|Year : 2017 | Volume
| Issue : 4 | Page : 11-17
Effects of Shensongyangxin capsule on myocardial connexin 40 expression in diabetic rat model
Da-Bin Pan, Ji-Min Chen, Shi-You Gao, Lin-Tao Zha, Xiang-Rong Xie, Heng Cao
Department of Cardiology, Cardiovascular Disease Research Institute, Affiliated Yijishan Hospital of Wangnan Medical College, Wuhu 241001, Anhui, China
|Date of Web Publication||12-Mar-2018|
Dr. Da-Bin Pan
Department of Cardiology, Cardiovascular Disease Research Institute, First Affiliated Yijishan Hospital of Wannan Medical College, Wuhu 241001, Anhui
Source of Support: None, Conflict of Interest: None
Objective: To investigate the effects of Shensongyangxin (SSYX) capsule on myocardial expression of connexin 40 (Cx40) in diabetic rat model. Materials and Methods: Thirty-one male Sprague-Dawley rats (SD, 8 weeks old, weighing 220 g) were randomly divided into three groups: normal control group (NC, n = 10), diabetic group (diabetes mellitus [DM], n = 9), and diabetes plus SSYX group [SDM], n = 12). SD rats were injected intraperitoneally with streptozotocin (SZT, 65 mg/kg) to make the diabetic rat model. SDM group was given daily SSYX 1 g/kg by gavage and NC group and DM group were given a daily amount of distilled water by gavage. The expression levels of Cx40 in atrial and ventricular muscles were analyzed by Western blot techniques. Hematoxylin and eosin staining technique was used to observe the changes of myocardial structure; the distribution of myocardial Cx40 was analyzed by immunofluorescence technique. Results: Cx 40 levels in the atrial and ventricular muscle were lower in DM group than in the NC group (P < 0.05). The cardiac muscle cells were arranged in order in the NC group. Extensive necrosis and apoptosis of myocardial cells were noted accompanied by the disorder of permutation, adipose tissue, and fibrous tissue in DM group. In the SDM group, the cardiac muscle cells were arranged orderly, but fibrous tissue and vasculitis were observed. Immunostaining showed that the expression of Cx40 in the myocardium was distributed at the end of the long axis of myocardial cells with a ladder-like distribution in the NC group. The expression of Cx40 in the myocardium was mostly distributed on the side of the long axis of myocardial cells in the DM group. The expression level of Cx40 in atrial muscle was increased, and the distribution in the SDM group was primarily lateral. Conclusions: The expression of Cx40 in the myocardium was decreased and mainly distributed on the sides in DM group. SSYX capsule upregulated the expression of Cx40 in the atrium in diabetic rats. No significant effect was noted on the expression and distribution of Cx40 in the ventricular muscle.
Keywords: Connexin 40, diabetes, gap junction, rat, Shensongyangxin capsule
|How to cite this article:|
Pan DB, Chen JM, Gao SY, Zha LT, Xie XR, Cao H. Effects of Shensongyangxin capsule on myocardial connexin 40 expression in diabetic rat model. Cardiol Plus 2017;2:11-7
|How to cite this URL:|
Pan DB, Chen JM, Gao SY, Zha LT, Xie XR, Cao H. Effects of Shensongyangxin capsule on myocardial connexin 40 expression in diabetic rat model. Cardiol Plus [serial online] 2017 [cited 2018 Dec 12];2:11-7. Available from: http://www.cardiologyplus.org/text.asp?2017/2/4/11/227169
| Introduction|| |
Diabetic heart disease is one of the major causes of death in diabetic patients, with 70%–80% of diabetic patient deaths due to cardiovascular complications. Generalized diabetic heart disease includes coronary atherosclerotic heart disease, diabetic cardiomyopathy, and diabetic autonomic neuropathy., Hypertrophic cardiomyopathy, myocardial fibrosis, and intramyocardial arteriole lesions are characteristics of diabetic cardiomyopathy and are manifested as diastolic dysfunction and eventual systolic dysfunction. Animal experiments have shown that diabetes can lead to cardiac electrical remodeling and arrhythmias, and malignant arrhythmia is one of the major causes of death in patients with diabetic cardiomyopathy. At present, the pathogenesis of diabetic arrhythmias has not yet been fully elucidated and is lacking effective treatment. Under normal circumstances, the cardiac myocytes are short columnar, intercalated discs is connection between the cardiac myocytes. Gap junction (GJ) is a special way of connecting connexins (Cxs) on the membrane of cardiomyocytes. GJ acts as a bridge between cells and is mainly distributed between intercalated discs of cardiac muscle.
Na+, Ca2+, and other metal ions and small molecules can be exchanged between cardiomyocytes through GJ, which is the channel of electrical and chemical coupling between myocardial cells. This is beneficial to the rapid conduction of the action potential of cardiomyocytes and makes the myocardial contraction synchronously. Human myocardium mainly expresses three kinds of Cxs –Cx40, Cx43, and Cx45. Cx40 – and is mainly expressed in atrial myocardium, and Cx43 is mainly distributed in ventricular muscle, whereas Cx45 is mainly distributed in sinoatrial node and atrioventricular node. GJs composed of Cx43 or Cx40 have rapid electrical conductivity. However, the conductivity of the Cx45 component is much slower. Uncoupling between cardiomyocytes is closely related to the occurrence and development of arrhythmias. The decrease of Cxs expression and phosphorylation level in myocardium reduces the longitudinal conduction between myocardia, and the inhomogeneous distribution and lateral distribution of Cxs leads to an increase of the hemichannel. Increasing adenosine triphosphate (ATP) efflux and Na+ influx may cause myocardial cell edema and depolarization delay. Conversely, transverse velocity increase may form a reentrant loop, inducing rapid arrhythmia or slow arrhythmia due to conduction block. Reduction of Cx40 level may increase atrial electrical vulnerability, which is one of the risk factors of spontaneous atrial fibrillation. Gene knockout Cx40 homozygous mice may exhibit conduction block, resulting in neonatal death. Abnormal expression and distribution of Cx40 could lead to conduction abnormalities mainly manifesting as atrioventricular block and right bundle branch block, which are closely related to sudden cardiac death. Under physiological conditions, Cx40 is mainly distributed in intercalated discs at the ends of the myocardia with less on the cells' side surface. This leads to the electrical conductivity of the long axis of the atrial muscle, which is much greater than that of the transverse axis and beneficial to the electrical and signaling conduction between the atrial myocytes. It is beneficial to the synchronism of myocardial contraction and diastole. In pathological condition, the lateral distribution of Cx40 distribution increases leads to the increase of transverse conductivity, and decreased anisotropy of atrial muscle, which results in increased heterogeneous and atrial reentrant wavelet. These are the anatomical basis for the formation of atrial tachycardia, atrial fibrillation, and other atrial arrhythmias.
Shensongyangxin (SSYX) capsule is a compound composed of 12 kinds of Chinese herbal medicines such as ginseng, ophiopogon root, cornus officinalis, radix salviae miltiorrhizae, semen ziziphi spinosae, loranthaceae, red peony root, wood louse insects, nard, coptis chinensis, fructus schisandrae, keel. Many studies have shown that SSYX capsule has different blocking effects on ion channels of ICa,L, INa, IK1, and Ito and prolongs the action potential duration as well as reduces the myocardial excitability. The ion channel blocking effect can also reduce the risk of drug-induced arrhythmias. SSYX can also improve myocardial energy metabolism, reverse/inhibit myocardial remodeling, and myocardial fibrosis. In addition, SSYX also has the effect of inhibiting myocarditis and antioxidative stress. Based on this, the present study was to evaluate the effects of SSYX capsule on the expression and distribution of Cx40 in the myocytes in a diabetic rat model.
| Materials and Methods|| |
Adult male Sprague-Dawley (SD) rats of 8 weeks old, weighing 220 ± 28 g, were purchased from the Qinglong Mountain Animal Breeding Ground of the Nanjing Jiangning District, Nanjing, China. This study was approved by the hospital medical ethics committee.
Streptozotocin (STZ) from Sigma; SSYY capsule from Yiling Pharmaceutical; BCA and Western protein concentration assay kit, anti-mouse alpha-tubulin antibody, hematoxylin and eosin (HE) staining kit, immunofluorescence staining kit (rabbit), from Beyotime Biotechnology Co.; anti-Rabbit Cx40 antibody from EMD Millipore.
Preparation of diabetic rat model and grouping
Thirteen-one adult male SD rats (8 weeks old, weighing 220 ± 28 g) were randomly divided into three groups: normal control group (NC, n = 10), diabetic mellitus group (DM, n = 9), and diabetic rats fed with SSYX capsule group (SDM, n = 12). SD rats were injected intraperitoneally with SZT 65 mg/kg to make the diabetic rat model. SDM group was given daily SSYX capsule 1 g/kg by gavage. NC group and DM group were given a daily amount of distilled water by gavage. Tail vein blood glucose was measured after 72 h, and the rats were enrolled in the experiment if the random blood glucose level was >16.7 mmol/L. On the 2nd day, daily medication intervention was conducted and weighed weekly. The diabetic rats were fed with SSYY capsule 1 g/kg/day by gavage for 8 weeks in the SDM group, whereas the NC and DM group rats were fed distilled water 3 ml daily for 8 weeks.
Electrocardiogram recording and analysis
After 8 weeks, all rats were anesthetized with intraperitoneal injection of 25% urethane (6 ml/kg), and rats were fixed with four limbs in supine position. The acupuncture needles were inserted into the muscle of limbs, and the RM624OB/C biological signal acquisition system was connected to the computer terminal to record the electrocardiogram (ECG). The heart rate (HR), P-wave width, and QT interval were recorded by RM624OB/C biological signal acquisition system (Guizhou Xin Boya instrument & Instrument Co., Ltd). QTc was calculated.
Histological examination of myocardium
Atrial histological examination: harvested atrial tissue was fixed in 10% buffered formalin for 24 h and embedded in paraffin. By cutting continuously along the axis of the atrium, four slices in 5 μm thick tissues were collected every 1 mm atrium.
Hematoxylin and eosin staining (briefly)
Slices were xylene dewaxed, hematoxylin stained for 10 min, rinsed in running tap water 30 s, ethanol differentiation, light ammonia anti-blue, eosin dyeing solution for 1 min, dehydrated, cleared, and mounted.
Sections were incubated in 3% H2O2 at room temperature for 10 min to eliminate the activity of endogenous peroxidase; sections were rinsed in distilled water and soaked in phosphate-buffered saline (PBS)-Tween 20 for 5 min. Antigen repair was applied (0.4% pepsin soaked for 30 min at 37°C), rinsed in PBS-Tween 20 for 5 min, and incubated in bovine serum diluted with PBS-Tween 20 at room temperature for 20 min). Appropriate dilution of anti-rabbit-Cx40 antibody was added and then the sections were incubated overnight at 4°C. After rewarming, sections were rinsed in PBS-Tween 20 for 5 min, 3 times and then incubated with biotinylated secondary antibody (anti-rabbit) for 2 h at room temperature. Sections were rinsed in PBS-Tween 20 for 10 min × 3 then mounted in glycerol. Sections were examined under a fluorescence microscope and photographed. The expressions of Cx40 in ventricular and atrial muscles were analyzed using Western blot techniques. Tissue total protein extraction – A small amount of myocardial tissue was placed in the spherical part of the 1–2 ml homogenizer, and the tissue blocks were cut with scissors. The 400 ml lysate (PMSF) was homogenized in the homogenizer and placed on ice. The tissue block was ground every 3–5 min until milled. The lysate was transferred to 1.5 ml centrifuge tube and centrifuged at 12,000 rpm for 5 min. The supernatant was separated into 0.5 ml centrifuge tube and stored in a refrigerator below −20°C.
Determination of protein content
The standard curve of protein content was determined by the colorimetric analysis of biological spectrophotometer, and the protein content was determined.
The Statistical Package for Social Science, version 13.0 software (SPSS, Chicago, IL, USA)was used for data analysis. The measurement data were expressed by mean standard deviation (x– ± S); single-factor analysis of variance was used to compare the mean of multiple groups; SNK-q test was used for comparison between two groups. The difference of P < 0.05 was considered statistically significant.
| Results|| |
Compared with the NC group, diabetic rats had more symptoms (polyuria, polydipsia, and emaciation), irritability, and sparse hair. In the experimental 8 weeks, there were three rats with lousy tail, two rats with cataract, and one with red eye disease in DM group. There was only one rat with lousy tail in the SDM group.
Blood glucose and body weight
Compared with the NC group, the blood glucose levels were higher and body weight was decreased in the DM group and SDM group (P< 0.05). There was no significant difference in blood glucose between the DM and SDM groups (P > 0.05) [Table 1].
The basic characteristics of electrocardiogram and changes
The HR was decreased significantly in the DM group, while the HR was increased in the SDM group. The P-wave in the DM group had more width with wider QTc dispersion than in the NC group [Table 2].
The connexin 40 expression in ventricle and atrial muscle in normal control group
The expression of Cx40 in the ventricular myocardium was significantly lower than the atrial myocardium in the NC group (P< 0.05) [Figure 1].
|Figure 1: The expression of connexin 40 in the ventricular myocardium was lower than that in atrial myocardium in the normal control group (P < 0.05)|
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Hematoxylin and eosin staining of myocardia
- Atrial HE staining (Cx40 expression in ventricular myocardium): There was a large amount of fat infiltration in the myocardium, a vast area without nuclear staining, ventricular myocardial cell arrangement disorder, and vasculitis in DM group. The arrangement of myocardial cells was slightly irregular and there were a little nuclear pale staining area and mild vasculitis [Figure 2]
- Ventricular myocardium HE staining: The ventricular myocardial cell arrangement disorder, fatty infiltration, and vasculitis were observed in DM group. The myocardial cells were slightly irregular and mild vasculitis was observed in the SDM group [Figure 3].
|Figure 2: Atrial myocardial cell H and E (×400). (a) Normal control group, myocardial cells arrange regular and tight. (b) Diabetes mellitus group, myocardial disorder, fatty infiltration, vasculitis and without nuclear staining area. (c) Steroid-induced diabetes mellitus group, myocardial cells slightly irregular, a little nuclear pale staining and mild vasculitis|
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|Figure 3: Ventricular myocardium H and E (×400). (a) Normal control group, ventricular myocardial cells arrange regular and tight. (b) Diabetes mellitus group, ventricular myocardial cell arrange disorder, fatty infiltration, and vasculitis. (c) Steroid-induced diabetes mellitus group, myocardial cells slightly irregular and mild vasculitis|
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Connexin 40 expression in myocardia and comparison
- Expression and comparison of Cx40 protein in atrial myocytes by Western blot: The Cx40 expression decreased and a lateral distribution was observed in DM group. The Cx40 expression increased mainly by lateral distribution in SDM group than in the DM group [Figure 4]
- Expression of Cx40 in atrial myocardium by immunofluorescence staining:
Cx40 was mainly distributed in both ends of cardiac myocytes in the NC group. The expression of Cx40 decreased and the distribution showed a lateral distribution in DM group (P< 0.01). The expression of Cx40 was increased compared to DM group while the distribution in the SDM group was mainly by side compared with DM group, P < 0.05 [Figure 5]
- Expression of Cx40 in ventricular myocardia by immunofluorescence staining and Western blot: The Cx40 expression decreased and a lateral distribution was noted in both the DM and SDM groups (P< 0.01) [Figure 6] and [Figure 7].
|Figure 4: Expression of connexin 40 in atrial myocardium by immunofluorescence staining. (a) Normal control group: Connexin 40 mainly distributed in both ends of cardiac myocytes. (b) The connexin 40 expression decreased and a lateral distribution. (c) The connexin 40 expression increased than in diabetes mellitus group, mainly by side|
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|Figure 6: Expression of connexin 40 in ventricle by Immunofluorescence staining. Normal control group: connexin 40 mainly distributed in both ends of cardiac myocytes. Diabetes mellitus group: connexin 40 decreased and lateral distribution. The connexin 40 increased than in diabetes mellitus group, mainly by side|
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| Discussion|| |
DM is a major risk factor for cardiovascular disease events. Studies indicated that heart conduction abnormality increased in patients with DM, including bundle branch block, high-degree atrioventricular block, atrial fibrillation, as well as malignant arrhythmia and sudden death. In the present study, we observed a slower HR in the STZ-induced diabetic rat model as early as 7 days after intraperitoneal injection of STZ, which was consistent with the results reported by Joshi et al. Khandoker et al. recently confirmed that HR variability decreased in the diabetic population., Cx40 is widely distributed in myocardial tissues and the conduction system. In our experiments, we observed that the expression of Cx40 in myocardia in diabetic rats decreased significantly; the ECG showed that the P-wave width increased; the QRS duration shortened with the QT dispersion increased; and no significant difference was seen between QTc.
In normal rats, the myocardium has short columnar shape, and the intercalated disks are formed by GJs between adjacent myocardia. The intercalated disks of the heart are low resistance, which is beneficial to the conduction of electrical signals between the cardiomyocytes and makes the myocardial cells orderly and coordinated contracting and relaxing. GJ proteins (Cxs) play important roles among cells coupling in intercalated discs. It is reported that there are 21 kinds of Cxs in humans. Cx40, Cx43, and Cx45 are mainly expressed in the myocardia, and Cx40 is first observed in the atrium, and later the expression was also found in the ventricular myocardia. Cx43 is mainly distributed in the ventricular myocardia, and Cx45 is more common in the sinoatrial node and the cardiac conduction system. The homologous linkers made up of Cx40 and Cx43 have a fast electrical conductivity, while the conductivity in connectors formed by Cx45 is much slower. Cx40, Cx43, and Cx45 may be coexpressed, forming a heterologous linker and showing a complex synergistic effect. Cxs are mainly located in intercalated discs at both ends of cardiac myocytes. The lateral distribution and inhomogeneity of Cxs lead to the decrease of conduction velocity and the formation of reentrant loops. The main reason is that the Cxs formed by the lateral distribution exists in the half-channel form. These half channels are closed under physiological conditions. Under some pathological conditions, the lateral distribution of Cxs increased and the inhomogeneity of Cxs distribution led to the increase of the half channels and the decrease of myocardial longitudinal conductivity and the increase of the transverse conductivity. This leads to the increase of ATP outflow and Na+ influx, resulting in myocardial edema and depolarization delay. Decreased expression of Cx40 in the myocardia is a potential factor leading to cardiac conduction disturbances and fatal arrhythmias.
Several studies suggested that type 2 diabetes was associated with abnormal electrical conduction and sudden cardiac death, but the exact pathogenic mechanism remains unknown. Axelsen et al. found that in a diet-induced prediabetic rat model, high-fructose-fat diet caused electrophysiological changes, which leads to QRS prolongation, decreased conduction velocity, and increased arrhythmogenesis during reperfusion. In addition, the immunofluorescence demonstrated an increased fraction of Cx43 localized at the intercalated disks. In our STZ-induced diabetic rat model, the Cx40 in the atrial and ventricular muscles showed a downward trend after 8 weeks [Figure 5] and [Figure 7] with a lateral distribution [Figure 4] and [Figure 6]. In 2015, Joshi et al. observed evidence of ventricular conduction significant abnormalities (QRS complex, Q-T interval) as early as 7 days after STZ, which persisted throughout the study in STZ induced a diabetic rat model. Their data suggested that changes in Cx43 content and distribution occurred during experimental diabetes and are likely to contribute to alterations in cardiac function. In the present study, we found that SSYX capsule can upregulate the expression of Cx40 in atrial myocardium, but not in ventricular muscle. This may suggest that SSYX capsule mainly acts on the GJ remodeling of atrial muscle, while the effect on ventricular Cx40 is limited. Immunofluorescence showed that the expression of Cx40 was upregulated by SSYX capsule, but distribution remained on the side in ventricle [Figure 6].
SSYX is commonly used in clinical practice to improve the sinus node function. Studies have shown that SSYX capsule has different blocking effects on ICa, IL, INa, IK1, and Ito channels, prolonging the action potential duration and reducing the myocardial excitability, which can reduce the risk of drug-induced arrhythmias. Studies also indicated that SSYX capsule can improve myocardial energy metabolism and reverse/inhibit myocardial remodeling and myocardial fibrosis as well as inhibit myocarditis and antioxidative stress. Zhang reported that SSYX capsule inhibited the inflammatory response and oxidative stress reaction and reduced the atrial arrhythmias in diabetic rats.
| Conclusion|| |
Diabetic rats may suffer from various types of arrhythmias and decreased HR. SSYX capsule can increase HR, but it has limited influence on P-wave, QRS, and QTc. Cx40 expression of myocardia in diabetic rats was decreased and its distribution was by side. SSYX capsule increased the Cx40 expression in atrial myocytes in diabetic rats though it is still lower than the NC group rats. SSYX had no significant effects on the expression and distribution of Cx40 in ventricles of diabetic rats.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2]