Colchicine ameliorates myocardial injury induced by coronary microembolization through suppressing pyroptosis via the AMPK/SIRT1/NLRP3 signaling pathway.

BACKGROUND: Coronary microembolization(CME)is a common complication in acute coronary syndrome and percutaneous coronary intervention, which is closely related to poor prognosis. Pyroptosis, as an inflammatory programmed cell death, has been found to be associated with CME-induced myocardial injury. Colchicine (COL) has potential benefits in coronary artery disease due to its anti-inflammatory effect. However, the role of colchicine in pyroptosis-related CME-induced cardiomyocyte injury is unclear. This study was carried out to explore the effects and mechanisms of colchicine on myocardial pyroptosis induced by CME. METHODS: The CME animal model was constructed by injecting microspheres into the left ventricle with Sprague-Dawley rats, and colchicine (0.3 mg/kg) pretreatment seven days before and on the day of modeling or compound C(CC)co-treatment was given half an hour before modeling. The study was divided into 4 groups: Sham group, CME group, CME + COL group, and CME + COL + CC group (10 rats for each group). Cardiac function, serum myocardial injury markers, myocardial histopathology, and pyroptosis-related indicators were used to evaluate the effects of colchicine. RESULTS: Colchicine pretreatment improved cardiac dysfunction and reduced myocardial injury induced by CME. The main manifestations were the improvement of left ventricular systolic function, the decrease of microinfarction area, and the decrease of mRNA and protein indexes related to pyroptosis. Mechanistically, colchicine increased the phosphorylation level of adenosine monophosphate-activated protein kinase (AMPK), promoted the expression of silent information regulation T1 (SIRT1), and inhibited the expression of NOD-like receptor pyrin containing 3 (NLRP3) to reduce myocardial pyroptosis. However, after CC co-treatment with COL, the effect of colchicine was partially reversed. CONCLUSION: Colchicine improves CME-induced cardiac dysfunction and myocardial injury by inhibiting cardiomyocyte pyroptosis through the AMPK/SIRT1/NLRP3 signaling pathway.

Upregulation of miR-29b-3p alleviates coronary microembolization-induced myocardial injury via regulating BMF and GSK-3ß.

Coronary microembolization (CME) is an intractable complication results from acute coronary syndrome. CME-induced myocardial apoptosis was associated with progressive cardiac contractile dysfunction. miR-29b-3p has been reported implicated in variety cardiovascular diseases, but its function in CME-induced myocardial injury is yet unknown. Herein, a rat model of CME was established by injecting microspheres into the left ventricle and found that the expression level of miR-29b-3p was markedly decreased in the CME rat heart tissues. By using echocardiography, CD31 immunohistochemistry staining, hematoxylin basic fuchsin picric acid (HBFP) staining, TUNEL staining, and western blotting analysis after CME, it was found that upregulating miR-29b-3p improved cardiac dysfunction, promoted angiogenesis, decreased myocardial microinfarct area, and inhibited myocardial apoptosis. Additionally, miR-29b-3p inhibition can reverse the protective benefits of miR-29b-3p overexpression. Mechanistically, the target genes of miR-29b-3p were identified as glycogen synthase kinase 3 (GSK-3ß) and Bcl-2 modifying factor (BMF) by bioinformatics analysis and luciferase reporter experiment. Overall, our findings imply that induction of miR-29b-3p, which negatively regulates GSK-3ß and BMF expression, attenuates CME-induced myocardial injury, suggesting a novel potential therapeutic target for cardioprotective after CME.

Coronary microembolization sheep model by adjusting the number of microspheres based on coronary blood flow.

BACKGROUND: A heart failure (HF) model using coronary microembolization in large animals is indispensable for medical research. However, the heterogeneity of myocardial response to microembolization is a limitation. We hypothesized that adjusting the number of injected microspheres according to coronary blood flow could stabilize the severity of HF. This study aimed to evaluate the effect of microsphere injection based on the left coronary artery blood flow in an animal model. METHODS: Microembolization was induced by injecting different numbers of microspheres (polystyrene, diameter: 90 µm) into the left descending coronary artery of the two groups of sheep (400 and 600 times coronary blood flow [ml/min]). Hemodynamic parameters, the pressure-volume loop of the left ventricle, and echocardiography findings were examined at 0.5, 1.5, 3.5, and 6.5 h after microembolization. RESULTS: End-diastolic pressure and normalized heart rate increased over time, and were significantly higher in 600 × coronary blood flow group than those in 400 × coronary blood flow group (p = 0.04 and p < 0.01, respectively). The maximum rate of left-ventricular pressure rise and normalized stroke volume decreased over time, and were significantly lower in 600 × coronary blood flow group than those in 400 × coronary blood flow group (p < 0.01 and p < 0.01, respectively). The number of microspheres per coronary blood flow was significantly correlated with the decrease in stroke volume and the maximum rate of left ventricular pressure rise in 6.5 h (r = 0.74, p = 0.01 and r = 0.71, p = 0.02, respectively). CONCLUSIONS: Adjusting the number of injected microspheres based on the coronary blood flow enabled the creation of HF models with different degrees of severity.

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.

MicroRNA-136-5p protects cardiomyocytes from coronary microembolization through the inhibition of pyroptosis.

This study investigated how miR-136-5p partially affected cardiomyocyte pyroptosis in rats with coronary microembolization (CME). The cardiac function and structure of rats with CME were evaluated using echocardiography, hematoxylin and eosin staining, Masson staining, and troponin I level. Pyroptosis was induced by lipopolysaccharide (LPS) in isolated rat cardiomyocytes and evaluated by the expression of caspase-1, NOD-like receptor family pyrin domain-containing 3, interleukin-1ß, and gasdermin D-N. After cell transfection, the expression of Ataxin-1 like (ATXN1L), pyrin domain-containing 1 (PYDC1), and pyroptosis-related proteins was assessed. Dual-luciferase reporter and immunoprecipitation assays were used to verify the relationships among miR-136-5p, ATXN1L, and capicua (CIC). MiR-136-5p was under-expressed, whereas ATXN1L was overexpressed in rats with CME and in LPS-treated primary cardiomyocytes. MiR-136-5p targeted ATXN1L, and ATXN1L bound to CIC to suppress PYDC1 expression. MiR-136-5p overexpression suppressed pyroptosis by inhibiting the binding of ATXN1L with CIC and promoting PYDC1 expression, which was reversed by simultaneous elevation of ATXN1L. In conclusion, miR-136-5p suppressed pyroptosis by upregulating PYDC1 via ATXN1L/CIC axis, thereby attenuating cardiac damage caused by CME.

Tanshinone IIA reduces pyroptosis in rats with coronary microembolization by inhibiting the TLR4/MyD88/NF-κB/NLRP3 pathway.

Pyroptosis is an inflammatory form of programmed cell death that is linked with invading intracellular pathogens. Cardiac pyroptosis has a significant role in coronary microembolization (CME), thus causing myocardial injury. Tanshinone IIA (Tan IIA) has powerful cardioprotective effects. Hence, this study aimed to identify the effect of Tan IIA on CME and its underlying mechanism. Forty Sprague-Dawley (SD) rats were randomly grouped into sham, CME, CME + low-dose Tan IIA, and CME + high-dose Tan IIA groups. Except for the sham group, polyethylene microspheres (42 µm) were injected to establish the CME model. The Tan-L and Tan-H groups received intraperitoneal Tan IIA for 7 days before CME. After CME, cardiac function, myocardial histopathology, and serum myocardial injury markers were assessed. The expression of pyroptosis-associated molecules and TLR4/MyD88/NF-κB/NLRP3 cascade was evaluated by qRT-PCR, Western blotting, ELISA, and IHC. Relative to the sham group, CME group's cardiac functions were significantly reduced, with a high level of serum myocardial injury markers, and microinfarct area. Also, the levels of caspase-1 p20, GSDMD-N, IL-18, IL-1ß, TLR4, MyD88, p-NF-κB p65, NLRP3, and ASC expression were increased. Relative to the CME group, the Tan-H and Tan-L groups had considerably improved cardiac functions, with a considerably low level of serum myocardial injury markers and microinfarct area. Tan IIA can reduce the levels of pyroptosis-associated mRNA and protein, which may be caused by inhibiting TLR4/MyD88/NF-κB/NLRP3 cascade. In conclusion, Tanshinone IIA can suppress cardiomyocyte pyroptosis probably through modulating the TLR4/MyD88/NF-κB/NLRP3 cascade, lowering cardiac dysfunction, and myocardial damage.

Animal model of coronary microembolization under transthoracic echocardiographic guidance in rats.

The aim of the study was to develop a model of coronary microembolization (CME) in rats at a lower cost. We developed a novel rat model without thoracotomy and ventilation under the guidance of echocardiography. Rats were sacrificed at 3 h, 24 h and 1 month postoperatively in both the Echo-CME and Open-chest CME groups for the comparison of the modeling accuracy, mortality, cardiopulmonary circulation, pleural adhesion and ventilation-induced lung injury (VILI). Results showed that the coronary microthrombus formed at 3 h and reached its peak at 24 h postoperatively, which included platelet aggregation and fibrin web. The Echo-group increases success rates, decreased mortality, postoperative complications including pleural adhesion, cardiopulmonary dysfunction and VILI postoperatively than the Open-chest group at 1month postoperatively. The ejection fraction of the CME group decreased to 50% and obvious cardiac fibrosis formed at 3 months postoperatively. Our unique surgical method provided a platform to study molecular mechanisms and potential new pathways for CME treatment.

Puerarin pretreatment attenuates cardiomyocyte apoptosis induced by coronary microembolization in rats by activating the PI3K/Akt/GSK-3ß signaling pathway.

Coronary microembolization (CME) is associated with cardiomyocyte apoptosis and cardiac dysfunction. Puerarin confers protection against multiple cardiovascular diseases, but its effects and specific mechanisms on CME are not fully known. Hence, our study investigated whether puerarin pretreatment could alleviate cardiomyocyte apoptosis and improve cardiac function following CME. The molecular mechanism associated was also explored. A total of 48 Sprague-Dawley rats were randomly divided into CME, CME + Puerarin (CME + Pue), sham, and sham + Puerarin (sham + Pue) groups (with 12 rats per group). A CME model was established in CME and CME + Pue groups by injecting 42 µm microspheres into the left ventricle of rats. Rats in the CME + Pue and sham + Pue groups were intraperitoneally injected with puerarin at 120 mg/kg daily for 7 days before operation. Cardiac function, myocardial histopathology, and cardiomyocyte apoptosis index were determined via cardiac ultrasound, hematoxylin-eosin (H&E) and hematoxylin-basic fuchsin-picric acid (HBFP) stainings, and TdT-mediated dUTP nick-end labeling (TUNEL) staining, respectively. Western blotting was used to measure protein expression related to the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase-3ß (GSK-3ß) pathway. We found that, puerarin significantly ameliorated cardiac dysfunction after CME, attenuated myocardial infarct size, and reduced myocardial apoptotic index. Besides, puerarin inhibited cardiomyocyte apoptosis, as revealed by decreased Bax and cleaved caspase-3, and up-regulated Bcl-2 and PI3K/Akt/GSK-3ß pathway related proteins. Collectively, puerarin can inhibit cardiomyocyte apoptosis and thus attenuate myocardial injury caused by CME. Mechanistically, these effects may be achieved through activation of the PI3K/Akt/GSK-3ß pathway.

Protective effects and mechanism of curcumin on myocardial injury induced by coronary microembolization.

OBJECTIVE: Coronary microembolization (CME) is a common complication during the percutaneous coronary intervention (PCI). CME-induced local myocardial inflammation and myocardial apoptosis are the primary causes of progressive cardiac dysfunction. Curcumin exerts a protective role in various cardiovascular diseases; however, its effects in CME are yet to be clarified. Therefore, the current study investigated the effects of curcumin on myocardial inflammatory responses, myocardial apoptosis, and cardiac dysfunctions induced by CME in rats. METHODS: A total of 40 Sprague-Dawley rats were randomly divided into the following groups: Sham operation (sham group), CME group, curcumin, and control with 10 rats in each group. The ascending aortas were clamped, and the CME-model group was established by injecting microspheres into the apex of the left ventricle. An equivalent amount of normal saline was injected to establish the sham group. The cardiac functions, serum c-troponin I level, and apoptotic index was examined. Also, the levels of Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MYD88), nuclear factor κB (NF-κB) p65, BCL2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), cleaved caspase-3, tumor necrosis factor α (TNF-α), and interleukin-1ß (IL-1ß) were detected. RESULTS: Myocardial dysfunction enhanced serum c-troponin I, and apoptotic index were induced following CME. Moreover, CME elevated the expression of TLR4, MyD88, NF-κB p65, cleaved caspase-3, TNF-α, and IL-1ß, while the Bcl-2/Bax ratio decreased. Curcumin reversed these effects by CME, while the gastric lavage control did not exert any effect. CONCLUSION: Curcumin was responsible for the anti-CME-induced myocardial injury. The effector mechanism might be related to the reduction of cardiomyocyte apoptosis and inhibition of myocardial inflammatory responses mediated by TLR4/MyD88/NF-κB signaling pathway.

Role of high-mobility group B1 in myocardial injury induced by coronary microembolization in rats.

OBJECTIVE: This study aimed to explore the effects of high-mobility group B1 (HMGB1) on coronary microembolization (CME)-induced myocardial inflammation, myocardial apoptosis, and cardiac function injury in rats. METHODS: Forty Sprague-Dawley rats were divided into sham operation group (sham group), microembolization group (CME group), CME + HMGB1 siRNA (HMGB1 siRNA) group, and CME + scrambled siRNA (control siRNA) group (10 rats in each group). The CME model group was constructed by injecting microembolism spheres into the apex of the left ventricle after clamping the ascending aorta. The sham group was constructed by injecting the same amount of saline. The HMGB1 siRNA group was injected with HMGB1 siRNA transfection complex via the tail vein 72 hours before CME modeling. The control siRNA group was injected with the same amount of scrambled siRNA mixture through the tail vein 72 hours before CME modeling. The cardiac function, serum cardiac troponin I level, and apoptotic index were examined 12 hours after the surgery. The levels of HMGB1, nuclear factor-κB (NF-κB) p65, glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), cleaved caspase-12, cleaved caspase-3, tumor necrosis factor-α (TNF-α), and interleukin 1ß (IL-1ß) were detected. RESULTS: Myocardial dysfunction, enhanced serum cardiac troponin I level, and apoptotic index were induced following CME. Moreover, CME increased the expression of HMGB1, NF-κB p65, GRP78, CHOP, cleaved caspase-12, cleaved caspase-3, TNF-α, and IL-1ß. HMGB1 siRNA reversed these effects, whereas scrambled siRNA had no effect. CONCLUSIONS: Inhibition of HMGB1 expression reduced CME-induced myocardial injury and improved cardiac function. Hence, it may serve as a new target for preventing and treating the CME-induced myocardial injury.

Effects of the TLR4/Myd88/NF-κB Signaling Pathway on NLRP3 Inflammasome in Coronary Microembolization-Induced Myocardial Injury.

BACKGROUND/AIMS: Coronary microembolization (CME) is a common complication of acute coronary syndrome (ACS) and percutaneous coronary intervention (PCI); Myocardial inflammation, caused by CME, is the main cause of cardiac injury. TLR4/MyD88/NF-κB signaling plays an important role in the development of myocardial inflammation, but its effects on CME remain unclear. To assess the cardiac protective effects of TAK-242 (TLR4 specific inhibitor) on CME-induced myocardial injury, and explore the underlying mechanism. METHODS: Cardiac function, serum c-troponin I level, microinfarct were examined by cardiac ultrasound, myocardial enzyme assessment, HBFP staining. The levels of TLR4/MyD88/NF-κB signaling and NLRP3 inflammasome pathway were detected by ELISA, qRT-PCR and western blot. RESULTS: The results showed inflammatory responses in the myocardium after CME, with increased expression levels of pro-inflammatory factors TNF-α, IL-1ß and IL-18. Meanwhile, TLR4/MyD88/NF-κB signaling and the NLRP3 inflammasome were involved in the inflammatory process. TAK-242 administration before CME effectively inhibited the inflammatory response in the rat myocardium after CME and reduced myocardial injury, mainly by inhibiting TLR4/ MyD88/NF-κB signaling and reducing NLRP3 inflammasome activation. In addition, in vitro assays with neonatal rat cardiomyocytes further confirmed that TLR4/MyD88/NF-κB signaling was significantly activated in the inflammatory response of LPS-induced cardiomyocytes, via activation of the NLRP3 inflammasome. Inhibition of TLR4/MyD88/NF-κB signaling resulted in increased survival of cardiomyocytes mainly by reducing the release of inflammatory cytokines and decreasing NLRP3 inflammasome activation. CONCLUSIONS: TLR4/MyD88/NF-κB signaling participates in the inflammatory response of the myocardium after CME, activating the NLRP3 inflammasome, promoting the inflammatory cascade, and aggravating myocardial injury. Blocking TLR4/MyD88/NF-κB signaling may help reduce myocardial injury and improve cardiac function after CME.

The protective effect of nicorandil on cardiomyocyte apoptosis after coronary microembolization by activating Nrf2/HO-1 signaling pathway in rats.

BACKGROUND: Myocardial apoptosis is considered to be the chief cause of progressive cardiac dysfunction induced by coronary microembolization (CME), and the Nrf2/HO-1 signaling pathway is involved in CME-induced myocardial apoptosis. Nicorandil (NIC) has multiple beneficial cardiovascular effects on myocardial injury. Therefore, this study was undertaken to analyze the role of NIC pretreatment in the inhibiting myocardial apoptosis after CME in rats. METHODS: Forty rats were divided into Sham group, CME group, CME plus NIC (NIC) group, and CME plus AAV9-Nrf2 (AAV9-Nrf2) group (n = 10 per group). CME-induced myocardial apoptosis model was established through injecting plastic microspheres (42 µM) into the left ventricle except the Sham group. NIC group received nicorandil 3 mg/(kg.d) for 7 days before the operation. Cardiac function was assessed by echocardiography. The mRNA expression level of Nrf2 was detected by RT-PCR. The protein expression levels of Nrf2, HO-1, Bcl-2, Bax and cleaved caspase-3 were detected by Western blot. The size of the microinfarction area was measured by HBFP staining; myocardial apoptosis was analyzed by TUNEL staining. RESULTS: Compared with the sham group, the cardiac function and the expression level of Nrf2, HO-1 and Bcl-2were decreased, while myocardial apoptosis and the expression of Bax and cleaved caspase-3 were increased in the CME group. Compared with the CME group, cardiac function was significantly improved, the expression levels of Nrf2, HO-1, and Bcl-2 were increased, the expression of Bax and cleaved caspase-3 were decreased, and the myocardial apoptosis was attenuated in the NIC group and AAV9-Nrf2 group. CONCLUSION: NIC pretreatment effectively inhibit CME-induced myocardial apoptosis and improve cardiac function. The protective effects are mediated through the activation of the Nrf2/HO-1 signaling in cardiomyocytes.

TAK-242 Protects Against Apoptosis in Coronary Microembolization-Induced Myocardial Injury in Rats by Suppressing TLR4/NF-κB Signaling Pathway.

BACKGROUND/AIMS: Myocardial apoptosis is heavily implicated in the myocardial injury caused by coronary microembolization (CME), and toll-like receptor 4 (TLR4) is considered to be involved in this apoptotic cascade. Therefore, the present study was designed to investigate the role of TLR4/NF-κB signaling pathway regulated by TAK-242, a selective TLR4 signal transduction inhibitor, in the myocardial apoptosis after CME in rats. METHODS: Forty-five rats were randomized (random number) into three groups: sham, CME and CME + TAK-242 (n = 15 per group).CME was induced by injecting polyethylene microspheres (42µm) into the left ventricular except the sham group. CME + TAK-242 group was treated with TAK-242 (2mg/kg) via the tail vein 30 minutes before CME modeling. Cardiac function was evaluated 6 hours after operation. Tissue biopsy was stained with HBFP to measure the size of micro-infarction area. TUNEL staining was used to detect myocardial apoptosis. Western blot and qPCR were used to evaluate the expression of TLR4, MyD88, NF-κB p65, p-IκBα and Cleaved caspase-3. RESULTS: Cardiac function in the CME group and CME + TAK-242 group were significantly decreased compared with the sham group (P < 0.05) and the micro-infarction area, the apoptotic index, the expression of TLR4, NF-κB p65, p-IκBα and Cleaved caspase-3 were increased significantly (P < 0.05). Cardiac function in the CME + TAK-242 group was significantly improved compared with the CME group (P < 0.05) and the micro-infarction area, the apoptotic index, the expression of TLR4, MyD88, NF-κB p65, p-IκBα and Cleaved caspase-3 were decreased significantly (P < 0.05). CONCLUSIONS: TAK-242 can effectively improve CME-induced cardiac dysfunction by regulating TLR4/NF-κB signaling pathway and then reducing the myocardial apoptosis.

Levosimendan Pretreatment Inhibits Myocardial Apoptosis in Swine after Coronary Microembolization.

BACKGROUND/AIMS: In addition to its cardiotonic effect, levosimendan has been thought to have multiple cardiovascular benefits, including anti-inflammatory and anti-apoptotic. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) has been revealed to be up-regulated in circumstances of coronary microembolization (CME), and the PTEN signaling pathway mediates myocardial apoptosis in swine after CME. However, whether this functional protein could be modified by pretreatment of levosimendan in models of CME has not been disclosed yet. METHODS: Swine CME was induced by intra-coronary injection of inertia plastic microspheres (diameter 42µm) into left anterior descending coronary, with or without pretreatment of levosimendan or PTEN siRNA. Echocardiologic measurements, Terminal-deoxynucleotidyl Transferase Mediated Nick End Labeling (TUNEL) staining and western blotting were applied to assess their functional, morphological and molecular effects in CME. RESULTS: PTEN mRNA and protein were aberrantly up-regulated in cardiomyocytes following CME. Furthermore, down-regulation of PTEN in vivo via siRNA was associated with an improved cardiac function, attenuated myocardial apoptosis, and concomitantly inhibited expressions of key proapoptotic proteins such as caspase-3. Interestingly, levosimendan could markedly attenuate PTEN expression and inhibit myocardial apoptosis, therefore partially reverse cardiac dysfunction. CONCLUSION: Modulation of PTEN was probably as a potential mechanism involved in the beneficial effects of pretreatment of levosimendan to cardiac function and apoptosis in animal models of CME.

MiRNA Expression Profile of the Myocardial Tissue of Pigs with Coronary Microembolization.

BACKGROUND/AIMS: Coronary microembolization (CME) is a serious complication of coronary heart disease and is considered as a strong predictor of poor long-term prognosis and major cardiac adverse events. Here, we identified differentially expressed microRNAs (miRNAs) in the myocardial tissue of CME pigs, and predicted and analyzed the possible functions of their target genes. METHODS: Twelve Bama mini-pigs were randomly assigned to the sham and CME group (n = 6 in each group). The two groups were compared with regard to heart function, area of infarction, cardiomyocyte apoptosis, and myocardial expression of TNF-α, IL-1ß and IL-6. Further, miRNA chip analysis was used to screen for differentially expressed miRNAs, and the results were validated by real-time PCR. Bioinformatics methods were used to predict and analyze the functions of the target genes of the identified miRNAs. RESULTS: The model CME pigs showed significantly increased expression of TNF-α, IL-1ß and IL-6, as well as micro-infarction lesions and cell apoptosis in the myocardial tissue. Thus, the model was established successfully. In the myocardial tissue of the CME pigs, the expression of ssc-miR-92b-5p, ssc-miR-491, ssc-miR-874, ssc-miR-425-3p, ssc-miR-376a-5p, ssc-miR-370, ssc-miR-30c-3p, ssc-miR-493-5p and ssc-miR-323 was significantly increased, whereas the expression of ssc-miR-136 and ssc-miR-142-3p was significantly decreased. GO and KEGG pathway analysis indicated that the target genes of these miRNAs are mainly associated with cell proliferation, apoptosis, necrosis, inflammation, and fibrosis. CONCLUSION: The differentially expressed miRNAs identified in the myocardial tissue of CME pigs could be new biomarkers or potential treatment targets for CME.

Potential Involvement of MiR-30e-3p in Myocardial Injury Induced by Coronary Microembolization via Autophagy Activation.

BACKGROUND/AIMS: Coronary microembolization (CME) can lead to no-reflow or slow reflow, which is one of the important reasons for loss of clinical benefit from myocardial reperfusion therapy. MicroRNAs and autophagy are heavily implicated in the occurrence and development of almost all cardiovascular diseases. Therefore, the present study was designed to investigate the role of miR-30e-3p and autophagy in CME-induced myocardial injury rat model. METHODS: Sixty rats were randomly divided into six groups: sham, CME 1h,3h,6h,9h, and 12h (n = 10 per group). Our CME rat model was created by injecting polyethylene microspheres (42mm) into the left ventricle of the heart; the sham group was injected with same volume of normal saline. The cardiac function and serum cardiac troponin I (cTnI) level of each group was measured. HE staining and HBFP staining were used to evaluate the myocardial micro-infarction area of myocardium tissue samples. Then RT-qPCR and western blot were used to detect the expression of miR-30e-3p and, autophagy related protein LC3-II and p62, respectively. Transmission electron microscope (TEM) was used to identify autophagic vacuoles in tissue samples. RESULTS: The cardiac function of the CME 6h,9h, and 12h groups were significantly decreased compared to the sham group (P < 0.05) and the cTnI level in each group were also significantly increased (P < 0.05). The expression of miR-30e-3p in the CME 6h, 9h and 12h group were decreased significantly compared with the sham group (P < 0.05). Meanwhile, the expression of autophagy related protein LC3-II decreased significantly and p62 increased significantly in the CME 9h and 12h group (P < 0.05). TEM images showed typical autophagic vacuoles for each of the CME groups. CONCLUSIONS: Myocardial miR-30e-3p is down regulated after CME and is accompanied by inhibited autophagy and decreased cardiac function. Therefore, miR-30e-3p may be involved in CME-induced cardiac dysfunction by regulating myocardial autophagy.

Effects of Trimetazidine on PDCD4/NF-κB/TNF-α Pathway in Coronary Microembolization.

BACKGROUND/AIMS: The local inflammatory response caused by coronary microembolization (CME) is the primary cause of progressive cardiac dysfunction. The PDCD4/NF-κB/TNF-α signaling pathway plays a significant role in CME-induced myocardial Inflammation. Trimetazidine (TMZ) reduces myocardial injury, caused by percutaneous coronary intervention, through relieving the CME-induced myocardial systolic dysfunction. Therefore, the present study investigated the role of TMZ pre-treatment in the protection of myocardium after CME and PDCD4/NF-κB/TNF-α in mini pigs. METHODS: 20 Bama mini pigs were randomized into sham operation (sham), microembolization (CME), TMZ, and siRNA-PDCD4 groups (n = 5). The CME model was established by injecting polyethylene microspheres via microcatheter into the left anterior descending coronary artery. The TMZ group was injected 2.5 mg/kg drug via ear vein 30 min before CME; whereas, the siRNA-PDCD4 group was transfected with PDCD4 siRNA at the left anterior descending coronary artery via microcatheter 72h before CME. Cardiac function indexes were measured using cardiac echocardiography. The mRNA expression of PDCD4 and TNF-α in the myocardium was detected by quantitative fluorescence PCR, and the protein expression of PDCD4, NF-κB (p65), and TNF-α by Western blot. RESULTS: Echocardiographic parameters showed lower cardiac function and higher serum cTnI level in the CME group than sham, which was manifested as reduced left ventricular ejection fraction (LVEF), left ventricular fractional shortening (FS), cardiac output (CO), and increased left ventricular diastolic diameter (LVEDd). Compared to the CME group, the CME-induced cardiac function injury was reduced, and the serum cTnI level was decreased in the TMZ and siRNA-PDCD4 groups. The expressions of PDCD4, NF-κB (p65), and TNF-α were significantly increased in the CME than the sham groups (P < 0.05), and significantly decreased in the TMZ and siRNA-PDCD4 groups than the CME group (P < 0.05). CONCLUSION: TMZ pretreatment effectively reduced the myocardial damage caused by CME via inhibiting the PDCD4/NF-κB/ TNF-α pathway in cardiomyocytes.

The protective effect of activating Nrf2 / HO-1 signaling pathway on cardiomyocyte apoptosis after coronary microembolization in rats.

BACKGROUND: Myocardial apoptosis is closely related to myocardial injury caused by coronary microembolization (CME).Nuclear factor erythroid 2-like (Nrf2) has been taken into account as an inhibitor of apoptosis in various tissues. Thus, this research aims to investigate which part Nrf2/HO-1 signaling pathway plays in myocardial apoptosis process following the effect of CME on rats. METHODS: Separate 40 rats then form them into a group of shame, a group of CME, a group of CME plus AAV-Nrf2(AAV-Nrf2 (CME) group) and a group of CME plus AAV-control (AAV-control (CME) group) stochastically and averagely. Rat CME was established by injecting into the left ventricular chamber, with or without pretreatment of adeno-associated virus Nrf2 (AAV-Nrf2). Echocardiological measurements, using Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) to stain, conducting Quantitative PCR in real time (RT-PCR) as well as Western blotting to evaluate the impacts of them functionally, morphologically and molecularly in CME. RESULTS: Nrf2 decreased in cardiomyocytes after CME. Upregulation of Nrf2 inside an organism through AAV connect to improving the function of heart as well as attenuating myocardial apoptosis, following the restrain of proapoptotic mRNAs and proteins like caspase-3, caspase-9 and bax expressing as well as the increase of antiapoptotic mRNA and proteins like HO-1 and bcl-2 expressing. CONCLUSION: Activation of Nrf2/HO-1 pathway can improve CME-induced cardiac dysfunction effectively and also reduce the myocardial apoptosis.

Assessment of the acute effects of glucocorticoid treatment on coronary microembolization using cine, first-pass perfusion, and delayed enhancement MRI.

PURPOSE: To assess the acute effects of methylprednisone treatment (MPT) on coronary microembolization (CME) by cardiac cine, first-pass perfusion, and delayed gadolinium enhancement magnetic resonance imaging (DE-MRI) in an experimental swine model. MATERIALS AND METHODS: Microembolization was established by intracoronary infusion of microspheres into the left anterior artery. Swine received placebo (n = 12) or methylprednisolone (n = 10, 30 mg/kg) intravenously 30 minutes before microembolization. Perfusion and DE-MRI was performed 6 hours after microembolization. Cine MR images of pre-/post-CME were obtained using 1.5T scanner. RESULTS: Cine MRI demonstrated relative amelioration of the post-CME myocardial contractile dysfunction in the glucocorticoid-treated group compared to the placebo group (P < 0.001). Post-CME target myocardial perfusion parameters decreased in both groups after microembolization. The extent of these decreases were the same for the embolized-to-control area ratio of maximum upslope (P = 0.245; 95% confidence interval of the difference [CID], -0.041/0.148) and time to peak ratio (P = 0.122; 95% CID, -0.201/0.026); however, the maximum signal intensity was higher in the glucocorticoid-treated group (P = 0.012; 95% CID, 0.023/0.156). DE-MRI revealed patchy hyperenhancement in all placebo pigs (n = 12/12) after microembolization, but no hyperenhanced regions in the glucocorticoid-pretreated pigs (n = 0/10). CONCLUSION: Standard, readily available, cardiac MRI techniques are useful in demonstrating post-CME myocardial dysfunction and the acute effects of glucocorticoid treatment on CME. Glucocorticoid pretreatment improves myocardial contractile dysfunction, prevents hyperenhancement, and partially ameliorates the decline of myocardial perfusion in the embolized area.

Effects of atorvastatin on PDCD4/NF-κB/TNF-α signaling pathway during coronary microembolization of miniature pigs.

OBJECTIVE: In this study, we explore the effects of pretreatment with atorvastatin on the cardiac function of piglets after coronary microembolization (CME). METHODS: Twenty Bama miniature pigs were randomized to sham surgery group (sham group), CME Group, conventional dose group and intensive-dose group, with 5 miniature pigs in each group. Pigs in the CME group received a total dosage of 100,000 microspheres (42 µm) suspended in 10 ml normal saline within 40 min, while animals in the sham group received the same dosage of normal saline. In the conventional dose group, atorvastatin (1.5mg/kg) was given once daily starting 7d before microembolization. In the intensive-dose group, atorvastatin (1.5mg/kg) was also given once daily 7d before intervention, and an additional 3mg/kg 4h before percutaneous coronary intervention. Cardiac function indices were determined by echocardiography; and infarct size was determined histopathologically. PDCD4 mRNA and TNF-α mRNA and protein expression were evaluated by quantitative fluorescence-polymerase chain reaction and Western blot, respectively. NF-κB activation was evaluated by electrophoretic mobility shift assay. RESULTS: (1) Cardiac function was significantly lower (P<0.05) in CME group compared with the sham group. CME reduced myocardial systolic dysfunction and left ventricular dilatation. The conventional and intensive-dose groups showed improved CME-induced cardiac function when compared with the CME Group (P<0.05). (2) Compared with the conventional group, the dose-intensive group showed lower PDCD4 and TNF-α expression and NF-κB activation as well as improved cardiac function (P<0.05). CONCLUSIONS: Atorvastatin effectively improved CME-induced cardiac damage with intensive-dose therapy showing better outcome. The protective effects are mediated via suppression of PDCD4/NF-κB/TNF-α signaling in cardiomyocytes.