Coronary Microembolization Group Research Articles

Atorvastatin attenuates ferroptosis-dependent myocardial injury and inflammation following coronary microembolization via the Hif1a/Ptgs2 pathway.

Objectives: Coronary microembolization (CME) represents a serious periprocedural complication after percutaneous coronary intervention. Ferroptosis has been identified in multiple cardiovascular diseases. In this study, we aimed to investigate the effects of atorvastatin (ATV) on ferroptosis and inflammation following CME and elucidate the underlying mechanism. Methods: We established a rat model of CME by injecting microspheres into the left ventricle. Deferoxamine (DFO), a selective ferroptosis inhibitor, or ATV was pretreated before modeling. Cardiac function and cardiac troponin T (cTnT) levels were detected. Levels of ferroptosis-associated genes, malondialdehyde (MDA), glutathione (GSH), and ferrous iron (Fe2+) were measured to validate ferroptosis. Levels of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) were assayed to determine the inflammation. Chromatin immunoprecipitation was performed to determine the binding of hypoxia-inducible factor 1 subunit alpha (Hif1a) to the promoter of prostaglandin-endoperoxide synthase-2 (Ptgs2). Results: Ferroptosis and inflammation were induced following CME with increased levels of MDA (∼2.5 fold, p < 0.01), Fe2+ (∼1.5 fold, p < 0.01), TNF-α, and IL-1β and decreased GSH levels (∼42%, p < 0.01). Meanwhile, the level of Ptgs2 was significantly increased, while those of glutathione peroxidase 4 (Gpx4) and solute carrier family 7 member 11 (Slc7a11) were decreased. The level of cTnT was increased by 7-fold (p < 0.01). Left ventricular ejection fraction (LVEF) was significantly reduced (∼85% in the sham group versus ∼45% in the CME group, p < 0.01). DFO or Ptgs2 silencing inhibited the increase of MDA, Ptgs2, TNF-α, and IL-1β, and induced the levels of GSH and Gpx4, followed by reduction in cTnT levels by approximately 50% (p < 0.01). LVEF was improved by approximately 2 fold (p < 0.01). Mechanistically, the transcription factor Hif1a bound to the promoter of Ptgs2 and upregulated its expression. In addition, ATV inhibited the activation of the Hif1a/Ptgs2 axis and attenuated cardiac ferroptosis and inflammation, thus ameliorating CME-induced myocardial injury (LVEF, ∼34% elevation; cTnT, ∼1.8 fold decrease, p < 0.01). Conclusion: Atorvastatin ameliorates ferroptosis-mediated myocardial injury and inflammation following CME via the Hif1a/Ptgs2 pathway.

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-1β, 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.

Long non-coding RNA Sox2OT promotes coronary microembolization-induced myocardial injury by mediating pyroptosis.

ObjectiveAs a common complication of coronary microembolization (CME), myocardial injury (MI) implies high mortality. Long non‐coding RNAs (lncRNAs) are rarely studied in CME‐induced MI. Herein, this study intended to evaluate the role of lncRNA Sox2 overlapping transcript (Sox2OT) in CME‐induced MI.MethodsThe CME rat models were successfully established by injection of microemboli. Rat cardiac functions and MI were observed by ultrasonic electrocardiogram, HE staining, and HBFP staining. Functional assays were utilized to test the inflammatory responses, oxidative stress, and pyroptosis using reverse transcription quantitative polymerase chain reaction, Western blotting, immunohistochemistry, immunofluorescence, and ELISA. Dual‐luciferase reporter gene assay and RNA immunoprecipitation were conducted to clarify the targeting relations between Sox2OT and microRNA (miRNA)‐23b and between miR‐23b and toll‐like receptor 4 (TLR4).ResultsRat CME disrupted the cardiac functions and induced inflammatory responses and oxidative stress, and activated the nuclear factor‐kappa B (NF‐κB) pathway and pyroptosis (all P < 0.05). An NF‐κB inhibitor downregulated the NF‐κB pathway, reduced pyroptosis, and relieved cardiomyocyte injury and pyroptosis. Compared with the sham group (1.05 ± 0.32), lncRNA Sox2OT level (4.41 ± 0.67) in the CME group was elevated (P < 0.05). Sox2OT acted as a competitive endogenous RNA (ceRNA) of miR‐23b to regulate TLR4. Silencing of Sox2OT favoured miR‐23b binding to 3′UTR of TLR4 mRNA leading to suppressed TLR4‐mediated NFKB signalling and pyroptosis in myocardial tissues harvested from CME rat models. In addition, miR‐23b overexpression could supplement the cytosolic miR‐23b reserves to target TLR‐4 and partially reverse Sox2OT‐mediated pyroptosis in LPS‐treated H9C2 cells.ConclusionsThis study supported that silencing Sox2OT inhibited CME‐induced MI by eliminating Sox2OT/miR‐23b binding and down‐regulating the TLR4/NF‐κB pathway. This investigation may provide novel insights for the treatment of CME‐induced MI.

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.

Alprostadil Injection Attenuates Coronary Microembolization-Induced Myocardial Injury Through GSK-3β/Nrf2/HO-1 Signaling-Mediated Apoptosis Inhibition.

ObjectiveCoronary microembolization (CME) results in progressive contractile dysfunction associated with cardiomyocyte apoptosis. Alprostadil injection improves microcirculation, which is effective in treating various cardiovascular disorders. However, the therapeutic effects of alprostadil in CME-induced myocardia injury remain unknown. Therefore, we evaluated the effects of alprostadil injection on cardiac protection in a rat model of CME and explored the underlying mechanisms.MethodsA rat model of CME was established by injecting polyethylene microspheres into the left ventricle. After injection of microspheres, rats in the alprostadil group received alprostadil via tail vein within 2 minutes. Cardiac function, histological alterations in myocardium, serum c-troponin I (cTnI) levels, myocardium adenosine triphosphate (ATP) concentrations, the activity of superoxide dismutase (SOD) and malondialdehyde (MDA) content in myocardium, and myocardial apoptosis-related proteins were detected 12 hours after CME modeling.ResultsCompared with the Sham group, ATP concentrations, SOD activity in the myocardium, and cardiac function were significantly decreased in a rat model of CME. In addition, serum cTnI levels, MDA content, expression levels of pro-apoptotic proteins, and the number of TUNEL-positive nuclei were remarkably higher in CME group than those in the Sham group. However, alprostadil treatment notably reduced serum cTnI levels and expression levels of pro-apoptotic proteins, while noticeably improved cardiac function, and accelerated SOD activity in the myocardium following CME. Additionally, it was unveiled that the protective effects of alprostadil injection inhibit CME-induced myocardial apoptosis in the myocardium potentially through regulation of the GSK-3β/Nrf2/HO-1 signaling pathway.ConclusionAlprostadil injection seems to significantly suppress oxidative stress, alleviate myocardial apoptosis in the myocardium, and improve cardiac systolic and diastolic functions following CME by regulating the GSK-3β/Nrf2/HO-1 signaling pathway.

Shexiang Tongxin Dropping Pill () Reduces Coronary Microembolization in Rats via Regulation of Mitochondrial Permeability Transition Pore Opening and AKT-GSK3β Phosphorylation.

To investigate the protective effects of Shexiang Tongxin Dropping Pill (, STDP) following sodium laurate-induced coronary microembolization (CME) in rats. Forty rats were divided into 4 groups: the control (sham) group, CME group, low-dose STDP pretreatment group (20 mg·kg-1·d-1), and high-dose STDP pretreatment group (40 mg·kg-1·d-1). The rats were intragastric administrated with STDP 2 weeks before operation. Moreover, the histopathological alterations were observed using optical microscopy and transmission electron microscopy. Antioxidant biomarkers were analyzed by enzyme-linked immunosorbent assay. Mitochondrial functions including the mitochondrial permeability transition pore (mPTP) mtDNA copy number were determined and proteins of AKT/GSK3β were analyzed by Western blot. The rats in the CME group showed a significant increase in the fibrinogen-like protein 2 expression level and mitochondrial dysfunction and a decrease in the expression level of antioxidant biomarkers (superoxide dismutase and catalase, P<0.01 for all). In contrast, the rats in the low- and high-dose STDP pretreatment groups showed a significant decrease in coronary microthrombi (P<0.05); moreover, STDP restored the antioxidant-related protein activities and mitochondrial function, inhibited mPTP opening, decreased AKT-Ser473 phosphorylation, and increased GSK3β-Ser9 phosphorylation (P<0.05 or P<0.01). STDP may be useful for treatment of CME, possibly via regulation of mPTP opening and AKT/GSK3β phosphorylation.

Levosimendan Attenuates Myocardial Injury Induced by Coronary Microembolization in Swine by Inhibiting Myocardial Inflammation and Apoptosis

Aims: In addition to its cardiotonic effect, Levosimendan (Levo) has been thought to have multiple cardiovascular benefits, including anti-inflammatory and anti-apoptotic effects. The present study was undertaken to determine whether the Levo pretreatment could attenuate myocardial apoptosis and inflammation and improve cardiac function in a swine model of coronary microembolization (CME). Materials and Methods: A total of 15 swine were randomly and equally divided into a sham-operated (control) group, CME group, and CME plus Levo group. Swine CME was induced by intracoronary injection of inertia plastic microspheres (42 μm diameter) into the left anterior descending (LAD) coronary artery, with or without pretreatment of Levo. Echocardiological measurements, a pathological examination, Terminal-deoxynucleoitidyl Transferase-Mediated dUTP Nick End-Labeling (TUNEL) staining, H and E staining, and Western blotting were performed to assess the functional, morphological, and molecular effects in CME. Results: The expression levels of caspase-3 and tumor necrosis factor-α (TNF-α) were increased in cardiomyocytes following CME. Downregulation of caspase-3 and TNF-α with Levo pretreatment was associated with improved cardiac troponin I (cTnI) and high sensitivity C-reactive protein. In addition, through Pearson correlation analysis, the left ventricular ejection fraction was negatively correlated with caspase-3, TNF-α, and cTnI. Conclusion: This study demonstrated that Levo pretreatment could significantly inhibit CME-induced myocardial apoptosis and inflammation and improve cardiac function. The data generated from this study provide a rationale for the development of myocardial apoptosis and inflammation-based therapeutic strategies for CME-induced myocardial injury.

Dynamic Changes and the Relationship between Cardiac Function Damage and Glycogen Synthase Kinase-3 β Expression Induced by Coronary Microembolization in Rats

Background: Coronary microembolization (CME) is a severe complication in the treatment of acute coronary syndrome (ACS) and percutaneous coronary intervention (PCI). It is a distal microvascular embolism caused by the shedding of atherosclerotic plaque debris, which can directly lead to “no blood flow” or “slow blood flow”. Aims: This study investigates the dynamic changes and the relationship between cardiac function damage and GSK-3β expression induced by coronary microembolization (CME) in rats. Materials and Methods: Specific Sprague Dawley rats (SD rats) were evenly divided into a microembolization group (CME group) and a sham operation group (Sham group). Each group of rats was randomly subdivided into 0h, 3h, 9h, and 24h groups. Results: Echocardiographic parameters showed that the left ventricular ejection fraction (LVEF) of the CME group was significantly lower than that of the sham group with the exception of the 0h time point (P

Resveratrol Attenuates Cardiomyocyte Apoptosis in Rats Induced by Coronary Microembolization Through SIRT1-Mediated Deacetylation of p53.

Coronary microembolization (CME)-induced cardiomyocyte apoptosis is the primary factor in causing cardiac dysfunction. Resveratrol (RES) is known to play a protective role in a variety of cardiovascular diseases, yet it is not known whether RES has a protective role in CME. Therefore, the effect of RES on cardiomyocyte apoptosis and cardiac function damage which are induced by CME in rats was investigated in this study. Fifty Sprague-Dawley rats were separated into 5 groups randomly (10 rats were included in each): sham group, CME group, RES+CME group, RES+CME+Sirtuin-1 (SIRT-1) inhibitor EX527 (RES+CME+EX) group, and CME+EX group. Cardiac function, serum c-troponin I (cTnI) level, apoptotic index, and microinfarct were measured by cardiac ultrasound, myocardial enzyme assessment, TdT-mediated dUTP Nick-end labeling and hematoxylin-basic fuchsin-picric acid staining. The levels of p53, p53 acetylation, SIRT-1, Bax, Bcl-2, and cleaved caspase-3 were detected by Western blot. Myocardial dysfunction, enhanced apoptotic index as well as cTnI were caused after the operation of CME. Coronary microembolization induced increased expression of p53 acetylation and cleaved caspase-3, while the SIRT-1 and Bcl-2/Bax ratio was reduced. The CME effect was reversed by RES while EX527 attenuated this protective effect. Resveratrol can improve cardiac function, in the sense that it attenuates CME-induced cardiomyocyte apoptosis, which is perhaps associated with its inhibition pro-apoptotic pathway of p53 which is transcription-independent.

Egr-1 is involved in coronary microembolization-induced myocardial injury via Bim/Beclin-1 pathway-mediated autophagy inhibition and apoptosis activation.

Coronary microembolization (CME) substantially reduces the clinical benefits of myocardial reperfusion therapy. Autophagy and apoptosis participate in the pathophysiological processes of almost all cardiovascular diseases, including CME-induced myocardial injury, but the precise underlying mechanisms remain unclear. In the present study, we observed that Egr-1 expression was substantially increased after CME modeling. Inhibition of Egr-1 expression through the targeted delivery of rAAV9-Egr-1-shRNA improved cardiac function and reduced myocardial injury. The microinfarct size was also significantly smaller in the Egr-1 inhibitor group than in the CME group. These benefits were partially reversed by the autophagy inhibitor 3-MA. As shown in our previous study, autophagy in the myocardium was impaired after CME. Inhibition of Egr-1 expression in vivo restored the autophagy flux and reduced myocardial apoptosis, at least partially, by inhibiting the Egr-1/Bim/Beclin-1 pathway, as evidenced by the results of the western blot, RT-qPCR, and TUNEL staining. At the same time, TEM showed a dramatic increase in the number of typical autophagic vacuoles in the Egr-1 inhibitor group compared to the CME group. Based on these findings, the Egr-1/Bim/Beclin-1 pathway may be involved in CME-induced myocardial injury by regulating myocardial autophagy and apoptosis, and this pathway represents a potential therapeutic target in CME.

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

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. 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 Xprotein (Bax), B-cell lymphoma 2 (Bcl-2), cleaved caspase-3, tumor necrosis factor α (TNF-α), and interleukin-1β (IL-1β) were detected. 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-1β, while the Bcl-2/Bax ratio decreased. Curcumin reversed these effects by CME, while the gastric lavage control did not exert any effect. 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.

Effects of pretreatment with nicorandil on PDCD4/NF-κB/TNF-α pathway in coronary microembolization

Objective To investigate the role of nicorandil pretreatment on protecting myocardium after coronary microembolization (CME) and on the PDCD4/NF-κB/TNF-α signaling pathway in miniature pigs. Methods Fifteen Bama miniature pigs were randomly(random number) divided into the sham operation group (sham group), microembolization group (CME group) and CME plus nicorandil group, with 5 pigs in each group. The CME model was constructed by injecting polyethylene microspheres via microcatheter into the left anterior descending artery, and pigs in the sham group were injected with the same amount of saline. Pigs in the CME plus nicorandil group were injected intravenously with nicorandil (150 μg/kg) via ear vein 30 min before CME. Cardiac function indexes were measured using cardiac ultrasonography. The expression of PDCD4 and TNF-α mRNA in myocardium were detected by fluorescence quantitative PCR, and the protein expression of PDCD4 and TNF-α in myocardium were detected by Western blotting. NF-κB activation was evaluated by electrophoretic mobility shift assay. Results (1) Cardiac function was significantly lower and the level of serum cTnI was significantly higher in the CME group compared with the sham group. CME reduced myocardial systolic dysfunction and left ventricular dilatation. The CME plus nicorandil group showed improved CME-induced cardiac function and reduced serum cTnI level when compared with the CME Group (P < 0.05). (2) Compared with the CME group, the CME plus nicorandil group showed lower PDCD4 and TNF-α expression and NF-κB activity as well as improved cardiac function (P < 0.05). Conclusions The pretreatment of nicorandil effectively reduced the myocardial damage caused by CME, mainly through inhibiting the PDCD4/NF-κB/TNF-α pathway in cardiomyocytes. Key words: Coronary vessels; Embolism; Nicorandil; Programmed cell death 4; Nuclear facotr-κappa B; Tumor necrosis factor α; Inflammation; Myocardial damage

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

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. 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 1β (IL-1β) were detected. 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-1β. HMGB1 siRNA reversed these effects, whereas scrambled siRNA had no effect. 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.

Nicorandil pretreatment inhibits myocardial apoptosis and improves cardiac function after coronary microembolization in rats.

BackgroundNicorandil (NIC) is a vasodilatory drug used to treat angina. However, its efficacy of cardioprotection in coronary microembolization (CME) is largely unknown. This study was undertaken to determine whether nicorandil pretreatment could attenuate myocardial apoptosis and improve cardiac function after CME in rats.MethodsForty-five rats were randomly divided into a Sham group, a CME group and a CME + NIC (NIC) group (n = 15 per group). CME was established by injecting plastic microspheres (42 µm in diameter) into the left ventricle of the rats in all of the groups except the Sham group. The NIC group received nicorandil at 3 mg/kg per day for seven days before the operation. Cardiac function was assessed by echocardiography, the expression levels of cleaved caspase-9/8/3 were detected by Western blot, microinfarction area was measured by haematoxylin-basic fuchsin picric acid staining, and myocardial apoptosis was detected by TUNEL staining.ResultsCompared to that in the Sham group, cardiac function in the CME group was significantly decreased (P < 0.05). However, compared to the CME group, the NIC group showed improved cardiac function (P < 0.05). The expression levels of cleaved caspase-9/8/3 protein and myocardial apoptosis were dramatically increased in the CME group compared to those in the Sham group (P < 0.05), while the NIC pretreatment group had significantly decreased expression levels of cleaved caspase-9/8/3 protein as well as a decreased apoptotic rate (P < 0.05).ConclusionsNIC pretreatment inhibited CME-induced myocardial apoptosis and improved cardiac function through blockade of the mitochondrial and death receptor-mediated apoptotic pathways.

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

BackgroundMyocardial 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. MethodsForty 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. ResultsCompared 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. ConclusionNIC 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.

Expression of p53 in myocardium following coronary microembolization in rats and its significance.

BackgroundCardiomyocyte apoptosis is a primary cause for coronary microembolization (CME)-induced cardiac dysfunction. p53 induces cell growth retardation and apoptosis through stress pathway. The present study investigated the mechanism of p53-induced myocardial apoptosis and cardiac dysfunction by activating the mitochondrion apoptotic pathway following CME.MethodsForty SD rats were equally divided into microembolization (CME), sham operation (sham), CME+siRNA-p53, and CME+control-p53 groups. The CME rat model was established by injecting microembolization spheres via the left ventricle. Cardiac ultrasound, TUNEL, fluorescence quantitative PCR, and Western blot were used to assess the cardiac function indicators, cardiomyocyte apoptosis, and the expressions of mRNA and protein in myocardial tissues, respectively.ResultsEchocardiography revealed a significantly reduced cardiac function of the CME group than the sham group while the CME-induced cardiac dysfunction was improved in the CME+siRNA-p53 group. The indicators of myocardial apoptosis in the CME group increased significantly than the sham group; those of the CME+siRNA-p53 group decreased significantly than the CME group. Fluorescence quantitative PCR and Western blot demonstrated that p53, Bbc3 (PUMA), and cleaved caspase-3 expressions were significantly increased, and BCL-2 expression was declined in myocardial tissues of the CME group compared to the sham group. A contrasting result was observed in the CME+siRNA-p53 group as compared to the CME group.ConclusionsP53 is involved in the CME-induced cardiac dysfunction, which may up-regulate Bbc3 to activate BCL-2/caspase3 mitochondrial apoptotic pathway and induce myocardial apoptosis. Inhibiting the p53 expression can effectively suppress this pathway, thereby reducing myocardial apoptosis and cardiac dysfunction.

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.

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.

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-1β 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-1β 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.

Atorvastatin Pretreatment Inhibits Myocardial Inflammation and Apoptosis in Swine After Coronary Microembolization.

In addition to its cholesterol-lowering effect, atorvastatin (ATV) has been thought to have multiple cardiovascular benefits, including anti-inflammatory and anti-apoptotic properties. The present study was undertaken to determine whether ATV pretreatment could attenuate myocardial apoptosis and inflammation and improve cardiac function in a swine model of coronary microembolization (CME). Twenty-four swine were randomly and equally divided into a sham-operated (control) group, CME group, and CME plus ATV group. Swine CME was induced by intracoronary injection of inert plastic microspheres (diameter 42 μm) into the left anterior descending coronary, with or without pretreatment of ATV. Echocardiographic measurements, a pathological examination, terminal deoxynucleotidyl transferase-mediated nick end labeling staining, and Western blotting were performed to assess the functional, morphological, and molecular effects in CME. The expression levels of caspase 3 and tumor necrosis factor-α (TNF-α) were aberrantly upregulated in cardiomyocytes following CME. Downregulation of caspase 3 and TNF-α with ATV pretreatment was associated with improved cardiac function and attenuated serum cardiac troponin I (cTnI) and high-sensitivity C-reactive protein. In addition, through a Pearson correlation analysis, the left ventricular ejection fraction negatively correlated with caspase 3, TNF-α, and cTnI. This study demonstrated that ATV pretreatment could significantly inhibit CME-induced myocardial apoptosis and inflammation and improve cardiac function. The data generated from this study provide a rationale for the development of myocardial apoptosis and inflammation-based therapeutic strategies for CME-induced myocardial injury.