IKKε protects against starvation-induced NLRP3 inflammasome and pyroptosis in H9c2 cells by alleviating mitochondrial injury.

The deprivation of myocardial nutrition causes cardiomyocyte death and disturbance of energy metabolism. IKKε plays an important regulatory role in many biological events such as inflammation, redox reaction, cell death, etc. However, the more in-depth mechanism by which IKKε contributes to cardiomyocytes death in nutrition deprivation remains poorly understood. IKKε expression was knocked down by siRNA in H9c2 cells, and cells were cultured under starvation conditions to simulate ischemic conditions. Starvation triggered greater NLRP3 activation, accompanied by more IL-1ß, IL-18 and caspase-1 release in the siIKKε H9c2 cells compared with the control H9c2 cells. Western blot and immunofluorescence showed that the IKKε konckdown promoted NLRP3 expressions and ROS release under starvation conditions. Furthermore, electron micrography and JC-1 analysis revealed that IKKε konckdown resulted in aggravated mitochondrial damage and more mitochondrial ROS (mtROS) released in vitro. Notably, Western blot analysis showed that IKKε deficiency activated the TBK1 and IRF3 signaling pathways to promote pyroptosis in vitro. Collectively, our results indicate that IKKε protects against cardiomyocyte injury by reducing mitochondrial damage and NLRP3 expression following nutrition deprivation via regulation of the TBK1/IRF3 signaling pathway. This study further revealed the mechanism of IKKε in inflammation and myocardial nutrition deprivation.

Stomatin-like protein-2 attenuates macrophage pyroptosis and H9c2 cells apoptosis by protecting mitochondrial function.

Myocytes undergoing hypoxia condition can recruit macrophages and cause pro-inflammation initiation around the injury area. Mitochondrial dysfunction is related to macrophage pyroptosis. Stomatin-like protein-2 (SLP-2) can regulate mitochondrial biogenesis and function. Whether SLP-2 could affect macrophage pyroptosis remains unclear. In this study, bone marrow derived macrophages (BMDMs) were extracted from WT and SLP-2 knocked out mice, then stimulated by LPS/Nigericin. Western blot showed that SLP-2-/- promoted the expression of NLRP3, GSDMD-N, caspase-11 in macrophages, which means the deficiency of SLP-2 augments macrophage pyroptosis. Higher fluorescence intensity of dihydroethidium and TUNEL represented the increased ROS releasing and macrophage programmed death in SLP-2 deficiency groups. The immunofluorescence intensity of MtioTracker Red decreased and that of mitochondrial ROS (mtROS) increased in SLP-2 deletion groups with LPS/Nigericin stimulation, representing the increased mitochondrial damage. The expression level of HIF-1α increased in SLP-2 deletion macrophages with LPS and Nigericin stimulation. The level of Parkin and the ratio of LC3II/I decreased in SLP-2 deficiency macrophages after stimulated by LPS/Nigericin, compared with untreated macrophages. H9c2 cells were cultured in hypoxia condition before being cocultured with macrophage supernatant. The cocultured H9c2 cells were injured due to the serious pyroptosis of SLP-2 deficiency macrophages. According to these results, we suggest that SLP-2 can reduce macrophage pyroptosis and relieve hypoxia H9c2 cells injury through protecting mitochondrial function.

MiR-937-3p promotes metastasis and angiogenesis and is activated by MYC in lung adenocarcinoma.

BACKGROUND: Non-small cell lung cancer (NSCLC) is still one of the diseases with the highest mortality and morbidity, and lung adenocarcinoma (LUAD) accounts for more than half of all NSCLC cases in most countries. miRNA can be used as a potential biological marker and treatment for lung adenocarcinoma. However, the effect of miR-937-3p to the invasion and metastasis of LUAD cells is not clear. METHODS: miRNA microarray is used to analyze the expression of miRNA in lung adenocarcinoma tissue. Transwell migration, Wound-healing assay and Western blot analysis are used to analyze cell migration, invasion and epithelial-mesenchymal transition (EMT) capabilities. Tube formation is used to assess angiogenesis ability. In addition, dual luciferase reporter gene detection is used to identify the potential binding between miRNA and target mRNA. In vivo experiments were performed on male NOD/SCID nude mice by tail vein injection to establish a transplanted tumor model. The CHIP experiment is used to verify the transcription factors of miRNA. RESULT: In our study, miR-937-3p was high-regulated in LUAD cell lines and tissues, and its expression level was related to tumor progression. We found that miR-937-3p high-expression has an effect on cell invasion and metastasis. In molecular mechanism, miR-937-3p causes SOX11 reduction by directly binding to the 3'-UTR of SOX11.In addition, MYC affects miR-937-3p transcription by binding to its promoter region. CONCLUSIONS: Our research shows that miR-937-3p is mediated by MYC and can control the angiogenesis, invasion and metastasis of LUAD by regulating SOX11, thereby promoting the progress of LUAD. We speculate that miR-937-3p can be used as a therapeutic target and potential biomarker for LUAD.

Targeting translocator protein protects against myocardial ischemia/reperfusion injury by alleviating mitochondrial dysfunction.

Ischemic heart disease (IHD) remains a leading cause of mortalities worldwide, necessitating timely reperfusion to reduce acute mortality. Paradoxically, reperfusion can induce myocardial ischemia/reperfusion (I/R) injury, which is primarily characterized by mitochondrial dysfunction. Translocator protein (TSPO) participates in multiple cellular events; however, its role in IHD, especially in the process of myocardial I/R injury, has not been well determined. The aim of the present study was to investigate the functional role of TSPO in myocardial I/R injury and dissect the concomitant cellular events involved. This study utilized small interfering RNA (siRNA) technology to knock down TSPO expression. The I/R process was simulated using an anoxia/reoxygenation (A/R) model. The role of TSPO in H9c2 cardiomyocytes was assessed using various techniques, such as Western blotting, Flow cytometry, Reverse transcription-quantitative PCR (RT-qPCR), Immunofluorescence, Co-immunoprecipitation (co-IP) and similar methods. It was found that A/R markedly upregulated the expression of TSPO in cardiomyocytes. Inhibition of TSPO improved myocardial cell apoptosis and damage following A/R stimulation. Additionally, targeting TSPO alleviated mitochondrial damage, reduced mitochondrial ROS release and enhanced ATP synthesis following A/R stimulation. It was further confirmed that A/R stimulation induced a significant increase in the expression of pivotal markers [phosporylated-PKR-like ER kinase (PERK)/PERK, activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1] involved in the adaptive unfolded protein response, which is accompanied by downstream signaling during endoplasmic reticulum (ER) stress. Notably, TSPO knockdown increased the expression of the aforementioned markers and, subsequently, TSPO was confirmed to interact with ATF6, suggesting that TSPO might play a role in ER stress during myocardial I/R injury. Finally, inhibition of TSPO upregulated mitophagy, as indicated by further decreases in P62 and increases in Parkin and PINK1 levels following A/R stimulation. Together, the results suggest that TSPO plays a multifaceted role in myocardial I/R injury. Understanding TSPO-induced cellular responses could inform targeted therapeutic strategies for patients with IHD.

Stomatin-like protein 2 deficiency exacerbates adverse cardiac remodeling.

Myocardial fibrosis, oxidative stress, and autophagy both play key roles in the progression of adverse cardiac remodeling. Stomatin-like protein 2 (SLP-2) is closely related to mitochondrial function, but little is known about its role and mechanism in cardiac remodeling. We developed doxorubicin (Dox), angiotensin (Ang) II, and myocardial ischemia-reperfusion (I/R) injury induced cardiac remodeling model and Dox treated H9C2 cell injury model using SLP-2 knockout (SLP-2-/-) mice and H9C2 cells with low SLP-2 expression. We first examined cardiac functional and structural changes as well as levels of oxidative stress, apoptosis and autophagy. We found that SLP-2 deficiency leads to decreased cardiac function and promotes myocardial fibrosis. After Dox and Ang II treatment, SLP-2 deficiency further aggravated myocardial fibrosis, increased myocardial oxidative stress and apoptosis, and activated autophagy by inhibiting PI3K-Akt-mTOR signaling pathway, ultimately exacerbating adverse cardiac remodeling. Similarly, SLP-2 deficiency further exacerbates adverse cardiac remodeling after myocardial I/R injury. Moreover, we extracted cardiomyocyte mitochondria for proteomic analysis, suggesting that SLP-2 deficiency may be involved in myocardial I/R injury induced adverse cardiac remodeling by influencing ubiquitination of intramitochondrial proteins. In addition, the oxidative stress, apoptosis and autophagy levels of H9C2 cells with low SLP-2 expression were further enhanced, and the PI3K-Akt-mTOR signaling pathway was further inhibited under Dox stimulation. Our results suggest that SLP-2 deficiency promotes myocardial fibrosis, disrupts normal mitochondrial function, overactivates autophagy via PI3K-Akt-mTOR signaling pathway, affects the level of ubiquitination, leads to irreversible myocardial damage, and ultimately exacerbates adverse cardiac remodeling.

CARMA3 Deficiency Aggravates Angiotensin II-Induced Abdominal Aortic Aneurysm Development Interacting Between Endoplasmic Reticulum and Mitochondria.

BACKGROUND: Abdominal aortic aneurysm (AAA) is life threatening and associated with vascular walls' chronic inflammation. However, a detailed understanding of the underlying mechanisms is yet to be elucidated. CARMA3 assembles the CARMA3-BCL10-MALT1 (CBM) complex in inflammatory diseases and is proven to mediate angiotensin II (Ang II) response to inflammatory signals by modulating DNA damage-induced cell pyroptosis. In addition, interaction between endoplasmic reticulum (ER) stress and mitochondrial damage is one of the main causes of cell pyroptosis. METHODS: Male wild type (WT) or CARMA3-/- mice aged 8 to 10 weeks were subcutaneously implanted with osmotic minipumps, delivering saline or Ang II at the rate of 1 µg/kg/min for 1, 2, and 4 weeks. RESULTS: We discovered that CARMA3 knockout promoted formation of AAA and prominently increased diameter and severity of the mice abdominal aorta infused with Ang II. Moreover, a significant increase in the excretion of inflammatory cytokines, expression levels of matrix metalloproteinases (MMPs) and cell death was found in the aneurysmal aortic wall of CARMA3-/- mice infused with Ang II compared with WT mice. Further studies found that the degree of ER stress and mitochondrial damage in the abdominal aorta of CARMA3-/- mice was more severe than that in WT mice. Mechanistically, CARMA3 deficiency exacerbates the interaction between ER stress and mitochondrial damage by activating the p38MAPK pathway, ultimately contributing to the pyroptosis of vascular smooth muscle cells (VSMCs). CONCLUSIONS: CARMA3 appears to play a key role in AAA formation and might be a potential target for therapeutic interventions of AAA.

Macrophage, a potential targeted therapeutic immune cell for cardiomyopathy.

Cardiomyopathy is a major cause of heart failure, leading to systolic and diastolic dysfunction and promoting adverse cardiac remodeling. Macrophages, as key immune cells of the heart, play a crucial role in inflammation and fibrosis. Moreover, exogenous and cardiac resident macrophages are functionally and phenotypically different during cardiac injury. Although experimental evidence has shown that macrophage-targeted therapy is promising in cardiomyopathy, clinical translation remains challenging. In this article, the molecular mechanism of macrophages in cardiomyopathy has been discussed in detail based on existing literature. The issues and considerations of clinical treatment strategies for myocardial fibrosis has also been analyzed.

I-κB kinase-ε deficiency improves doxorubicin-induced dilated cardiomyopathy by inhibiting the NF-κB pathway.

Dilated cardiomyopathy (DCM) can lead to heart expansion and severe heart failure, but its specific pathogenesis is still elusive. In many cardiovascular diseases, I-κB kinase-ε (IKKε) has been recognized as a pro-inflammatory molecule. In this study, wild-type mice (WT, n = 14) and IKKε knockout mice (IKKε-KO, n = 14) were intraperitoneally injected with a cumulative dose of 25 mg/kg with Dox or Saline five times in 30 days. Finally, the experimental mice were divided into WT + Saline group、WT + DOX group、IKKε-KO + Saline group and IKKε-KO + Dox group. Echocardiography was performed to assess cardiac structure and function. Moreover, the mechanism was validated by immunohistochemistry and western blotting. Our results demonstrated that compared to WT + Dox mice, IKKε-KO + Dox mice exhibited attenuation of dilated cardiomyopathy-related morphological changes and alleviation of heart failure. Additionally, compared to the WT mice after Dox-injected, the expression of fibrosis and proinflammatory were decreased in IKKε-KO mice, and the expression of cardiac gap junction proteins was much higher in IKKε-KO mice. Further testing found that pyroptosis and apoptosis in the myocardium were also ameliorated in IKKε-KO mice compared to WT mice after Dox was injected. Mechanistically, our results showed that deficiency of IKKε might inhibit the phosphorylation of IκBα, p65, RelB, and p100 in mouse heart tissues after Dox stimulation. In summary, our research suggests that IKKε might play an essential role in the development of Dox-induced dilated cardiomyopathy and may be a potential target for the treatment of dilated cardiomyopathy in the future.

I-κB Kinase-ε Deficiency Attenuates the Development of Angiotensin II-Induced Myocardial Hypertrophy in Mice.

I-κB kinase-ε (IKKε) is a member of the IKK complex and a proinflammatory regulator that is active in many diseases. Angiotensin II (Ang II) is a vasoconstricting peptide hormone, and Ang II-induced myocardial hypertrophy is a common cardiovascular disease that can result in heart failure. In this study, we sought to determine the role of IKKε in the development of Ang II-induced myocardial hypertrophy in mice. Wild-type (WT) and IKKε-knockout (IKKε-KO) mice were generated and infused with saline or Ang II for 8 weeks. We found that WT mouse hearts have increased IKKε expression after 8 weeks of Ang II infusion. Our results further indicated that IKKε-KO mice have attenuated myocardial hypertrophy and alleviated heart failure compared with WT mice. Additionally, Ang II-induced expression of proinflammatory and collagen factors was much lower in the IKKε-KO mice than in the WT mice. Apoptosis and pyroptosis were also ameliorated in IKKε-KO mice. Mechanistically, IKKε bound to extracellular signal-regulated kinase (ERK) and the mitogen-activated protein kinase p38, resulting in MAPK/ERK kinase (MEK) phosphorylation, and IKKε deficiency inhibited the phosphorylation of MEK-ERK1/2 and p38 in mouse heart tissues after 8 weeks of Ang II infusion. The findings of our study reveal that IKKε plays an important role in the development of Ang II-induced myocardial hypertrophy and may represent a potential therapeutic target for the management of myocardial hypertrophy.

IKKε knockout alleviates angiotensin II-induced apoptosis and excessive autophagy in vascular smooth muscle cells by regulating the ERK1/2 pathway.

Inhibitor of nuclear factor-κB kinase subunit ε (IKKε) is an important signal regulator in the formation of abdominal aortic aneurysm (AAA). However, the underlying mechanism remains to be elucidated. Therefore, the present study aimed to investigate the mechanism underlying IKKε function in AAA formation by studying apoptosis and autophagy in angiotensin II (Ang II)-induced vascular smooth muscle cells (VSMCs). AngII was used to stimulate VSMCs for 24 h to simulate the process of AAA formation. VSMCs were transfected with IKKε small interfering RNA to investigate the effect of IKKε on AAA formation, cell apoptosis and autophagy. IKKε deficiency led to reduced mitochondrial damage and apoptosis in VSMCs in the early stage of apoptosis in vitro, as demonstrated using a JC-1 probe. IKKε deficiency also reduced autophagy and decreased the formation of autophagic vacuoles in VSMCs, demonstrated using transmission electron microscopy. The decrease in apoptosis caused by IKKε knockdown was reversed when the autophagic flow was blocked using bafilomycin A1. Western blot analysis further revealed that IKKε deficiency negatively regulated the ERK1/2 signaling pathway to reduce autophagy. Collectively, the results of the present study revealed that IKKε played a key role in apoptosis by inducing excessive autophagy, thereby potentially contributing to AAA formation. These findings further revealed the mechanism underlying IKKε function in the formation of AAA.

De Ritis ratio as a significant prognostic factor of international normalized ratio ≥4 in the initial 10 days of warfarin therapy.

Aim: To assess the relationship between the De Ritis ratio on admission and warfarin sensitivity in the initial 10 days of anticoagulation therapy. Methods: We retrospectively reviewed data from 906 patients who underwent heart valve replacement surgery. Results: A De Ritis ratio of 1.19 was identified as the optimal cutoff according to the ROC curve. Patients with a high De Ritis ratio achieved an international normalized ratio (INR) ≥4 more easily and earlier than those with a low De Ritis ratio in the initial 10 days of warfarin therapy. Multivariate analysis showed that a high De Ritis ratio was an independent predictor of an INR ≥4 (HR: 2.567; p < 0.001). Conclusion: A De Ritis ratio ≥1.19 on admission was significantly associated with an INR ≥4 in the initial 10 days of warfarin therapy among patients underwent heart valve replacement surgery.