced the severity of myocardial injury after CME by improving cardiac dysfunction, decreasing serum myocardial injury markers, decreasing microinfarct area, and decreasing cardiomyocyte pyroptosis, primarily by blocking the signaling of TLR4/MyD88/NF-κB and also reducing the NLRP3 inflammasome activation. Resveratrol may be able to alleviate CME-induced myocardial pyroptosis and cardiac dysfunction by impeding the activation of NLRP3 inflammasome and the signaling pathway of TLR4/MyD88/NF-κB.

The role of ROS-induced pyroptosis in CVD.

Cardiovascular disease (CVD) is the number one cause of death in the world and seriously threatens human health. Pyroptosis is a new type of cell death discovered in recent years. Several studies have revealed that ROS-induced pyroptosis plays a key role in CVD. However, the signaling pathway ROS-induced pyroptosis has yet to be fully understood. This article reviews the specific mechanism of ROS-mediated pyroptosis in vascular endothelial cells, macrophages, and cardiomyocytes. Current evidence shows that ROS-mediated pyroptosis is a new target for the prevention and treatment of cardiovascular diseases such as atherosclerosis (AS), myocardial ischemia-reperfusion injury (MIRI), and heart failure (HF).

MicroRNA-30e-3p reduces coronary microembolism-induced cardiomyocyte pyroptosis and inflammation by sequestering HDAC2 from the SMAD7 promoter.

This study investigates the mechanism by which microRNA (miR)-30e-3p reduces coronary microembolism (CME)-induced cardiomyocyte pyroptosis and inflammation. Cardiac function tests, histological staining, and transmission electron microscopy were performed on CME-model rats injected with adeno-associated viral vectors. Cardiomyocytes were transfected 24 h before a cellular model of pyroptosis was established via treatment with 1 μg/mL lipopolysaccharide (LPS) for 4 h and 5 mM ATP for 30 min. Pyroptosis, inflammation, and Wnt/β-catenin signaling in cardiomyocytes were detected. Dual-luciferase reporter assays and/or RNA pull-down assays were performed to verify the binding of miR-30e-3p to HDAC2 mRNA or HDAC2 to the SMAD7 promoter. Chromatin immunoprecipitation was used to assess the level of H3K27 acetylation at the SMAD7 promoter. miR-30e-3p and SMAD7 expression levels were downregulated and HDAC2 expression was upregulated with CME. The overexpression of miR-30e-3p restored cardiac functions in CME-model rats and reduced serum cTnI, IL-18, and IL-1β levels, microinfarcts, inflammatory cell infiltration, apoptosis, collagen content, and GSDMD-N, cleaved caspase-1, and NLRP3 expression in the myocardium, but these effects were reversed by SMAD7 knockdown. The overexpression of miR-30e-3p or knockdown of HDAC2 reduced LDH, IL-18, and IL-1β secretion, propidium iodide intake, and GSDMD-N, NLRP3, cleaved caspase-1, Wnt3a, Wnt5a, and β-catenin expression in the cardiomyocyte model. miR-30e-3p inhibited the expression of HDAC2 by binding HDAC2 mRNA. HDAC2 repressed the expression of SMAD7 by catalyzing H3K27 deacetylation at the SMAD7 promoter. miR-30e-3p, by binding HDAC2 to promote SMAD7 expression, reduces CME-induced cardiomyocyte pyroptosis and inflammation.

Ticagrelor reduces doxorubicin-induced pyroptosis of rat cardiomyocytes by targeting GSK-3β/caspase-1.

Doxorubicin (Dox) is a widely used clinical drug whose cardiotoxicity cannot be ignored. Pyroptosis (inflammatory cell death) has gradually gained attention in the context of Dox-induced cardiotoxicity. In addition to the inhibition of platelet activation by ticagrelor, little is known about its other pharmacological effects. Glycogen synthase kinase 3β (GSK-3β) has been shown to contribute to the pathological process of pyroptosis, but whether it is related to the potential role of ticagrelor is unclear. In this study, we investigated the effects of ticagrelor on Dox-induced pyroptosis in cardiomyocytes. Rats were treated with ticagrelor (7.5 mg/kg, i.g.) 1 h before intravenous injection of Dox (2.5 mg/kg), once every 3 days, six times in total. Hearts were collected for histochemical analysis and western blot detection 8 weeks after the last administration. Ticagrelor was shown to significantly improve cardiac function by inhibiting GSK-3β/caspase-1/GSDMD activation. In vitro experiments were conducted using rat cardiac myocytes (RCMs) and rat embryonic cardiac-derived H9c2 cells. Pretreatment with ticagrelor (10 μm) significantly inhibited Dox (1 μm)-induced hypertrophy and reversed the upregulation of GSDMD-NT expression. We showed that ticagrelor suppressed the activation of Akt caused by Dox in the heart tissue as well as in RCMs/H9c2 cells caused by Dox. When GSK-3β expression was absent in H9c2 cells, the inhibitory effect of ticagrelor on Dox-induced caspase-1/GSDMD activation was weakened. These data showed that ticagrelor reduced Dox-induced pyroptosis in rat cardiomyocytes by targeting GSK-3β/caspase-1.

CPAL, as a New Mediator of Cardiomyocyte Metabolic Alterations and Pyroptosis, Regulates Myocardial Infarction Injury in Mice

Myocardial infarction (MI), the most serious of the ischemic heart diseases, is accompanied by myocardial metabolic disorders and the loss of cardiomyocytes. Increasing evidence has shown that Long noncoding RNAs (lncRNAs) are involved in various pathological conditions such as cancer and cardiovascular diseases (CVDs), and are emerging as a novel biomarker for these disorders. This study aims to investigate the regulatory role and mechanisms of lncRNAs in myocardial remodeling in the setting of MI. We find that post-infarcted hearts exhibit a reduction of adenosine triphosphate (ATP) and an alteration of the glucose and lipid metabolism genes Cluster of differentiation 36 (CD36) , Hexokinase ( HK ), and Glucose transporter 4 (GLUT 4), accompanied by cardiomyocyte pyroptosis. We then identify a previously unknown conserved lncRNA, AK009126 (cardiomyocyte pyroptosis-associated lncRNA, CPAL), which is remarkably upregulated in the myocardial border zone of MI mice. Importantly, the Adeno-associated virus 9 (AAV9)-mediated silencing of endogenous CPAL by its short hairpin RNA (shRNA) partially abrogates myocardial metabolic alterations and cardiomyocyte pyroptosis during MI in mice. Mechanistically, CPAL is shown to bind directly to nuclear factor kappa B (NFκB) and to act as an activator of NFκB to induce NFκB phosphorylation in cardiomyocytes. We also find that CPAL upregulates caspase-1 expression at the transcriptional level and consequently promotes the release of interleukin (IL)-18 and IL-1β from cardiomyocytes. Collectively, our findings reveal the conserved lncRNA CPAL as a new regulator of cardiac metabolic abnormalities and cardiomyocyte pyroptosis in the setting of MI and suggest CPAL as a new therapeutic target to protect cardiomyocytes against ischemic injury in infarcted hearts.

Effects of tropomyosin 3 on pyroptosis of cardiomyocytes and fibroblast activation induced by hypoxia/reoxygenation in rats

To explore the role of tropomyosin 3 (TPM3) in hypoxia/reoxygenation (H/R)-induced cardiomyocyte pyroptosis and fibroblast activation. Rat cardiomyocytes (H9c2 cells) were treated with H/R method to simulate myocardial ischemia/reperfusion (I/R) injury, and cell proliferation activity was evaluated with cell counting kit-8 (CCK8). The expression of TPM3 mRNA and protein was detected by quantitative real-time polymerase chain reaction (RT-qPCR) and Western blotting. H9c2 cells with stable TPM3-short hairpin RNA (shRNA) expression were constructed and treated with H/R (hypoxia for 3 hours, and reoxygenation for 4 hours). The expression of TPM3 was measured by RT-qPCR. The expressions of TPM3, pyroptosis-related proteins including caspase-1, NOD-like receptor protein 3 (NLRP3) and Gasdermin family proteins-N (GSDMD-N) were measured by Western blotting. The expression of caspase-1 was also observed by immunofluorescence assay. The levels of human interleukins (IL-1β, IL-18) in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA) to elucidate the effect of sh-TPM3 on pyroptosis of cardiomyocytes. Rat myocardial fibroblasts were incubated with the above cell supernatant, and the expressions of human collagen I, collagen III, matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase inhibitor 2 (TIMP2) were detected by Western blotting to determine the effect of TPM3-interfered cardiomyocytes on the activation of fibroblasts under H/R conditions. Compared with the control group, H/R treatment for 4 hours significantly decreased the survival rate of H9c2 cells [(25.81±1.90)% vs. (99.40±5.54)%, P < 0.01], promoted the expression of TPM3 mRNA and protein [TPM3/GAPDH (2-ΔΔCt): 3.87±0.50 vs. 1, TPM3/β-Tubulin: 0.45±0.05 vs. 0.14±0.01, both P < 0.01], and promoted the expressions of caspase-1, NLRP3, GSDMD-N, and the enhanced release of cytokines IL-1β and IL-18 [cleaved caspase-1/caspase-1: 0.89±0.04 vs. 0.42±0.03, NLRP3/β-Tubulin: 0.39±0.03 vs. 0.13±0.02, GSDMD-N/β-Tubulin: 0.69±0.05 vs. 0.21±0.02, IL-1β (μg/L): 13.84±1.89 vs. 4.31±0.33, IL-18 (μg/L): 17.56±1.94 vs. 5.36±0.63, all P < 0.01]. However, compared with the H/R group, sh-TPM3 significantly weakened the promoting effects of H/R on these proteins and cytokines [cleaved caspase-1/caspase-1: 0.57±0.05 vs. 0.89±0.04, NLRP3/β-Tubulin: 0.25±0.04 vs. 0.39±0.03, GSDMD-N/β-Tubulin: 0.27±0.03 vs. 0.69±0.05, IL-1β (μg/L): 8.56±1.22 vs. 13.84±1.89, IL-18 (μg/L): 9.34±1.04 vs. 17.56±1.94, all P < 0.01]. In addition, the expressions of collagen I, collagen III, TIMP2, and MMP-2 in myocardial fibroblasts were significantly increased by the cultured supernatants from the H/R group (collagen I/β-Tubulin: 0.62±0.05 vs. 0.09±0.01, collagen III/β-tubulin: 0.44±0.03 vs. 0.08±0.00, TIMP2/β-tubulin: 0.73±0.04 vs. 0.20±0.03, TIMP2/β-Tubulin: 0.74±0.04 vs. 0.17±0.01, all P < 0.01). However, these boosting effects were weakened by sh-TPM3 (collagen I/β-Tubulin: 0.18±0.01 vs. 0.62±0.05, collagen III/β-Tubulin: 0.21±0.03 vs. 0.44±0.03, TIMP2/β-Tubulin: 0.37±0.03 vs. 0.73±0.04, TIMP2/β-Tubulin: 0.45±0.03 vs. 0.74±0.04, all P < 0.01). Interference with TPM3 can alleviate H/R-induced cardiomyocyte pyroptosis and fibroblast activation, suggesting that TPM3 may be a potential target of myocardial I/R injury.

LOXL1-AS1 Aggravates Myocardial Ischemia/Reperfusion Injury Through the miR-761/PTEN Axis.

Myocardial ischemia and reperfusion injury (MIRI) has high morbidity and mortality worldwide. We aimed to explore the role of long noncoding RNA lysyl oxidase like 1 antisense RNA 1 (LOXL1-AS1) in cardiomyocyte pyroptosis. Hypoxia/reoxygenation (H/R) injury was constructed in human cardiomyocyte (HCM). The level of LOXL1-AS1, miR-761, phosphatase and tensin homolog (PTEN) and pyroptosis-related proteins was monitored by quantitative real-time polymerase chain reaction or western blot. Flow cytometry examined the pyroptosis level. Lactate dehydrogenase (LDH), creatine kinase-MB and cardiac troponin I levels were detected by test kits. Enzyme-linked immunosorbent assay measured the release of inflammatory cytokines. Dual-luciferase assay validated the binding relationship among LOXL1-AS1, miR-761, and PTEN. Finally, ischemia/reperfusion (I/R) animal model was constructed. Hematoxylin and eosin staining assessed morphological changes of myocardial tissue. NOD-like receptor pyrin domain-containing protein 3 (NLRP3) and casepase-1 expression was determined by immunohistochemistry. After H/R treatment, LOXL1-AS1 and PTEN were highly expressed but miR-761 level was suppressed. LOXL1-AS1 inhibition or miR-761 overexpression increased cell viability, blocked the release of LDH and inflammatory cytokines (interleukin [IL]-1β, IL-18), inhibited pyroptosis level, and downregulated pyroptosis-related proteins (ASC, cleaved caspase-1, gasdermin D-N, NLRP3, IL-1β, and IL-18) levels in HCMs. LOXL1-AS1 sponged miR-761 to up-regulate PTEN. Knockdown of miR-761 reversed the effect of LOXL1-AS1 down regulation on H/R induced HCM pyroptosis. LOXL1-AS1 aggravated the MIRI by regulating miR-761/PTEN axis in vivo. LOXL1-AS1 targeted miR-761 to regulate PTEN expression, then enhance cardiomyocyte pyroptosis, providing a new alternative target for the treatment of MIRI.

The protective effect of thiolutin on doxorubicin-induced H9c2 cardiomyocyte injury.

The use of doxorubicin (DOX) may contribute to cardiotoxicity, limiting its clinical application. Thiolutin (THL) has been found to exert protective roles in various biological activities, while its effects on DOX-induced cardiotoxicity are still uncovered. Cell counting kit 8 assay was utilized to detect cell viability and half maximal inhibitory concentration of THL in H9c2 cardiomyocytes. The level of lactate dehydrogenase (LDH), adenosine triphosphate (ATP), interleukin (IL)-18 and IL-1 beta (IL-1β) were measured using the corresponding detection kits, and flow cytometry determined cell apoptosis rate. The reactive oxygen species (ROS) accumulation was evaluated by utilizing immunofluorescence or flow cytometry assay. The protein levels of NLR family Pyrin domain 3 (NLRP3), pro-Caspase1, cleaved-Caspase1, gasdermin D (GSDMD) and cleaved-GSDMD (GSDMD-N) in H9c2 cells were detected by immunoblotting assay. The treatment of THL reduced H9c2 cell viability in a gradient-dependent manner. THL treatment reversed the DOX-induced inhibition of proliferation, decrease of ATP, up-regulation of LDH, IL-18, IL-1β and production of ROS, activation of NLRP3 and inflammasome-mediated pyroptosis in H9c2 cells. Additionally, NLRP3 knockdown abolished the effects of THL in DOX-treated H9c2 cells remarkably. This investigation proved that THL notably ameliorated DOX-induced apoptosis, oxidative stress, and pyroptosis in H9c2 cardiomyocytes. Besides, THL effectively inactivated DOX-induced NLRP3 inflammasome in H9c2 cells. These findings revealed a promising drug to assist DOX in its anti-cancer effects and protect the heart of patients.

Quercetin Inhibits Pyroptosis in Diabetic Cardiomyopathy through the Nrf2 Pathway.

The present study investigated whether quercetin promotes the nuclear translocation of nuclear factor erythroid-2-related factor 2 (Nrf2) to inhibit pyroptosis progression and ameliorate diabetic cardiomyopathy. We evaluated the protective effects of quercetin against diabetic cardiomyopathy by analyzing the expression of pyroptosis pathway proteins, myocardial cell apoptosis rate, degree of myocardial fibrosis, and serum inflammatory indices in the hearts of model rats with diabetes. We evaluated the expression of Nrf2 in the nucleus of cardiomyocytes and H9C2 cells to clarify the role of quercetin in promoting the nuclear translocation of Nrf2. In addition, we coincubated cardiomyocytes with the Nrf2 inhibitor ML385 to confirm that quercetin inhibits the diabetes-induced cardiomyocyte pyroptosis via the Nrf2 pathway. We found that quercetin promoted the nuclear translocation of Nrf2 in cardiac cells of diabetic rats, increased the expression of the antioxidant proteins HO-1, GCLC, and SOD, reduced the accumulation of ROS and the degree of cardiomyocyte apoptosis, and alleviated diabetes-induced cardiac fibrosis. The therapeutic effects of quercetin were further validated in H9C2 cardiomyocytes. Interestingly, ML385 prevented the beneficial effects of quercetin on diabetic cardiomyopathy, further indicating that the quercetin-mediated inhibition of pyroptosis requires the participation of the Nrf2 pathway. In conclusion, quercetin promoted the nuclear translocation of Nrf2, increased the expression of antioxidant factors in cells, and inhibited the progression of cell pyroptosis, thereby alleviating diabetic cardiomyopathy.

Exogenous 8-hydroxydeoxyguanosine attenuates doxorubicin-induced cardiotoxicity by decreasing pyroptosis in H9c2 cardiomyocytes

Doxorubicin (DOX), which is widely used in cancer treatment, can induce cardiomyopathy. One of the main mechanisms whereby DOX induces cardiotoxicity involves pyroptosis through the NLR family pyrin domain containing 3 (NLRP3) inflammasome and gasdermin D (GSDMD). Increased NAPDH oxidase (NOX) and oxidative stress trigger pyroptosis. Exogenous 8-hydroxydeoxyguanosine (8-OHdG) decreases reactive oxygen species (ROS) production by inactivating NOX. Here, we examined whether 8-OHdG treatment can attenuate DOX-induced pyroptosis in H9c2 cardiomyocytes. Exposure to DOX increased the peroxidative glutathione redox status and NOX1/2/4, toll-like receptor (TLR)2/4, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) expression, while an additional 8-OHdG treatment attenuated these effects. Furthermore, DOX induced higher expression of NLRP3 inflammasome components, including NLRP3, apoptosis-associated speck-like protein containing a c-terminal caspase recruitment domain (ASC), and pro-caspase-1. Moreover, it increased caspase-1 activity, a marker of pyroptosis, and interleukin (IL)-1β expression. All these effects were attenuated by 8-OHdG treatment. In addition, the expression of the cardiotoxicity markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was increased by DOX, whereas the increase of ANP and BNP induced by DOX treatment was reversed by 8-OHdG. In conclusion, exogenous 8-OHdG attenuated DOX-induced pyroptosis by decreasing the expression of NOX1/2/3, TLR2/4, and NF-κB. Thus, 8-OHdG may attenuate DOX-induced cardiotoxicity through the inhibition of pyroptosis.

M2 macrophage-derived exosomal miR-145-5p protects against the hypoxia/reoxygenation-induced pyroptosis of cardiomyocytes by inhibiting TLR4 expression.

Exosomes carrying micro ribonucleic acids (miRNAs) protect against myocardial ischemic injury. In the study, we sought to investigate the protective effect mechanism of M2 macrophage-derived exosome miR-145-5p in hypoxia-reoxygenation (H/R)-induced cardiomyocytes. M2 macrophages were isolated and induced from blood donated by healthy donors. M2 macrophages were transfected with or without miR-145-5p. Exosomes derived from M2 macrophages were isolated and identified by flow cytometry, nanoparticle tracking analysis, and transmission electron microscopy (TEM). AC16 cells were used to establish an H/R model, and cell activity was detected using a Cell Counting Kit 8 (CCK-8). Western blot was used to detect the expression of gasdermin D (GSDMD), nucleotide-binding domain-like receptor protein 3 (NLRP3), and caspase-1 in the H/R-induced AC16 cells to evaluate pyroptosis. Immunofluorescence staining was used to detect the positive rates of PKH26 and caspase-1. Combined with database prediction, dual luciferase reporter assays were used to validate toll-like receptor 4 (TLR4) as a downstream target molecule of miR-145-5p. A real-time quantitative polymerase chain reaction (RT-qPCR) analysis and western blot were used to detect the expression of TLR4 in the AC16 cells. Flow cytometry, western blot, nanoparticle tracking and TEM results confirmed the successful isolation of M2 macrophage-derived exosomes. CCK-8 results showed M2 macrophage-derived exosomes decreased the viability of the H/R-induced cells. Western blot results showed the expressions of GSDMD, caspase-1, and NLRP3 were significantly downregulated in the H/R group. Moreover, CCK-8 results showed the M2 macrophage-derived exosome miR-145-5p significantly ameliorated H/R-induced AC16 cellular activity. Western blot results confirmed the expressions of GSDMD, NLRP3, and caspase-1 were significantly downregulated in the macrophage-derived exosome miR-145-5p group compared to the M2 macrophage-derived exosome NC (normal control) group. Immunofluorescence staining results displayed the same trend in terms of the caspase-1 positivity rate. Further, we demonstrated overexpression of TLR4 partially reversed the protective effect of M2 macrophage-derived exosome miR-145-5p in the H/R-induced AC16 cells. Additionally, overexpression of TLR4 reversed the protein expression associated with pyroptosis in M2 macrophage-derived exosome miR-145-5p in the H/R-induced AC16 cells. Our study indicated M2 macrophage-derived exosomes carrying miR-145-5p inhibited H/R-induced cardiomyocyte pyroptosis by downregulating the expression of TLR4.

Oridonin Protects against Myocardial Ischemia-Reperfusion Injury by Inhibiting GSDMD-Mediated Pyroptosis.

Pyroptosis serves a crucial function in various types of ischemia and reperfusion injuries. Oridonin, a tetracycline diterpene derived from Rabdosia rubescens, can significantly inhibit the aggregation of NLRP3-mediated inflammasome. This experiment is aimed at investigating the effect of oridonin on pyroptosis in mice cardiomyocytes. Based on the models of myocardial ischemia/reperfusion (I/R) and hypoxia/reoxygenation (H/R), Evans Blue/TTC double staining, TUNEL staining, and Western blotting were applied to determine the effects of oridonin on myocardial damage, cellular activity and signaling pathways involved in pyroptosis. During I/R and H/R treatments, the extent of gasdermin D-N domains was upregulated in cardiomyocytes. Apart from that, oridonin improved cell survival in vitro and decreased the myocardial infarct size in vivo by also downregulating the activation of pyroptosis. Finally, the expression levels of ASC, NLRP3 and p-p65 were markedly upregulated in cardiomyocytes after H/R treatment, whereas oridonin suppressed the expression of these proteins. The present experiment revealed that myocardial I/R injury and pyroptosis can be alleviated and inhibited by oridonin pretreatment via NF-κB/NLRP3 signaling pathway, both in vivo and in vitro. Therefore, oridonin may serve as a potentially novel agent for the clinical treatment of myocardial ischemia-reperfusion injuries.