| Myocardial infarction (MI) is a common clinical critical disease. Epidemiolo-gical data from recent years indicates that MI is a serious threat to human health as the morbidity and mortality of MI has been rising sharply, and there has been a trend of increasingly younger people suffering MI year by year. MI can cause a variety of arrhythmia, and malignant ventricular arrhythmia is the main cause of death after MI. Foreign epidemiological survey showed that 75% of sudden cardiac death was owing to cardiac arrhythmia caused by MI. So correct assessment and treatment of ventricular arrhythmia after MI have important significance to improve the patients survival rate and quality of life.After MI, ischemic myocardial necrosis, hypertrophy and fibrosis changes become severe with time prolonged, which can lead to ventricular expansion, tension increase, ventricular wall motion abnormalities. Research shows that the pathological remodeling after MI sustained long duration (average one year). So how to effectively intervene ventricular remodeling and reducing myocardial fibrosis after MI will contribute to improve cardiac function and reduce the occurrence of arrhythmia.The myocardial microvasculature is the primary location of material exchange, energy metabolism and information regulation in myocardial tissue. Microvascular dysfunction secondary to MI plays an important role in cardiac remodeling. Clinical evidence also suggests that the microcirculation might change at the early stage in patients who are at a high risk of coronary heart disease. This pathologic change is responsible for various types of complications, such as arrhythmia, which increases risk of morbidity and mortality during the healing stage after MI. At present, With the improvement of percutaneous coronary interention (PCI) and coronary artery bypass grafting (CABG), a high recanalization rate and survival rate of patients with acute myocardial infarction (AMI) was presented. However, no reflow phenomenon after PCI or CABG often occurs, studies have demonstrated that the microcirculation dysfunction is the main reason. Therefore, some clinical studies also put the microcirculation function as a prediction of long-term high-quality for patients with MI. Thus, therapy to improve microvascular function following MI is reasonable and required for mproving cardiac function and reversing/preventing myocardial remodeling.Thus far, anti-arrhythmic drug treatment has received primary focus because ventricular arrhythmia following MI is the primary concern in regards to sudden cardiac death. Conservative anti-arrhythmia medication therapies mainly include sodium channel blockers, beta blockers, potassium channel blockers, calcium channel blockers and digitalis. However, these pharmacological interventions proven to be so ineffective, In fact, several initially promising antiarrhythmic agents were found to increase rather than decrease mortality in patients recovering from MI. Beta-blockers was limited its application because of the negative heart rate and negative inotropic effect. Clearly, targeting single ion channels (using either isolated ion channels or single myocyte preparations) has proven to be less than effective. We also speculate that an ideal drug for arrhythmia after MI should have multiple anti-arrhythmic efficacy and long-term safety.The traditional Chinese medicine ShensongYangxin (SSYX) consists of Panax ginseng, Ophiopogon japonicus, Fructus Corni, Sal via miltiorrhiza Radix et Rhizoma, parched semen of Ziziphi Spinosa, HerbaTaxilliChinensis, Paeonia lactiflora Pall, Eupolyphaga sinensis Walker, Nardostachys chinensis Batal, Coptis chinensis Franch, SchisandrasphenantheraRehd.EtWits and Polypodiodes chinensis. SSYX was prepared based on the theory of Chinese medicine of "homeostasis, compensatory autoadaptation, regulation and equilibrium" and the experience of "reinforcing and unblocking" to make sure the synergistic effects of different constituents. The SSYX capsule was approved by the State Food and Drug Administration (SFDA) of China in 2003 (File No. Z20030058) and has been widely used in the treatment of ventricular premature complexes (VPCs) and atrial premature complexes (APCs) in China. Clinical studies have shown that SSYX capsules can effectively treat premature ventricular contraction and alleviate premature ventricular contraction-related symptoms when compared with the western medicine groups. Previous study with animal models showed that SSYXsignificantly inhibited arrhythmias induced by ischemia-reperfusion injury or chemical compounds. Whole cell clamping experiments had revealed that SSYX was a multiple ion channel blocker. Recently, some studies demonstrated that there are at least 10 major active constituents in rat urine collected during different periods after oral administration of SSYX by using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) with multiple reaction monitoring (MRM) scanning mode. So we speculate that SSYX have multiple intervention efficacies in preventing arrhythmia after MI. However, until now, there is little study about the specific regulation and mechanism of SSYX. Based on the above, we carried out the experimental study.The left circumflex artery was ligated to produce an ischemic area and subsequent infarction in adult Japanese rabbits. The monophasic action potential technique in vivo was performed to study cardiac electrophysiological changes and microelectrode array (MEA) technology was used in vivo for extracellular electrophysiological recordings of the infarct border zone, to examine the electrophysiological effects of SSYX on 2-week infarcted rabbit hearts and the underlying mechanism.2-dimensional echocardiography (2DE) was performed in vivo for cardiac function measurement. Masson’s trichrome staining was used to observe myocardial fibrosis. Low-energy real-time myocardial contrast echocardiography (RT-MCE) was carried out to assess the microcirculatory perfusion of the ischemic region. Immunofluorescence double staining was used to measure the capillary density. Myocardium and microvascular endothelial ultrastructure were observed with a transmission electron microscope. Quantitative real-time polymerase chain reaction (Real-time PCR) and Western blotting were performed to evaluate the mRNA and protein expression levels of related proteins to found the underlying mechanism of SSYX.MethodsThe study includes three sections:1 the electrophysiological effect and mechanism of SSYX on the hearts of 2-week myocardial-infarcted rabbitsThe left circumflex artery was ligated to produce an ischemic area and subsequent infarction in adult Japanese rabbits. The experiments were randomly assigned to four groups:sham operation (Sham) group, myocardial infarction (MI) group, MI with amiodarone administration (MI+AD) group and MI with SSYX administration (MI+SSYX) group. Animals in MI+AD group were medicated with amiodarone 0.05g/kg/d dissolved in saline by oral administration for two weeks, and animals in MI+SSYX group with SSYX powder 0.4g/kg/d dissolved in saline. Those animals in the sham and MI groups were treated with the same dose of saline. After two weeks administration and fasted 12h, MEA technology was utilized in vivo for extracellular electrophysiological recording and excitation transmission observation, The field potential (FP) indexes include the first negative peak of FP (FPmin), the duration of the FP(FPdur), the total activation time (TAT), the dispersion of TAT, and the conduction velocity (CV) of the excitation transmission within the MEA registration area. The monophasic action potential duration (MAPD) of the 3 layers(Endo, Mid and Epi) of myocytes and the effective refractory period (ERP) were recorded in the border zone by using a constant voltage stimulator (LEAD2000B, Jinjiang Ltd). The transmural dispersion of repolarization (TDR) was calculated and the ventricular fibrillation threshold (VFT) was also tested. Quantitative real-time polymerase chain reaction (Real-time PCR) and Western blotting were performed to evaluate the mRNA and protein expression levels of CX43 and KV6.2.2 the intervention of SSYX on the hearts with pathological ventricular remodeling of 2-week myocardial-infarcted rabbitsIt was the same with part 1 in experimental animals, MI modeling method, grouping, and administration manner.2-dimensional echocardiography (2-DE) was performed in vivo for cardiac function measurement, includingleft ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), left ventricular fractional shortening (LVFS), left ventricular ejection fraction (LVEF) and E/A value. The HE staining was applied to observe myocardial morphology changes and transmission electron microscope was used to observe the ultra-structural changes in myocardium. Masson’s trichrome staining was used to observe myocardial fibrosis. Real-time PCR was performed to evaluate sarcoplasmic reticulum Ca2+-ATPase2a(SERCA2a), ryanodine receptor 2 (RYR2), phospholamban (PLB), Na+/Ca2+exchanger 1 (NCX1), transforming growth factor-β1 (TGF-β1), matrix metalloprotein 2 (MMP2) and matrix metalloprotein 9 (MMP9) mRNA expression levels. Western blotting was performed to evaluate the protein expression levels of SERCA2a, RYR2, PLB, NCX1, collagen Ⅰ (COL Ⅰ) and collagen Ⅲ (COL Ⅲ).3 The intervention of SSYX on the hearts with myocardial microangiopathy of 2-week myocardial-infarcted rabbitsIt was the same with part 1 in experimental animals, MI modeling method, grouping, and administration manner. Low-energy real-time myocardial contrast echocardiography (RT-MCE) was carried out respectively before and after surgery, and 2 weeks after operation to assess the microcirculatory perfusion of the ischemic region. After a brief burst (flash) at a high mechanical index during steady-state enhancement, the A, β and A×β values were extracted to assess the microcirculation perfusion in each group. the plasma levels of ET-1, TXA2, NO and vWF were examined with enzyme-linked immunosorbent assays (ELISAs). The endothelial ultrastructure was observed with a transmission electron microscope. Immunofluorescence double staining was used to measure the capillary density. The mRNA expression levels of vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), prostaglandin 12 (PGI2) and endothelial nitric oxide synthase (eNOS) were measured by Real-time PCR. Western blotting was performed to evaluate the protein expression levels of thrombomodulin (TM).Results1 the electrophysiological effect and mechanism of SSYX on the hearts of 2-week myocardial-infarcted rabbitsResults of MEA recording in vivo in each group:Compared with Sham group, FPmin, FPdur and CV of the myocardial tissue of the border zone in MI group were decreased significantly, TAT and the dispersion of TAT were increased significantly (P<0.05), the excitation propagation markedly disordered and the isopotential curves were chaotic and spacing became visibly denser. Compared with MI group, FPmin and CV were decreased significantly, and FPdur was increased significantly in MI+AD group (P<0.05), while TAT and the dispersion of TAT shown no significant differences. Amiodarone treatment exhibited a similar disordered conduction as MI experiments, moreover, the isopotential spacing was futher denser than MI group. Except that, the ectopic excited focus occured simultaneously in discontinute place within the recording area. Compared with MI+AD group, SSYX treatment significantly decreased FPdur, TAT and the dispersion of TAT, and significantly increased FPmin and CV(P<0.05). Furthermore, SSYX also alleviated the unfavorable changes of the excitation transmission followed MI, and the transmission pattern became regular as well as the isochrones was sparser compared with either MI group or MI+AD group.Results of electrophysiology recording in vivo in each group:Compared with Sham group, the APD90.Epi, APD90.Mid and APD90.Endo in the MI group significantly shortened, ERP and TDR significantly increased, and VFT significantly reduced (P<0.05). Compared with MI group, the three layers of myocardial MAPD90 (PD90.Epi, APD9o.Mid and APD90.Endo) as well as ERP were all considerably prolonged in MI+AD group and MI+SSYX group (P<0.05). Compared with MI+AD group, the three layers of MAPD90 were shorter in MI+SSYX group (P<0.05), No differences in ERP, TDR and VFT were detected between MI+AD group and MI+SSYX group.The mRNA and protein expression levels of CX43 and KV6.2:Compared with Sham group, the CX43 mRNA and protein expression levels of myocardium in the border zone significantly decreased, and the KV6.2 mRNA and protein expression levels significantly increased in MI group (P<0.05). Compared with either MI group or MI+AD group, SSYX treatment significantly increased the CX43 mRNA and protein expression levels, the KV6.2 mRNA and protein expression levels significantly decreased (P<0.05).2 the intervention of SSYX on the hearts with pathological ventricular remodeling of 2-week myocardial-infarcted rabbitsResults of Cardiac ultrasound examination in rabbits:Compared with Sham group, LVESD, LVEDD, LVEDV and LVESV were significantly raised, whereas, LVFS, LVEF and E/A ratio were significantly reduced in MI group. Compared with MI group, LVESD and LVESV significantly decreased, and E/A ratio notably recovered in MI+AD group (P<0.05); LVESD, LVEDD, LVEDV and LVESV significantly decreased, in the meanwhile, LVFS, LVEF and E/A significantly increased in MI+SSYX group (P<0.05). Compared with MI+AD group, LVESD, LVEDV and LVESV significantly decreased, while LVEF and E/A significantly increased in MI+SSYX group.The changes of myocardial tissue morphological morphology:Compared with Sham group, myocardial tissue in the border zone diaplayed myocardial fiber arranged disorder, analosis visible, the muscle fiber fracture, partly local apparent focal necrosis in MI group. Transmission electron microscopy test showed the structure of myocardial cells significantly changed in MI group:the basement membranes were not intact, the pericapillary space broadened, the basal lamina thickened and the lumen was irregular. Additionally, there were obvious changes in the mitochondrial structure. Compared with MI group, there were little changes in MI+AD group, however, Necrosis of myocardial fiber was significantly reduced in MI+SSYX group, and the SSYX treatment rabbits exhibited a better endothelial structure.The changes of collagen fibers hyperplasia:Masson staining results show that the myocardial fiber bundles are close, no obvious collagen fibers hyperplasia was observed in Sham rabbits’hearts. In MI group, there were visible collagen fiber hyperplasia accumulated and net-like distribution between myocardial fiber bundles. In MI+AD group and MI+SSYX group, the myocardial tissue in the border zone displayed different degree of collagen fiber hyperplasia eased, especially in SSYX treatment rabbits’hearts, the fiber hyperplasia reduced more significantly.The mRNA expression levels of SERCA2a, RYR2, PLB, NCX1, TGF-β1 and MMP-2:Compared with Sham group, the SERCA2a, RYR2, PLB and NCX1 mRNA expression levels of myocardium in the border zone significantly decreased, the TGF-β1 and MMP-2 mRNA expression levels significantly increased in MI group (P<0.05). Compared with MI group, RYR2 and NCX1 mRNA expression levels significantly increased in MI+AD group (P<0.05), however, there were no obvious changes in SERCA2a, PLB, TGF-β1 and MMP-2. The SERCA2a, RYR2, PLB and NCX1 mRNA expression levels in MI+SSYX group significantly increased, while TGF-Pand MMP-2 mRNA expression levels significantly decreased in SSYX treatment rabbits (P<0.05).The protein expression levels of SERCA2a, RYR2, PLB, NCX1, COL I and COLⅢ:Compared with Sham group, the SERCA2ATPase, RYR2, PLB and NCX1 protein expression levels of myocardium in the border zone significantly decreased, the COL Ⅰ and COLⅢ protein expression levels significantly increased in MI group (P<0.05). Compared with MI group, The SERCA2a, RYR2, PLB and NCX1 protein expression levels significantly increased and the COL Ⅰ and COLⅢ protein expression levels significantly decreased both in MI+AD group and MI+SSYX group. These indexes ameliorate more in SSYX treatment rabbits versus MI+AD group (P<0.05).3 The intervention of SSYX on the hearts with myocardial microangiopathy of 2-week myocardial-infarcted rabbitsCharacteristic variation of the myocardial microvascular perfusion:Compared with Sham group, the A, β and A*β values of the ligated groups (MI, AD and SSYX groups) were decreased markedly after surgery. At 2 week after operation. The A, β and A*β values in the ischemic area were still significantly decreased in either MI rabbits’hearts or amiodarone administration rabbits’hearts when compared with Sham group (P<0.05). However, The A, β and A*β values increased notably in MI+ SSYX group (P<0.05).The ultrastructure changes of myocardial microvasculature:The structure of endothelial cells significantly changed in MI group:the basement membranes were not intact, the pericapillary space broadened, the basal lamina thickened and the lumen was irregular. Additionally, there were obvious changes in the mitochondrial structure. Compared with MI group, there were little changes in MI+AD group, however, Necrosis of myocardial fiber was significantly reduced in MI+SSYX group, and the SSYX treatment rabbits exhibited a better endothelial structure.The microvessel density of the marginal cardiac tissue:Compared with the Sham group, the CD34 positive count and microvessel density of the cardiac tissue in the border zone significantly decreased in both MI and amiodarone administration rabbits’hearts. Compared with the Control group, the density of CD34 positive count significantly decreased in SSYX treatment rabbits. The result indicated that SSYX treatment prevented myocardial microvascular occlusion or even promoted neovascularization by increasing the capillary density after myocardial infarction,plasma TXA2, ET-1, vWF and NO levels:Compared with the Sham group, the plasma levels of TXA2, ET-1, vWF and NO levels significantly increased in MI group and MI+AD group (P<0.05). Compared with MI group and MI+AD group, the plasma levels of TXA2, ET-1 and vWF contents significantly decreased in the SSYX group (P<0.05).The mRNA expression levels of VEGF, ET-1, PGI2 and eNOS:Compared with Sham group, VEGF and ET-1 mRNA expression levels of myocardium in the border zone significantly increased, while PGI2 and eNOS mRNA expression levels significantly decreased in MI group and MI+AD group (P<0.05). Compared with MI and MI+AD groups, the ET-1 mRNA expression levels of myocardium in the border zone significantly decreased in MI+SSYX group, in the meanwhile, the VEGF, PGI2 and eNOS mRNA expression levels significantly increased significantly increased (P<0.05).The protein expression levels of TM:Compared with sham group, the TM protein expression levels of myocardium in the border zone significantly decreased in MI group and MI+AD group (P<0.05). Compared with MI group, The TM protein expression levels significantly increased in MI+SSYX group (P<0.05).Conclusion1 The study shows that SSYX has the same electrophysiological efficiency as amiodarone on the cadiocyte of the border zone of 2-week myocardial-infarcted rabbits’hearts. However, SSYX has favorable advantages in ameliorating the disordered electron excitation transmission caused by MI, which might be closely associated with up-regulating CX43 mRNA and protein expression levels and down-regulating KV6.2 mRNA and protein expression levels.2 The study reveals that SSYX can improve left ventricular function by up-regulating myocardial calcium regulation related genes SERCA2ATPase, RYR2, PLB and NCX1, and ameliorate left ventricular pathological remodeling by down-regulating TGF-β, MMP-2 and MMP-9 expression, which inhibited myocardial collagen fibers pathologic hyperplasia.3 The study indicates that SSYX has favorable advantages in ameliorating the impaired myocardial microcirculation after infarction in rabbit hearts. SSYX can balance the endogenous diastole and contraction active substances, and up-regulate VEGF expression to promote the border zone myocardial microvascular angiogenesis. |
