|Year : 2019 | Volume
| Issue : 3 | Page : 91-96
Dynamic changes and the relationship between cardiac function damage and glycogen synthase kinase-3 β expression induced by coronary microembolization in rats
Binghui Kong, Zhenbai Qin, Jing Zheng, Manyun Long
Department of Cardiology, The First Affiliated Hospital of Guangxi Medical University, Nanning, China
|Date of Submission||11-Jul-2019|
|Date of Acceptance||11-Sep-2019|
|Date of Web Publication||30-Sep-2019|
Department of Cardiology, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021
Source of Support: None, Conflict of Interest: None
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 < 0.05). The results of ELISA showed that the levels of TNF-α and IL-1β in myocardial tissue of the CME group began to increase at 3h, reached a peak at 9h, and decreased at 24h (P < 0.05). Western blot analysis showed that the protein expression of NF-κB p65 in the CME group began to increase at 3 h, peaked at 9h, and decreased at 24h. The protein expression of phospho-GSK-3β(Ser 9) began to decrease at 3 h, reached a low peak at 9 h, and increased at 24 h (P < 0.05). Conclusion: GSK-3βis involved in the damage of cardiac function induced by CME and shows obvious time-variation, which may be achieved by regulating the expression of TNF-α in myocardium.
Keywords: Cardiac function, coronary microembolism, glycogen synthase kinase-3 β, myocardial inflammation
|How to cite this article:|
Kong B, Qin Z, Zheng J, Long M. Dynamic changes and the relationship between cardiac function damage and glycogen synthase kinase-3 β expression induced by coronary microembolization in rats. Cardiol Plus 2019;4:91-6
|How to cite this URL:|
Kong B, Qin Z, Zheng J, Long M. Dynamic changes and the relationship between cardiac function damage and glycogen synthase kinase-3 β expression induced by coronary microembolization in rats. Cardiol Plus [serial online] 2019 [cited 2019 Dec 10];4:91-6. Available from: http://www.cardiologyplus.org/text.asp?2019/4/3/91/268295
| Introduction|| |
Coronary microembolization (CME) is a severe complication in the treatment of acute coronary syndrome 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.” It is also a strong predictor of a poor long-term prognosis and major adverse cardiac events., The current research indicates that the mechanism of CME-induced cardiac dysfunction is mainly related to local myocardial inflammation, especially to tumor necrosis factor (TNF)-α, but its specific mechanism of regulation is still unknown. Li et al. found that extensive activation of nuclear factor (NF)-κB and the resulting massive release of inflammatory mediators such as TNF-α and interleukin (IL)-1 β play an important role in the development of progressive cardiac insufficiency and advanced heart failure caused by CME. Glycogen synthase kinase-3 β (GSK-3 β) belongs to the highly conserved silk/threonine phosphokinase family and is present in all eukaryotic organisms. It is known to regulate the action of GSK-3 β by GSK-R phosphorylation (Ser or Tyr terminus), intracellular localization, and interaction with cell-binding proteins. In the cardiovascular system, GSK-3 β plays a key role in inflammatory injury, ischemia-reperfusion injury, energy metabolism, and myocardial necrosis., GSK-3 β is a collection point of host inflammatory response, mainly through two pathways: innate immune response and adaptive immune response. Yu et al. found that GSK-3 β was activated in the myocardium of dilated cardiomyopathy (DCM) rats and that oral treatment with curcumin could inhibit the expression of inflammatory factors such as IL-1 β and TNF-α in DCM rats. The anti-inflammatory effect of curcumin may be achieved by increasing the phosphorylation level of GSK-3 β and by decreasing the expression of GSK-3 β. Therefore, GSK-3 β is likely to be involved in the damage of CME-induced cardiac function. This study aims to establish a CME model by introducing a microembolized ball through the left ventricular injection and using Sprague Dawley (SD) rats as experimental patients. Investigating the dynamic changes of GSK-3 β expression after CME and its relationship with cardiac function changes after CME will provide a new understanding of the molecular mechanism of CME-induced cardiac dysfunction.
| Materials and Methods|| |
Model establishment and grouping
A total of 64-specific SD rats with no specific pathogen grade were divided evenly into a CME group (32 rats) and a sham operation group (32 rats). The rats were 7-8 weeks old, weighed 250-300 g. The rats were randomized grouping regardless of gender and were provided by the Experimental Animal Center of Guangxi Medical University. To establish the CME model, SD rats were anesthetized by propofol injection at 10 mg/kg through the tail vein, and anesthesia was maintained by tail vein pumping at 0.8 mg/kg/min. Then, intubation through the mouth was performed, and the following ventilator parameters were used: frequency 70 times/min, tidal volume 20 ml/kg, suction ratio 1:1, and air pressure 1.5 kPa. The preparation ranged from the neck to the upper abdomen, and the lateral direction was to the bilateral midline. Rats were placed on a sterile operating table, connected to an electrocardiogram monitor, restrained, and disinfected. The skin was cut along the left rim of the sternum, and the fascia and muscle layers were separated. Then, the ribs were opened, and the pericardium was removed. Next, 0.1 ml of sodium dodecyl sulfate (SDS) physiological saline suspension containing 45 μm of polystyrene microspheres 4000 U (microspheres suspended in normal saline containing 1.5 g/L SDS) was injected into the left ventricle, and 20 cardiac cycles of the ascending aorta were clamped (approximately 10 s). In the sham operation group, an equal amount of physiological saline replaced the microsphere suspension. At the end of the experiment, the arterial clip was loosened, the chest was closed, and 10,000 U penicillin was injected into the tail vein to prevent infection. Anesthesia was removed, and the rats were placed in a constant temperature blanket at 30°C. After the rats recovered spontaneously, the tracheal tube was withdrawn, and the rats were returned to their cages. The diets, drinking water, activity, and irritation of the rats were observed every hour. Rats in the CME group and the sham operation group were randomly subdivided into 0 h, 3 h, 9 h, and 24 h groups (eight rats in each group) according to the different observation time points.
Detection of cardiac function in rats (Philips Sonos 7500, USA)
The left ventricular ejection fraction (LVEF), left ventricular end-diastolic diameter (LVEDd), left ventricular short-axis shortening, fractional shortening (FS), and cardiac output (CO) were measured at 0h, 3h, 9h, and 24h after the surgery. The probe frequency was 12 MHz. All echocardiographic examinations were performed by a specialist with extensive experience in echocardiography.
Organizational materials and sample processing
After detecting cardiac function, 2–3 mL of 10% potassium chloride was injected into the tail vein of the rat to stop the heart in the ventricular diastolic phase. Immediately afterward, the hearts were excised, and the atrial appendage was removed. Parallel to the atrioventricular groove and divide the ventricle into the apex and the bottom of the heart at the midpoint of the long axis of the left ventricle. The apex was immediately frozen in liquid nitrogen and then transferred to a −80°C freezer until immunoblotting. The bottom of the heart was fixed with 4% paraformaldehyde for 12 h, embedded in paraffin, and serially sectioned (4 μm each) for hematoxylin-eosin (HE) staining and hematoxylin basic fuchsin picric acid (HBFP) staining, which was used to detect myocardial microinfarct size.
Measurement of myocardial microinfarction range
HBFP staining is an important staining method for the diagnosis of early myocardial ischemia. The ischemic myocardium and red blood cells are red stained, the normal myocardial cytoplasm is yellow stained, and the nucleus is blue stained. The DMR+Q550 pathology image analyzer was used to randomly select five fields of view (i100) for each HBFP-stained slice. The infarct area was measured using the Leica Qwin analysis software plane method. The area was expressed as the area percentage of the total analysis slice and was then averaged.
Detections of inflammatory cytokines in serum by enzyme-linked immunosorbent assay
The serum levels of IL-1 β and TNF-α were examined using a specific enzyme-linked immunosorbent assay kit (R and D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.
Western Blot analysis
A total protein obtained from the cardiac tissue and cardiomyocytes was separated by 10%–15% SDS-PAGE and was electrotransferred to a PVDF membrane (Millipore, Atlanta, GA, US). The membrane was blocked with 5% bovine serum albumin or skim milk for 1.5 h at room temperature and then incubated overnight at 4°C with the primary antibodies NF-κB p65 and p-GSK-3 β. The primary antibodies specific for NF-κB p65 and p-GSK-3 β were obtained from Cell Signaling Technology (Beverly, MA, USA). After washing five times with tris-buffered saline containing 0.1% Tween 20 (TBST), the membrane was incubated with horseradish peroxidase-conjugated secondary antibody in TBST for 2 h at room temperature. Signals were detected using an enhanced chemiluminescence detection system (Pierce, Rockford, IL, USA). The protein bands were evaluated and quantified using Bio-Rad's Image Lab software.
Measurement data were expressed as the mean ± standard deviation. A group t-test was used for comparison between groups. A one-way ANOVA was used for comparison between groups. An LSD test was used for comparison between groups. Correlation analysis was performed using linear correlation analysis. Differences were considered statistically significant when P < 0. 05.
| Results|| |
Changes in heart function indicators in rats
Rat heart function was evaluated by echocardiographic detection of LVEF, FS, CO, and LVEDd values. The results showed the following. Compared with the sham operation group, except for at 0 h, the cardiac function of the CME group was significantly decreased at each time point. This result was characterized by myocardial contractile dysfunction and left ventricular dilatation, that is, LVEF, FS, and CO decreased (P < 0.05) and LVEDd increased (P < 0.05). The cardiac function of the CME group decreased progressively at 0 h, 3 h, and 9 h, such that LVEF, FS, and CO decreased and LVEDd increased (both P < 0.05), and there was a recovery trend at 24 h [P < 0.05, [Table 1].
Coronary microembolization pathology observation
HE and HBFP staining results: the sham operation group occasionally saw subendocardial ischemia, and no obvious infarct was found. Multiple microinfarctions were seen in the CME group at 3 h, 9 h, and 24 h. They were primarily wedge-shaped, with focal distribution and were more common in the subendocardial and left ventricles. HE staining showed that the myocardial cell nucleus dissolved or disappeared in the microinfarction. Cytoplasmic red staining showed peripheral myocardial edema, degeneration, peripheral inflammatory cell infiltration and red blood cell exudation, and microembolism in the arteriole [Figure 1]. HBFP staining showed early ischemic myocardium: the red blood cells were red stained, a small amount of red blood cells was exuded in the gap, and inflammatory cells were infiltrated [Figure 2]. The infarct size of CME at 3 h, 9 h, and 24 h was (8.72 ± 3.23%), (9.11 ± 3.42%), and (8.92 ± 2.98%), respectively. There was no significant difference between the groups (P > 0.05).
|Figure 1: Histopathologic examination of myocardial microinfarction. Hematoxylin-eosin staining revealed myocardial infarction in the coronary microembolization E group. Microspheres in the samples of myocardium from the coronary microembolization group are indicated by arrows (×400). Coronary microembolization|
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|Figure 2: Histopathologic examination of coronary microembolization myocardial microinfarction. Hematoxylin basic fuchsin picric acid staining shows the area of myocardial microinfarction in red. The arrow indicates the presence of a microsphere (i200). (a) Hematoxylin basic fuchsin picric acid staining at 0 h in coronary microembolization group (n = 8) (b) Hematoxylin basic fuchsin picric acid staining at 3 h in coronary microembolization group (n = 8) (c) Hematoxylin basic fuchsin picric acid staining at 9 h in coronary microembolization group (n = 8) (d) Hematoxylin basic fuchsin picric acid staining at 24 h in coronary microembolization group (n = 8). Coronary microembolization|
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Changes in the expression of interleukin-1 β and tumor necrosis factor-α in rat coronary microembolization cardiomyocytes
Changes in the expression levels of IL-1 β and TNF-α in each group included the following. Compared with the sham operation group, the expression levels of IL-1 β and TNF-α in the cardiomyocytes of the CME group were significantly increased (P < 0.05). The expression levels of IL-1 β and TNF-α in the CME group began to increase at 3 h, peaked at 9 h, and decreased at 24 h (both P < 0.05) [Figure 3].
|Figure 3: Dynamic changes in tumor necrosis factor-α and interleukin-1 β in the myocardium detected by enzyme-linked immunosorbent assay analysis in the coronary microembolization and control groups. Data are presented as mean ± standard deviation (n = 8). The (*) indicates a P < 0.05 compared to the control group|
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Changes of nuclear factor-κB p65 and p-glycogen synthase kinase-3 β protein expression in rat coronary microembolization cardiomyocytes
Western Blot quantitative analysis showed the following. Compared with the sham operation group, and except for the 0 h group, the expression of NF-κB p65 relative protein in the cardiomyocytes of the CME group increased significantly at the corresponding time points. The relative protein expression of p-GSK-3 β was decreased (both P < 0.05). The protein expression of NF-κB p65 in the CME group began to increase at 3 h, peaked at 9 h, and decreased at 24 h. The protein expression of p-GSK-3 β began to decrease at 3 h, reached a low peak at 9 h, and increased at 24 h (both P < 0.05) [Figure 4].
|Figure 4: Dynamic changes of nuclear factor-κB p65 and p-glycogen synthase kinase-3 β protein expression in the myocardium of the coronary microembolization and control groups as detected by western blotting. Nuclear factor-κB p65 and p-glycogen synthase kinase-3 β normalized against GAPDH. Data are presented as mean ± standard deviation (n = 8). The (*) indicates a P < 0.05 compared to the control group|
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Correlation analysis between p-glycogen synthase kinase-3 β and tumor necrosis factor-α expression and cardiac function left ventricular ejection fraction in myocardial cells after coronary microembolization in rats
The relative expression of p-GSK-3 β in cardiomyocytes was significantly positively correlated with LVEF (r = 0.796, P ≤ 0.001) [Figure 5]a. TNF-α expression was negatively correlated with LVEF (r = −0.638, P ≤ 0.001) [Figure 5]b. The relative expression of p-GSK-3 β was negatively correlated with the TNF-α level (r = −0.629, P ≤ 0.001) [Figure 5]c.
|Figure 5: Correlation between expression of p-glycogen synthase kinase-3 β and tumour necrosis factor-α and left ventricular ejection fraction of cardiac function in rat cardiomyocytes after coronary microembolization. (a) The correlation between p-glycogen synthase kinase-3 β levels and left ventricular ejection fraction (%) (n = 8); (b) The correlation between tumor necrosis factor-α levels and left ventricular ejection fraction (%) (n = 8); (c) The correlation between p-GSK-3 β levels and tumor necrosis factor-α (n = 8)|
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| Discussion|| |
CME is a severe complication in the treatment of PCI. Once CME occurs, it can significantly affect the prognoses of patients. There is currently no effective prevention or treatment method. Studies have shown that microinfarction occurs in the local myocardium in the acute phase after CME, and the cardiac function progressively decreases. Skyschally et al. found that by injecting a microembolization into the coronary artery of the dog, the transient reduction of coronary blood flow immediately returned to normal, but the contractile function of the local myocardium progressively decreased. The explanation may be that the mechanism of CME-induced cardiac dysfunction is not a reduction in coronary blood flow. Recent studies have found that TNF-α plays a key role in CME-induced cardiac dysfunction. Li et al. found that the mRNA expression levels of inflammatory factors such as TNF-α, IL-1 β, and IL-10 were dynamically changed after CME and were closely related to the left ventricular function changes. Dörge et al. intervened with TNF-α and its antibodies before intracoronary injection of microembolism in dogs. The results showed that TNF-α can aggravate myocardial contractile dysfunction caused by microemboli and that anti-TNF-α antibody can enhance the tolerance of cardiomyocytes to microvascular embolization, and thereby reducing myocardial contractile dysfunction. Therefore, elevated levels of TNF-α are central to cardiac function damage after CME, but the exact upstream regulatory mechanisms are still unclear.
In this study, we also found that the expression level of TNF-α protein increased significantly after CME and showed a dynamic change, that is, the expression level began to rise at 3 h, reached a peak at 9 h, and showed a downward trend at 24 h. This change was significantly negatively correlated with changes in cardiac function, indicating that TNF-α plays a key role in CME-induced cardiac dysfunction. In addition, the study found no significant difference in microinfarct size between CME groups at each time point and that the size of the microinfarct size was not related to the time of infarction. Therefore, a progressive decline in cardiac function due to a difference in the size of the microinfarct size can be excluded.
GSK-3 β is a collection point of the host inflammatory response and plays an important role in the development of inflammation. Gao et al. showed that GSK-3 β inhibitor Thiadiazolidinones-8 can reduce neutrophil infiltration in acute myocardial ischemia-reperfusion injury. It reduces NF-κB levels and TNF-α and IL-1 β gene expression, inhibits the inflammatory response, reduces myocardial infarct size, and protects the myocardium in rats with acute myocardial infarction. Song et al. found that polyadenosine diphosphate-ribose polymerase inhibitors can reduce the expression of NF-κB, intercellular adhesion molecule, cyclooxygenase-2, and matrix metalloproteinases in rat hearts. It can also reduce myocardial cell apoptosis while improving cardiac function. This protection is achieved by activating GSK-3 β phosphorylation. GSK-3 β is involved in the regulation of TNF-α in myocardial inflammation, and elevated levels of TNF-α are a major factor in CME-induced cardiac dysfunction. Therefore, we speculate that GSK-3 β is likely to be involved in the regulation of TNF-α levels after CME which, in turn, leads to impairment of cardiac function.
In this study, we found that the level of p-GSK-3 β protein decreased significantly after CME compared with the sham operation group and that the level showed dynamic changes, which began to decrease at 3 h, reached a low peak at 9 h, and increased at 24 h. There was a significant negative correlation with the expression of TNF-α in the myocardium and a significant positive correlation with the changes of cardiac function. Therefore, these findings confirm that GSK-3 β is involved in the damage of cardiac function after CME, and it is likely to be achieved by regulating the expression of myocardial TNF-α.
The cardioprotective mechanisms involved in GSK-3 β are not isolated from each other. Studies have shown that growth arrest and DNA damage-induced protein 153 (GADD153) can simultaneously regulate apoptosis and inflammation. GSK-3 β also provides a mechanical link for cells to express GADD153 and the inflammatory response. Stress loading can aggravate cardiomyocyte death in ischemia-reperfusion injured rats through GSK-3 β-dependent mechanisms, including inflammatory responses (e.g., decreased IL-10 expression), DNA damage, and GADD153 expression pathways. Increasing attention has been paid to the important role of GSK-3 β in myocardial protection and more research on coronary artery diseases, and heart failure has begun to use it as a therapeutic target. GSK-3 β inhibitors have also been used in cell and animal experiments. In-depth study of the changes in GSK-3 β under differing physiological and pathological conditions and the effects of drugs on GSK-3 β provide a new method for the treatment of heart disease.
One shortcoming of this research includes the microembolic agent used in CME modeling is a plastic microsphere, which is different from the biologically active embolic material such as platelets and red blood cells formed by an actual clinical atherosclerotic plaque rupture. There may be some differences between the actual pathophysiological changes of CME caused by plaque rupture. In addition, in this study, interventions such as related inhibitors of GSK-3 β were not used; therefore, the mechanisms and pathways require further study.
| Conclusions|| |
In summary, GSK-3 β is involved in the damage to cardiac function induced by CME and shows obvious time variation, which may be achieved by regulating the expression of myocardial TNF-α. The results of this study indicate that increasing the GSK-3 β phosphorylation level within 9 h after CME may be an important means to reduce CME-induced cardiac dysfunction, and it is expected to become a new drug development target for CME-induced cardiac dysfunction prevention.
Financial support and sponsorship
This study was financially supported by CMVD Fund of XinXin Heart (SIP) Foundation – China Cardiovascular Association (NO. 2018-CCA-CMVD-09).
Conflicts of interest
There are no conflicts of interest.
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