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Objective To investigate the role of cathepsin K (CatK) in cardiac dysfunction in alcoholic cardiomyopathy (ACM) mice and its damage mechanism. Methods Forty-eight C57BL/6J male mice (8 weeks old) were randomly divided into control group (n=12, fed with isocaloric maltodextrin control liquid diet) and model group (n=36, fed with 4% ethanol Lieber-DeCarli liquid diet). After 8 weeks, the mice in the model group identified with decreased cardiac systolic function by echocardiography were regarded as ACM mice, and they were randomly divided into ACM group, sh-NC group, and sh-CatK group, with 12 mice in each group. The mice in the sh-NC group and the sh-CatK group were injected with 1×109 vg (100 μL) of recombinant adenoviruses containing scramble-shRNA or CatK-shRNA via tail vein, respectively. At the end of the 12th week, cardiac function was measured by M-mode echocardiography. The expression of CatK and Nox4 was detected by immunohistochemical assay. In in vitro study, H9c2 cardiomyocytes were used to evaluate the effect of CatK silencing on alcohol-induced cardiomyocyte injury. Western blotting was used to analyze the protein expression of AMPK/Nox4 signaling pathway. Flow cytometry was employed to analyze the apoptosis of H9c2 cells. The levels of reactive oxygen species in mouse myocardium and H9c2 cells cultured were measured using fluorescent dye dihydroethidium. Results The surface area of myocardial cells, area of myocardial fibrosis, protein levels of CatK and Nox4 and production of superoxide were significantly increased in the ACM group compared with the control group (P < 0.05), and those indicators in the sh-CatK group were significantly decreased than the ACM group (P < 0.05). Sh-CatK transfection significantly reversed the above changes in ACM mice (P < 0.05). In vitro results showed that the expression of CatK protein in H9c2 cells was increased with the prolonged exposure to ethanol (P < 0.05). CatK inhibition significantly reduced the apoptostic rate and oxidative stress levels (P < 0.05), and up-regulated AMPK phosphorylation in H9c2 cells (P < 0.05), resulting in decreased expression of Nox4 protein, collagen COL-1 and TGF-β (P < 0.05). Conclusion CatK is involved in the pathological process of cardiac dysfunction in the ACM mice, and its mechanism might be related to the promotion of oxidative stress mediated by the AMPK/Nox4 signaling pathway.

Objective To investigate whether myeloid-derived growth factor (Mydgf) can improve cardiomyocyte pyroptosis induced by hyperglycemia by regulating nucleotide-binding domain-like receptor protein 3 (NLRP3). Methods Cultured rat cardiomyocyte H9c2 cells were divided into 4 groups: normal group (Nor), high glucose group (HG, 50 mmol/L glucose for 24 h), Mydgf group (25 ng/mL recombinant Mydgf protein for 12 h, followed by 50 mmol/L glucose for 24 h), and necrosulfonamide group (NSA, 32 μmol/L necrosulfonamide for 0.5 h, followed by 50 mmol/L glucose for 24 h). Western blotting was used to detect the protein levels of cleaved Caspase-1, NLRP3, apoptosis-related speckle-like proteins (ACS) and gasdermin D (GSDMD). The secretions of IL-1β and IL-18 in cell culture medium were detected by ELISA. CCK-8 assay was used to detect the effect of Mydgf or NLRP3 on the viability of H9c2 cells. Lentiviral vector carrying NLRP3 and enhanced green fluorescent protein (EGFP) (NLRP3-EGFP) and empty viral vector (EGFP) were transfected into H9c2 cells respectively, and the changes of pyroptosis related indexes were detected by recovery experiments. Results Western blot results showed that the expression levels of pyroptosis related proteins, cleaved Caspase 1, ACS, NLRP3 and GSDMD were significantly higher in the HG group than the Nor group (P < 0.05). Mydgf treatment effectively inhibited the expression of above proteins (P < 0.05), but the inhibitory efficiencies were lower than that in the NSA group (P < 0.05). ELISA demonstrated that the secretions of IL-1β and IL-18 were increased in the HG group (P < 0.05), but decreased in the Mydgf group (P < 0.05), and more significantly reduced in the NSA group (P < 0.05). CCK-8 assay indicated that Mydgf at a concentration of below 25 ng/mL had no significant effect on the viability of H9c2 cells, while when the concentration above 25 ng/mL, the treatment significantly decreased the cell viability (P < 0.05). Compared with the control group, overexpression of NLRP3 significantly decreased H9c2 cells viability under the condition of HG. Transfection of lentiviral vector NLRP3-EGFP resulted in obvious increases in the mRNA and protein expression of NLRP3 (P < 0.05), but that of empty vector had no such effects (P>0.05). In the recovery experiment, Western blot results showed that in the high-glucose environment, the expression level of NLRP3, ASC, and cleaved Caspase-1 were significantly increased (P < 0.05), and the secretions of IL-1β and IL-18 were also significantly enhanced in the Mydgf +NLRP3-EGFP group compared with the Mydgf group (P < 0.05). Conclusion Mydgf may ameliorate cardiomyocyte pyroptosis induced by hyperglycemia by inhibiting NLRP3.

Background: Stent malapposition (SM) following percutaneous coronary intervention (PCI) for myocardial infarction continues to present significant clinical challenges. In recent years, machine learning (ML) models have demonstrated potential in disease risk stratification and predictive modeling. Hypothesis: ML models based on optical coherence tomography (OCT) imaging, laboratory tests, and clinical characteristics can predict the occurrence of SM. Methods: We studied 337 patients from the Affiliated Hospital of Zunyi Medical University, China, who had PCI and coronary OCT from May to October 2023. We employed nested cross‐validation to partition patients into training and test sets. We developed five ML models: XGBoost, LR, RF, SVM, and NB based on calcification features. Performance was assessed using ROC curves. Lasso regression selected features from 46 clinical and 21 OCT imaging features, which were optimized with the five ML algorithms. Results: In the prediction model based on calcification features, the XGBoost model and SVM model exhibited higher AUC values. Lasso regression identified five key features from clinical and imaging data. After incorporating selected features into the model for optimization, the AUC values of all algorithmic models showed significant improvements. The XGBoost model demonstrated the highest calibration accuracy. SHAP values revealed that the top five ranked features influencing the XGBoost model were calcification length, age, coronary dissection, lipid angle, and troponin. Conclusion: ML models developed using plaque imaging features and clinical characteristics can predict the occurrence of SM. ML models based on clinical and imaging features exhibited better performance. [ABSTRACT FROM AUTHOR]

Objective To investigate the mechanism of exosomes released from bone marrow stem cells (BMSCs) on the proliferation in cardiac microvascular endothelial cells (CMVECs) by miR-214 regulation. Methods Rat CMVECs were cultured by enzyme digestion combined with differential adherence. Ultra-high speed centrifugation was used to extract exosomes from normal-oxygen or hypoxic-pretreated BMSCs. Immunofluorescence assay was employed to track the internalization of exosomes in CMVECs. Then the BMSCs were divided into 4 groups: ①control group, ②free exosomes (Free-Exos), ③normal exosomes (Nor-Exos), and ④hypoxia exosomes (Hypoxia-Exos, 400 μg/μL exosome co-cultured with CMVECs for 24 h). The cell proliferation was measured by Edu assay and flow cytometry. The miR-214 levels in exosomes or CMVECs were assessed using qRT-PCR, and the levels were measured again after miR-214 in exosomes were inhibited. And the cell proliferation was observed again by the above methods. Finally, after liposome 2000 was employed to transfect miR-214 mimics into CMVECs, the cell proliferation ability was measured by the Edu assay. Results Transmission electron microscopy and immunoblotting indicated that the extracted vesicles were exosomes. Immunofluorescence assay revealed that DiI-labeled exosomes could be internalized by CMVECs. Edu assay and cell cycle assay showed that Hypoxic-Exos had stronger potential to promote cell proliferation than Nor-Exos in CMVECs. qRT-PCR results showed that miR-214 was highly expressed in Hypoxic-Exos compared with Nor-Exos (P < 0.05). In CMVECs, the expression of miR-214 in Hypoxia-Exos group was significantly higher than that in Control group and Nor-Exos group (P < 0.05). In addition, Hypoxia-Exos's ability to promote cell proliferation was partially blocked by down-regulation of miR-214 in Hypoxia-Exos. However, after overexpression of mir-214 in CMVECs, cell proliferation ability was enhanced. Conclusion Exosomes released from hypoxic-pretreated BMSCs can promote CMVECs proliferation by medicating miR-214.

Objective To explore the regulative effect of miR-214 on oxidative stress injury induced by hypoxia reoxygenation (H/R) in H9c2 myocardial cells though targeted inhibition of CaMKⅡ. Methods Rat myocardial H9c2 cells were randomly divided into 4 groups, ①control group: H9c2 cells without any treatment; ② H/R group: the myocardial cells were cultured in hypoxia for 24 h and then reoxygenation for 6 h; ③ mimics+H/R group: the myocardial cells were transfected with miR-214 and then cultured with H/R as above; ④ mimics negative control (MNC)+H/R group: the myocardial cells were transfected with miR-214's mimics negative control and then cultured with H/R as above. CCK-8 assay was used to detect the viability of cells. qPCR was adopted to detect the expression of miR-214. Flow cytometry, TUNEL assay, and SOD or MDA detect kits were used to determine the reactive oxygen species (ROS) levels and cell apoptosis. The association of miR-214 with CaMKⅡ was examined by double-luciferase reporter assay, and their changes at mRNA and protein levels were detected by qPCR and Western blotting. After H9c2 cells were transfected with recombinant adenovirus carrying CaMKⅡ gene or the control adenovirus, the regulative effect of miR-214 on oxidative stress of cells was examined by the above methods. Results CCK-8 assay showed that compared with normal group, the cell viability of H/R group was significantly decreased (P < 0.05), while miR-214 overexpression could obviously increase the cell viability (P < 0.05). qPCR results showed that the expression level of miR-214 was markedly downregulated in the H/R group than the control group, while notable increase of miR-214 was observed in the miR-214 mimics group (P < 0.05). Compared with the normal group, the ROS production, MDA level and TUNEL positive cells were obviously increased, while the SOD level was significantly decreased in H/R group (P < 0.05), but opposite trends were observed when the HR group compared to and the miR-214 mimics group (P < 0.05). TargetScan server indicated that there was binding site of miR-214 and CaMKⅡ. The dual luciferase reporter assay also confirmed that miR-214 could bind to CaMKⅡ in H9c2 cells. In addition, qPCR and Western blot results showed that H/R treatment increased the expression of CaMKⅡ at mRNA and protein levels, but miR-214 significantly inhibited the enhanced expression induced by H/R. Overexpression of CaMKⅡ in H9c2 cells resulted in partially blocked effects of miR-214 on oxidative stress and apoptosis induced by H/R treatment. Conclusion Overexpression of miR-214 can regulate oxidative stress and apoptosis of H9c2 cells by inhibiting CaMKⅡ at the transcription level.

Abstract: The aim of this study was to explore the relationship between in-stent neoatherosclerosis (ISNA) and the neutrophil-to-lymphocyte ratio (NLR) in patients with in-stent restenosis (ISR) following drug-eluting stent (DES) implantation. We divided 216 patients into 3 groups based on the NLR tertile. We performed a comparative analysis of baseline, angiographic, and features of optical coherence tomography (OCT) between the NLR groups and performed univariate and multivariate logistic regression analyses to assess the association of the NLR with ISNA and in-stent thin-cap fibroatheroma (TCFA). Patients in the third tertile NLR group had a higher incidence of ISNA and in-stent TCFA compared with those in the first tertile. Multivariate logistic regression analysis showed that the hazard ratios and 95% confidence intervals for ISNA and TCFA were 2.673 (1.257-5.684; P = 0.038) and 4.272 (1.740-10.488; P = 0.004), respectively, for patients in the highest tertile compared with those in the lowest tertile. Our study showed that an increased NLR was associated with ISNA and in-stent plaque fragility in patients with ISR following DES implantation., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)