MicroRNA-486-5p targeting PTEN Protects Against Coronary Microembolization-Induced Cardiomyocyte Apoptosis in Rats by activating the PI3K/AKT pathway.

Coronary microembolization (CME) is responsible for a substantial fraction of microvascular obstruction (MVO), which are strongly associated with mortality and hospitalization for heart failure within 1 year after primary percutaneous coronary intervention (PCI) in ST-segment elevation myocardial infarction (STEMI). However, the effect of miRNA on cardiomyocyte apoptosis in a CME model has been less well-studied. miRNA sequencing analysis was performed to examine differentially expressed miRNAs induced by CME in rats. Phosphatase and tensin homologue (PTEN) 3 'RACE and dual-luciferase reporter assays were performed to confirm that PTEN is a direct target gene of miR-486-5p. miRNA-486-5p overexpression was established by injecting AAV into rats via the tail vein. The CME model was established by injecting microspheres into the left ventricle of rats. 6h after surgery, cardiac function, microinfarct area, and the apoptotic index were determined. RT-PCR was used to evaluate mRNA level and Western blotting was used to evaluate protein expression. miRNA sequencing data showed that there were 5 upregulated and 8 downregulated miRNAs, and the relative expression of miRNA-486-5p was significantly downregulated. PTEN 3'RACE and dual-luciferase reporter assays confirmed that miR-486-5p directly targets the rat PTEN gene. The expression of miR-486-5p gradually declined, however, the expression of PTEN mRNA rapidly increased at early time points after CME. Overexpression of miR-486-5p reduced cardiomyocyte apoptosis and improved cardiac function through inhibition of PTEN and activation of the PI3K/Akt pathway in rat CME models. Overexpression of miR-486-5p, which targets PTEN, protects against CME-induced cardiomyocyte apoptosis and improves cardiac function in rats by activating the PI3K/Akt pathway.

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.

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

BACKGROUND: Nicorandil (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. METHODS: Forty-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. RESULTS: Compared 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). CONCLUSIONS: NIC pretreatment inhibited CME-induced myocardial apoptosis and improved cardiac function through blockade of the mitochondrial and death receptor-mediated apoptotic pathways.

Pim1 Overexpression Prevents Apoptosis in Cardiomyocytes After Exposure to Hypoxia and Oxidative Stress via Upregulating Cell Autophagy.

BACKGROUND/AIMS: Microvascular obstruction (MVO), an undesirable complication of percutaneous coronary intervention, is independently associated with adverse left ventricle remodeling and poor prognosis after acute myocardial infarction. Hypoxia and oxidative stress major roles in the pathophysiology of MVO. Pim1 serves an important protective role in the ischemic myocardium, but the underlying mechanisms remain poorly defined. Autophagy in early hypoxia or during moderate oxidative stress has been demonstrated to protect the myocardium. In this study, we investigated the association between the protective effect of Pim1 and autophagy after hypoxia and oxidative stress. METHODS: Ventricular myocytes from neonatal rat heart (NRVMs) were isolated. NRVMs were exposed to hypoxia and H2O2. Rapamycin and 3-methyladenine (3-MA) were used as an activator and inhibitor of autophagy, respectively. pHBAd-Pim1 was transfected into NRVMs. We assessed cardiomyocyte apoptosis by Annexin V-FITC/PI flow cytometry. Autophagy was evaluated by mRFP-GFP-LC3 adenovirus infection by confocal microscopy. Western blotting was used to quantify apoptosis or autophagy protein (caspase-3, LC3, P62, AMPK, mTOR, ATG5) concentrations. RESULTS: Autophagy and apoptosis in NRVMs significantly increased and peaked at 3 h and 6 h, respectively, after exposure to hypoxia and H2O2. The mTOR inhibitor rapamycin induced autophagy and decreased cardiomyocyte apoptosis, but the autophagy inhibitor 3-MA decreased autophagy and increased apoptosis at 3 h after exposure to hypoxia and H2O2. Pim1 levels in NRVMs increased at 3 h and decreased gradually after exposure to hypoxia and H2O2. Pim1 overexpression enhanced autophagy and decreased apoptosis. Pim1-induced promotion of autophagy is partly the result of activation of the AMPK/mTOR/ATG5 pathway after exposure to hypoxia and H2O2. CONCLUSION: Our results revealed that Pim1 overexpression prevented NRVMs from apoptosis via upregulating autophagy after exposure to hypoxia and oxidative stress, partly through activation of the AMPK/mTOR/ATG5 autophagy pathway.

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.

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.

The effects of simvastatin on left ventricular hypertrophy and left ventricular function in patients with essential hypertension.

This study is to evaluate the effects of Simvastatin on left ventricular hypertrophy and left ventricular function in patients with essential hypertension. Untreated or noncompliance with drug treatment patients with simple essential hypertension were treated with a therapy on the basis of using Telmisartan to decrease blood pressure (BP). There were 237 patients who had essential hypertension combined with left ventricular hypertrophy as diagnosed by echocardiography, taken after their BPs were decreased to meet the values of the standard normal. Among them, there were only 41 out of the original 237 patients, 17.3%, who had simple essential hypertension combined with left ventricular hypertrophy without any other co-existing disease. They were the patients selected for this study. All patients were randomly, indiscriminately divided into two groups: one was the control group (Group T), treated with the Telmisartan-based monotherapy; the other was the target group (Group TS), treated with the Telmisartan-based plus simvastatin therapy. The changes of left ventricular hypertrophy and left ventricular function were rediagnosed by echocardiography after 1 year. The results we obtained from this study were as follows: (i) The average BPs at the beginning of the study, of simple essential hypertension combined with left ventricular hypertrophy, were high levels (systolic blood pressure (SBP) 189.21 ± 19.91 mm Hg, diastolic blood pressure 101.40 ± 16.92 mm Hg). (ii) The Telmisartan-based plus simvastatin therapy was significantly effective in lowering the SBP (128.26 ± 9.33 mm Hg vs. 139.22 ± 16.34 mm Hg). (iii) After the 1-year treatment, the parameters of left ventricular hypertrophy in both groups were improved. Compared to group T, there were no differences in the characteristics of the subjects, including interventricular septum, left ventricular mass, left ventricular mass index, ejection fraction, left atrium inner diameter at baseline. The patients' interventricular septum (Group TS 10.30 ± 1.80 mm vs. Group T 10.99 ± 1.68 mm, P < .05), LVM (Group TS 177.43 ± 65.40 g vs. Group T 181.28 ± 65.09 g, P < .05), and LVMI (Group TS 100.97 ± 37.33 g/m(2) vs. Group T 106.54 ± 27.95 g/m(2), P < .05), all dropped more prominently (P < .05) in group TS; the ejection fraction rose more remarkably in group TS (Group TS: 57.50 ± 16.41% to 65.43 ± 11.60%, P < .01 while showing no change in Group T); the left ventricular hypertrophy reversed more significantly and the left ventricular systolic function improved more. (iv) The left atrium inner diameter of Group TS decreased (P < .01), the ratio of E/A, which indicates the left ventricular diastolic function, continued to drop further, showing no change to the trend of left ventricular diastolic function declination. Patients who have hypertension with left ventricular hypertrophy usually suffer other accompanying diseases at the same time. Telmisartan-based plus Simvastatin treatment can significantly reduce SBP, reverse left ventricular hypertrophy, improve the left ventricular systolic function, but it has no effect on reversing the left ventricular diastolic function. This experiment indicated that Simvastatin can reverse left ventricular hypertrophy and improve left systolic function.

The functional changes of the perivascular adipose tissue in spontaneously hypertensive rats and the effects of atorvastatin therapy.

The aim of this study was to examine the function of perivascular adiposa tissue (PVAT) on vascular relaxation response in spontaneously hypertensive rats (SHR) and the modulatory effects of the atorvastatin therapy on the PVAT functions. We investigated the mechanisms of the perivascular adipocyte-derived relaxation factor (PVRF) by using isolated rat's aortic rings and isometric contraction measurements. We found that contraction of the thoracic aorta induced by phenylephrine was significantly attenuated in the presence of PVAT from normotensive Wistar-Kyoto rats (WKY group) or the spontaneously hypertensive rats treated with atorvastatin (SHR-A group, atorvastatin 50mg/kg/day), whereas this effect was not observed in the thoracic aortic rings from the control SHR (SHR group). Transferring the solution incubated with PVAT-intact thoracic aorta to PVAT-free thoracic aorta, it induced a remarkable relaxation response in the WKY but not in the control SHR. Tetraethylammoniumchloride (TEA) could block the above relaxation. It was also shown that the PVRF function was likely, depending on the extracellular [Ca(2+)]; the anti-contractile effect of PVAT could be reduced by the inhibitor of the adenosine triphosphate (ATP)-dependent potassium channels, glibenclamide, and could be reduced by the inhibitor of cyclooxygenase by indomethacin. We thus infer that the PVAT function was distorted in hypertension rats, and the lipid-lowering treatment with atorvastatin could restore the PVAT function. The function of the PVRF may involve the Ca(2+)-activated potassium channels, the ATP-dependent potassium channels in vascular smooth muscle cell (SMC), and the release of PVRF from PVAT may involve prostaglandins (PGs) and the calcium metabolism. These results provide an insight into the pathological mechanisms of hypertension development, and indicate that the PVAT may be a potential new target for the hypertensive therapy.