| Heart failure (chronic heart failure, HF) is that the heart is unable to maintain adequate cardiac output to meet the body’s metabolic needs and the state venous reflux is blocked. Heart failure is always the end stage of many cardiovascular diseases, and now is becoming a major health problem around the world. European Society of Cardiology (ESC) estimated that among1billion people in Europe involves47countries, people with heart failure was about5percent of the total population. Framingham Heart Study showed that both the prevalence and incidence of heart failure increased significantly with the increasing age. The prevalence of heart failure was about1percent at the ages of50, and it easily raise to10percent when80years old. Moreover, the annual incidence of heart failure was0.2percent between the ages of45and54, however, it can rise to4percent between the ages of85and94. In2000the country’s large sample survey found that the incidence of heart failure was0.9percent, and the incidence of heart failure increased significantly with age. Heart failure is the disease featuring a poor prognosis and a high mortality. A great many of studies have shown that both the5-year mortality in patients with heart failure and the2-year mortality in patients of cardiac function between stage III and IV were higher than50percent, and30to40percent of these patients died of sudden death resulted from ventricular arrhythmias. Therefore, improving the heart function and preventing the ventricular arrhythmias after heart failure is the important measure to improve the prognosis of patients with heart failure.The myocardial contractility decreased obviously after heart failure, the basic parts of the decreased myocardial contractility is the abnormal cardiac calcium transient occurred among the excitement-contraction coupling. Ryanodine receptor (RyR) is located in the corresponding junction between the sarcoplasmic reticulum membrane and T-tube with L-type calcium channels. It composes the functional units of myocytes excitation-contraction coupling with L-type calcium channels in cardiac. Depending on the differences of encoding genes, RyR is divided into three subtypes:RyR1, RyR2and RyR3,and RyR2mainly exists in cardiac tissue. Studies have shown that in the development of heart failure, the structure and function of RyR2has occurred significant changes. Under physiological conditions, maintaining a rhythmic contraction of normal cardiac tissue relies on the calcium ion can be rapidly cleared from the cell after myocardial contractility, This is mainly depends on sarcoplasmic calcium ATP enzyme (SERCA2a) which can pump cytosolic calcium return to the sarcoplasmic reticulum. In addition, sodium-calcium exchanger (NCX), on the cell membrane, can also discharged cytosolic calcium to extracellular. The results of both SERCA2a and NCX could restore the cytosolic calcium ion concentration to the initial state of cardiac diastolic. The positive inotropic effect of myocardial calcium transient becomes weakened after heart failure heart, and hence, left ventricula ejection fraction obviously reduces.There are numerous studies confirm that the action potential duration (APD) of myocardial cells is extended during heart failure, and this change characterized chiefly intermittent dependence and extremely increased regional dispersion of repolarization. Meanwhile, the period of myocardial repolarization effective refractory is also obviously extended. The level of prolonged repolarization effective refractory is positively correlated with left ventricular end-diastolic pressure, that is, the more severe the heart failure is, the more longer the period of repolarization effective refractory is. As we all know, the current of cardiac repolarization is mediated by the cell current, which mainly contains potassium, calcium and sodium-calcium exchanger. Transient outward potassium current (Ito) is an important component of potassium currents, it mainly occurs in the phase1of the action potential repolarization. The reduction of Ito can have a significant effect on the action potential plateau. Such consequences shall largely lead to the action potential duration and the period of repolarization effective refractory significantly prolonged in heart failure. Sodium-calcium exchanger (NCX1) is a superfamily of the membrane proteins, sodium-calcium exchanger is a non ATP-dependent bidirectional transport protein. It has two types of transport modes, the forward transit and the reverse transit. By now, most studies have indicated that whether in patients with heart failure or in animal models of heart failure, the express of sodium-calcium exchanger protein1was elevated in myocardial cells. However, the mechanism of the sodium-calcium exchanger is not the same in different studies about chronic heart failure. But no matter the forward or the reverse transit transport of sodium-calcium exchanger, they all likely to lead to arrhythmia. The conduction of electrical excitability of myocardial cell via gap junctions between myocardial cells, and CX43is one major protein of the gap junction.Shen Song Yang Xin capsules are a12herbs preparations, including ginseng, dwarf lilyturf tuber, common macrocarpium fruit, salvia miltiorrbiza radix et rhizome, the parched semen ziziphi spinosae, herba taxilli chinensis, paeonia lactiflora Pall, eupolyphaga sinensis Walker, nardostachyschinensis batal, coptis chinensis franch, SchisandrasphenantheraRehd.EtWits and fossilized et al. These different constituents have mutual complementary effects as this pure Chinese herbal compound preparation had been confected according to the principle of Context theory and collaterals theory. From the perspective of traditional Chinese medical theory, diseases are caused by the maladjustment of the qi, and of blood circulation. Shen Song Yang Xin capsules have promoting blood circulation for removing obstruction in collaterals, supplementing qi and nourishing yin, and clearing away the heart fire and tranquillizing. Currently, many of researches about Shen Song Yang Xin capsules are mainly on clinical studies, what is directly to observe the effects in the treatment of arrhythmias of Shen Song Yang Xin capsules. Furthermore, there are also some researches about current changes of single ion channels recorded by patch-clamp technique. However, until recently there is little research on the upstream regulation of ion channels. Based on this, we build Japanese rabbits model of heart failure by using the technology of combining aortic insufficiency and aortic constriction in this research, aim to study the effects of Shen Song Yang Xin capsules on transient outward potassium current channel protein kv4.2, sarcoplasmic reticulum Ca2+release channel RyR2, sarcoplasmic reticulum calcium pump SERCA2a, sodium-calcium exchange (NCX1) protein and gap junction proteins (CX43) gene and protein expression changes. To explore the possible mechanisms of Shen Song Yang Xin capsules and provide an experimental basis for clinical treatment of heart failure.The first part The evaluation of chronic heart failure on rabbit model and the intervention of Shen Song Yang Xin capsulesObjective To investigate the effect of Shen song Yang xin capsules on rabbit ventricular function and structure after building Japanese rabbits model of heart failure by using the technology of combining aortic insufficiency and aortic constriction.Methods Healthy Japanese rabbits were divided into three groups, they are sham-operated group (SO group), heart failure in the control group (HF group), heart failure with Shen Song Yang Xin capsules intervention group (SSYX group) respectively. The survival and the heart function of rabbits in each group were observed after8weeks heart failure, the left ventricular end-systolic diameter (LVESD), the left ventricular end-diastolic diameter (LVEDD), the left ventricular shot ejection fraction (LVEF) and the left ventricular fractional shortening(LVFS) were examined after8weeks heart failure by using echocardiography. The heart weight in each group was accurately weighed after animals were immediately sacrificed, then the heart/body ratio was calculated.Results In the whole research process, there were two rabbits died in the sham group, six died in the heart failure control group and four died in heart failure with Shen Song Yang Xin capsules intervention group respectively, and the total mortality was33.33percent. There was no difference in mortality between the heart failure control group and the heart failure with Shen Song Yang Xin capsules intervention group. The LVESD, LVEDD and heart weight ratio were significantly increased when compared with the shame group, however, both LVEF and LVFS were significantly reduced (p<0.05). The LVESD, LVEDD and heart weight ratio were significantly reduced while the LVEF and the LVFS were significantly raised in the heart failure with Shen Song Yang Xin capsules intervention group when compared with the heart failure control group (p<0.05)Conclusions the technology of combining aortic insufficiency and aortic constriction methods to build rabbit model of chronic heart failure is reliable Shen Song Yang Xin capsules can inhibit the left ventricular expansion and improve the reduced function after rabbit heart failure.The second partThe effects of Shen Song Yang Xin capsules have on sarcoplasmic reticulum calcium regulation in ventricular myocardium after chronic heart failure in rabbitsObjective The decreased myocardial contractile function in heart failure is closely related to the sarcoplasmic reticulum calcium regulation. This study aims to observe the molecular mechanism of sarcoplasmic reticulum Ca2+regulation channel proteins remodeling in the pathological by determining Ryanodine receptor type2(RyR2) and sarcoplasmic reticulum calcium ATP enzyme (SERCA-2a) protein and mRNA expression level in ventricular myocardium in in rabbits heart failure, more importantly, to observe the effects of Shen Song Yang Xin capsules on sarcoplasmic reticulum Ca2+ regulation channel proteins remodeling and explore the possible molecular mechanisms of Shen Song Yang Xin capsules effect on cardiac function.Methods Rabbits were maintained for6weeks after operation of abdominal aortic coarctation, immune-blots were performed using myocardial fraction of ventricular myocardium from rabbit free ventricular wall. Anti-RyR2and anti-SERCA-2a were used as primary antibodies, anti-action antibody was used as internal loading control. Quantification of these signals was performed by densitometry, the protein bands were normalized to the action band in each model. The value was then averaged from all the different sets of samples. The mRNA expression of sarcoplasmic reticulum Ca2+regulation channel proteins RyR2, SERCA-2a in ventricular myocytes was evaluated by real-time PCR.Results1. Compared with the sham-operated group, quantification of sarcoplasmic reticulum Ca2+regulation channel proteins showed the amount of the proteins RyR2, SERCA-2a were both significantly decreased, they were42.68percent and40percent respectively (p<0.05), as well as the mRNA expression of RyR2and SERCA-2a were also significantly decreased, they were49.70percent and73.01percent respectively (p<0.05) in the heart failure group.2. Compared with the heart failure in the control group, the amount of the protein and the mRNA expression of RyR2were both significantly increased (p<0.05), and the mRNA expression of SERCA-2a was significantly increased in spite of they were still lower than sham-operated groupin the heart failure with Shen Song Yang Xin capsules intervention group (p<0.05). However, there were no significant differences in the amount of the SERCA-2a protein between the two groups despite there was still some increased in the heart failure with Shen Song Yang Xin capsules intervention group.Conclusions1. The decreased proteins and mRNA expression of RyR2and SERCA-2a in rabbit ventricular myocytes after chronic heart failure suggested that both RyR2and SERCA-2a involved the pathological process of sarcoplasmic reticulum calcium-regulating in the development of chronic heart failure.2. From the early stage of chronic heart failure in rabbits, giving Shen Song Yang Xin capsules intervention can improve the intracellular calcium regulation changes of the sarcoplasmic reticulum, and the mechanism of which may be related with Shen Song Yang Xin capsules can up-regulate RyR2and SERCA-2a mRNA and proteins. This results suggestinged that Shen Song Yang Xin capsules can improve left ventricular systolic function. The third part The impact of Shen Song Yang Xin capsules on ventricular electrical remodeling in rabbit heart failureObjective:Shensongyangxin capsule Shen Song Yang Xin capsules could treat ventricular arrhythmia, however, the exact mechanism has not yet been elucidated. In this study, heart failure rabbits were induced by volume overload combined with pressure overload, we made early intervention gaving Shen Song Yang Xin capsules as soon as the heart failure operation was carried out. Then the electrical remodeling of transient outward potassium current (Ito) channel subunit Kv4.2a, sodium-calcium exchanger (NCX1), connexin43(Cx43) were observed. Aim to explore the possible mechanisms that Shen Song Yang Xin capsules improve cardiac function and prevent ventricular arrhythmia after heart failure.Methods:Rabbits were maintained for6weeks after operation of abdominal aortic coarctation, the electrophysiological examination was performed in vivo. Immune-blots were performed using myocardial fraction of ventricular myocardium from rabbit free ventricular wall. Anti-Kv4.2, anti-NCX1and anti-Cx43were used as primary antibodies, anti-action antibody was used as internal loading control. Quantification of these signals was performed by densitometry, the protein bands were normalized to the action band in each model. The value was then averaged from all the different sets of samples. The mRNA expression of potassium channel Ito Kv4.2, Na+/Ca2+exchanger protein1and connexin43in ventricular myocytes was evaluated by real-time PCR.Results:1. Compared with the sham-operated group, the MAPD90, ERP and TDR were all significantly prolonged in left ventricular myocardium after heart failure in the control group, in the meanwhile, the ventricular fibrillation threshold (VFT) was significantly lower (p<0.05). quantification of electrical channel proteins showed the amount of the proteins Kv4.2a subnite, Cx43were both significantly decreased, they were61.25percent and49.83C respectively,while, the proteins NCX1increased39.24percent (p<0.05). The mRNA expression of Kv4.2and Cx43were also significantly decreased, there were27.77percent and48.31percent respectively, on the other hand, The mRNA expression of NCX1was significantly up-regulated in the heart failure group (p<0.05).2. Compared with the heart failure in the control group, the MAPD90, ERP and TDR were significantly shortened in left ventricular myocardium after heart failure in the heart failure with Shen Song Yang Xin capsules intervention group, and, the ventricular fibrillation threshold (VFT) was significantly larger (p<0.05). the amount of the protein and the mRNA expression of Kv4.2were both significantly increased (p<0.05), and the amount of the NCX1protein was significantly decreased in spite of they were still higher than sham-operated group in the heart failure with Shen Song Yang Xin capsules intervention group (p<0.05). However, there were no significant differences in the mRNA expression of NCX1between the two groups despite there was still some decreased in the heart failure with Shen Song Yang Xin capsules intervention group, the mRNA expression of Cx43was significantly increased in spite of they were still lower than sham-operated group in the heart failure with Shen Song Yang Xin capsules intervention group (p<0.05). However, there were no significant differences in the amount of the Cx43protein between the two groups despite there was still some increased in the heart failure with Shen Song Yang Xin capsules intervention group.Conclusions:1. The decreased proteins and mRNA expression of Kv4.2and Cx43in rabbit ventricular myocytes after chronic heart failure suggested that both Kv4.2and Cx43involved the pathological process of left ventricular electrical remodeling in the development of chronic heart failure, as well as the up-regulated proteins and mRNA expression of Kv4.2and Cx43.2. From the early stage of chronic heart failure in rabbits, giving Shen Song Yang Xin capsules intervention can inhibited the reconstitution of Ito、NCX1and Cx43in the ventricular myocardium, and the mechanism of which may be related with Shen Song Yang Xin capsules can up-regulate Ito and Cx43mRNA and proteins as well as down-regulate NCX1mRNA and proteins. From the results we may suggest that Shen Song Yang Xin capsules can inhibits electrical remodeling of the left ventricle in chronic heart failure and so reduce the occurrence and development of the ventricular arrhythmia. |
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