Discovery Of The Potential Biomarker Of Dengzhan Shengmai Capsule In The Intervention Of Heart Failure Caused By Myocardial Infarction

1 BackgroundAs an end stage of many cardiovascular diseases,heart failure(HF)is a complex disease with high morbidity and mortality.Myocardial infarction(MI)is one of the most common factors which induces HF.Several stages can be divided during the evolution of MI-induced HF which will help to find more effective ways of heterogeneous intervention.Dengzhan Shengmai Capsules(DZSMC)is a Chinese formula based on the classic prescription "Shengmai San" with adjustment of Erigeron breviscapus.DZSMC has been widely used in treating HF clinically.However,the mechanism of DZSMC intervention on HF remains vague,and the timing of its precise application remains to be further studied.2 Methods and results2.1 The research on the biological basis of DZSMC intervention on heart failure and the discovery of biomarkersBased on the proteomic data of rat myocardial tissue at 1,2,3,and 4 weeks after left anterior descending coronary artery ligation,combined with the verified components of DZSMCs obtained from the literature,the mechanism of DZSMC on the process of MI-induced HF was analyzed.The relationship between targets and pathways was reached by the network of DZSMC’s intervention on HF we constructed.In the meanwhile,the biological basis of DZSMC’s intervention in the course of HF caused by MI was excavated.Finally,the potential biomarkers and the possible mechanism of DZSMC’s intervention in the course of HF caused by MI were reached.(1)The DZSMC component identification results(measured by comprehensive multi-heart-cutting two-dimensional liquid chromatography combined with quadrupole time-of-flight mass spectrometry)were sorted,and 283 DZSMC compounds were collected.By using the BATMAN-TCM database to predict the action targets of DZSMC,548 drug action targets were collected.Finally,the DZSMC"component-target" data set was obtained.The mapping relationship is shown in Figure 1-1.(2)Using the proteomic data of the left anterior descending coronary artery ligation rat model(the data were obtained from the previous study of the subject and have not yet been published)to analyze the changes in the myocardial tissue proteome during the evolution of the HF caused by MI.Experiments were analyzed by BruKer NanoElute? liquid chromatography(LC)with a timsTOF Pro hybrid TIMS quadrupole time-of-flight mass spectrometer connected online with a CaptiveSpray nanoelectrospray ion source,using the National Center for Biotechnology Information(NCBI)RefSeq protein database as positive To the database,MaxQuant software(version 1.6.6.0)identified the peptides of MS/MS(FDR<1%),and clustered the expression of the same protein in different groups according to the K-means algorithm.Screening P<0.05 and a total of 6127 proteins were obtained at each time point in the evolution of the HF caused by MI in rats with FDR>20%.The number of left ventricular differential proteins was 1117,1183,826 and 860 in the left ventricle at 1 week,2 weeks,3 weeks and 4 weeks after the operation,respectively.(3)The proteomic data and the drug component data were integrated using bioinformatics technology.PPI analysis,KEGG Passway analysis,Veen analysis and other methods were applied to construct a dynamic network of DZSMC intervention in the course of HF caused by MI,from which we can explore potential mechanism of DZSMC intervention on HF and the biological connotation of intervention in the course of HF caused by MI.During this process,we constructed a potential target network for DZSMC to intervene in the course of HF caused by MI,and obtained long-term potential targets for DZSMC.The main target of DZSMC was analyzed at different time points during the evolution of MI-induced HF.Pathway analysis found that the main pathways of DZSMC at different time points in the evolution of MI-induced HF have the following differences:the main pathways with significant differences in 1 week are arginine and proline metabolism pathways;the main pathways with significant differences in 2 weeks is Myocardial contraction;while in 3 weeks the main difference significant pathway is myocardial contraction,phagosome;and in 4 week main difference significant pathway is the adrenergic signaling pathway in cardiomyocytes.(4)After integrating the related targets of HF in the Genecards database and the HPO database,1821 targets were accessed.Combined with the long-term potential target data set of DZSMC’s intervention in the course of HF caused by MI in step(3),the key target data set of DZSMC’s intervention in the course of MI-induced HF were selected.Combined with the significant differential pathways in the four weeks,we jointly constructed a "dynamic network of DZSMC’s intervention in the course of HF caused by MI".Seven potential targets of DZSMC intervention in the progression of HF caused by MI were obtained,namely CACNA2D2,IGF1,CAV3,ITGB3,CAMK2D,COX1,and COX2.CAMK2D,CACNA2D2 and CAV3 mainly regulate the cellular Ca2+channel.They participate in regulating the Ca2+homeostasis of cardiomyocytes,and stimulating contraction coupling.COX1 and COX2 are isozymes,both of which are cyclooxygenases,and are mainly involved in the oxidative phosphorylation pathway.IGF1 is an Insulin-like growth factor that regulates mitochondrial function,cardiac energy metabolism and the renin-angiotensin system.ITGB3 is an integrin that affects IGF 1 signaling and platelet aggregation.In the meanwhile,"Adrenergic signaling in cardiomyocytes","myocardial contraction","thyroid hormone signaling pathway",and "phagosome"are the main pathways involved in the above-mentioned genes involved in DZSMC’s intervention in the course of HF caused by MI.2.2 Pharmacodynamics evaluation of DZSMC on the course of HF caused by MI.Approved by the Animal Ethics Committee of the Chinese Academy of Chinese Medical Sciences,a rat model of acute MI was established by ligating the left anterior descending coronary artery in male Sprague Dawley rats.Massive literature shows that it takes 4 weeks from MI to HF after ligation of the left anterior descending coronary artery.Rats were randomly divided into sham operation group,model group,positive control group(with valsartan as the positive drug)and DZSMC(high,medium and low dose)groups.From the second day after the model establishment,rats in different groups were given saline,valsartan or DZSMC every day orally 7 weeks.The diet,activity and mental state of the rats were observed every day;and the cardiac function was observed by echocardiography at 1 week,3 weeks,5 weeks,and 7 weeks after the rat model was established.The ejection fraction and short-axis shortening rate of the ventricle were compared statistically.After 7 weeks of administration,the rats were sacrificed,and the left ventricle of the heart was selected for pathological staining analysis,including HE staining and Masson staining.The echocardiograms of rats at 1 week,3 weeks,5 weeks and 7 weeks after surgical ligation showed that the cardiac function of the rats in the sham-operated group did not change significantly;while in the model group compared with the sham-operated group,the left ventricular contraction,End-diastolic ventricular cavity dilation,ventricular anterior wall thinning,cardiac myocardial contractility weakened,the overall cardiac function decreased significantly;the positive control group and DZSMC group were significantly improved cardiac function.The results of left ventricular ejection fraction and left ventricular short-axis shortening rate of rats in each group showed that compared with the normal levels of the sham-operated group,the left ventricular ejection fraction and short-axis shortening rate of the rats in the model group were significantly lower.Gradually decreased to 50%(all satisfy P<0.001)and 25%(all satisfy P<0.001),respectively;the left ventricular ejection fraction and short-axis shortening rate in the valsartan administration group were compared with those in the model group.The values of left ventricular ejection fraction increased significantly at 3,5,and 7 weeks,and remained at the level of 50%(all satisfy P<0.01).Compared with the rats in the model group,the left ventricular ejection fraction and short-axis shortening rate of the DZSMC high-dose group were maintained at 57%(both met the P<0.05)level for 7 weeks.P<0.01)and 30%(both meet the level of P<0.01).The values of left ventricular ejection fraction increased significantly at 3,5,and 7 weeks,and remained at the level of 50%(all satisfy P<0.01).This indicates that DZSMC can improve cardiac function by delaying the thinning of the anterior wall of the left ventricle,increasing the left ventricular ejection fraction,and the short-axis shortening rate,thus achieving the effect of delaying HF in the treatment of HF caused by MI.In addition,the pathological staining results of the left ventricle of the experimental rats in each group showed that DZSMC also had a certain role in improving cardiac inflammation and myocardial fibrosis in HF.2.3 Validation on potential biomarkers of DZSMC in the intervention of HFBased on the dynamic network of DZSMC’s intervention in the course of MI-induced HF and the research basis of the pharmacodynamics of DZSMC in the treatment of MI-induced HF,we performed the ligation of the left anterior descending coronary artery in the pharmacodynamics study of DZSMC’s intervention on MI-induced HF.The serum of the rat model was used to verify the potential biomarkers of DZSMC’s intervention in the course of HF caused by MI.In the dynamic network of DZSMC’s intervention in the course of HF caused by MI,seven potential targets of DZSMC’s intervention in the evolution of the course of HF caused by MI were obtained.The main stage of the evolution of HF caused by MI mostly happened in 1-4 weeks after the ligation of the left anterior descending coronary artery.By taking the serum of rats 1,2,3,and 4 weeks after modeling for enzyme Linked immunosorbent assay,the expression changes of protein in the serum of MI rat model were detected.CACNA2D2 is a voltage-dependent calcium channel protein that regulates the balance of Ca2+in cardiomyocytes,the contraction of vascular smooth muscle and the function of the central nervous system.The experimental results show that the expression level of CACNA2D2 in rat serum changes during the evolution of MI-induced HF.Among them,compared with the sham-operated group,the expression level of CACNA2D2 in the serum of the rats in the model group showed a significant increase trend at 3 weeks and 4 weeks after MI(P<0.01).Compared with the model group,the expression level of CACNA2D2 in the serum of the DZSMC high-dose group showed a significant downward trend(P<0.01).This suggests that the up-regulation of CACNA2D2 expression level in serum may be a potential signal of DZSMC intervention in MI-induced HF,and also suggests that DZSMC may improve cardiac function by reducing the blood level of CACNA2D2.3 ConclusionIn this study,a multidimensional data integration strategy was adopted to integrate the proteomic data and the verified drug component data through bioinformatics technology.After using a rat MI model to explore the intervention of DZSMC on acute MI-induced HF disease,the biological basis and the potential biomarkers of DZSMC intervention in MI-induced HF was reached.It was found that DZSMC may affect "Adrenergic signaling in cardiomyocytes","Cardiac muscle contraction","thyroid hormone signaling pathway",and "phagosome" by intervening ITGB3,IGF1,CAMK2D,COX1,COX2,CAV3 and CACNA2D2 targets in different stages of MI-induced HF.DZSMC can regulate cardiac inflammation,myocardial fibrosis,and myocardial contraction through regulation of "energy signaling" and"myocardial contraction" pathways thereby playing a regulatory role in the evolution of HF.The research of efficacy of DZSMCs in the treatment of MI-induced HF found that DZSMCs can improve cardiac function by delaying the thinning of the anterior wall of the left ventricle,increasing the left ventricular ejection fraction,and the short-axis shortening rate.Improvements in cardiac inflammation and myocardial fibrosis also play a role in delaying and treating HF.The serum CACNA2D2 expression level increased after MI,which may indicate the evolution from MI to HF.The intervention of DZSMC significantly decreased the expression level of CACNA2D2 in serum.CACNA2D2 mainly regulates the balance of Ca2+and affects myocardial contractile function.It is suggested that CACNA2D2 may be a potential biomarker for DZSMC to interfere with MI-induced HF,and it can exert its anti-HF effect by reducing the expression level of CACNA2D2 and improving Ca2+channel-related myocardial contraction.At the same time,based on the proteomic data of the dynamic evolution of MI-induced HF,this study analyzed the possible biomarkers,CACNA2D2,of DZSMC intervention in MI-induced HF,and provided a new idea for the research on the intervention effect of traditional Chinese medicine(TCM)compounds.