Ye Du, Jian An, Yan-Qing Guo, Qing-Bo Bao, Xiao-Hong Li

1.Shanxi Medical University,Taiyuan,Shanxi,030000

2.Department of Cardiology, Shanxi Cardiovascular Hospital,Shanxi Medical University,Taiyuan,Shanxi,030024

Keywords:Acute myocardial infarction Angiotensin Receptor Neprilysin Inhibitor Ventricular remodeling

ABSTRACT Objective: To investigate the effect of Angiotensin Receptor Neprilysin Inhibitor (ARNI) on ventricular remodeling after AMI in rats. Methods: Sixty male SD rats were randomly divided into Control group, AMI group, AMI-valsartan group, AMI-ARNI group,15 rats in each group, ligating the left coronary anterior descending artery to establish the rat model of AMI. Valsartan group and ARNI group were treated with valsartan (34mg / kg / day) and ARNi (68mg / kg / day) for 6 weeks, the Control group and AMI group were given the same amount of normal saline. LVIDd, LVIDs, EF were measured by color doppler echocardiography before and 6 weeks after treatment. After 6 weeks, the rats were sacrificed, the hearts were weighed, then myocardial tissue Masson staining was performed to calculate the collagen volume fraction (CVF). Results: compared with the control group, LVIDs increased significantly with the reduction of EF in the AMI group, valsartan group and ARNI group (P＜0.05). After 6 weeks of treatment, compared with the AMI group, LVIDd and LVIDs both reduced significantly with the increase of EF in the ARNI group (P＜0.05). Compared with the control group, the left ventricular weight, right ventricular weight and atrial weight all increased significantly in the AMI group, valsartan group, and ARNI group (P＜0.05); compared with the AMI group, the left ventricular weight, right ventricular weight, atrial weight and CVF reduced significantly in the valsartan group and ARNI group (P＜0.05); compared with the valsartan group, the left ventricular weight and CVF further reduced in the ARNI group. (P＜0.05).Conclusions: ARNI has the effect of reversing ventricular remodeling after AMI in rats, which can reduce left ventricular volume, increase myocardial contractility, and inhibit myocardial cell hypertrophy and fibrosis.

1. Introduction

Acute myocardial infarction (AMI) is caused by a large number of myocardial cell necrosis due to acute or continuous coronary ischemia and hypoxia.AMI is an acute and dangerous disease in coronary heart disease. In recent years, the occurrence of AMI not only accounts for a large proportion of the elderly, but also gradually increases in the young adults. At present,with the continuous development of coronary intervention surgery[1],more patients with AMI have achieved revascularization after coronary recanalization within the effective time,avoiding heart pump failure caused by large-area myocardial cell necrosis,the mortality rate of AMI is reduced to a certain extent.Although the survival rate of AMI patients has been improved, the risk of heart failure (HF) in AMI patients continues to rise,and AMI has gradually become an important cause of HF,which because AMI can cause excessive activation of certain neuroendocrine hormones,causing ventricular remodeling,which leads to myocardial contraction and diastolic disfunction,and further develops HF[2].HF is a serious stage of the development of cardiovascular disease,and its mortality rate increases year by year[3],which seriously threatens human health.Ventricular remodeling can occur at the early stage of AMI,therefore,preventing the occurrence of ventricular remodeling effectively is of great significance to the prognosis of patients with AMI.

Ventricular remodeling is an important pathological mechanism in the HF.Ventricular remodeling causes corresponding pathophysiological changes due to the myocardial injury,mainly including myocardial cell hypertrophy,fibrosis,apoptosis, and inflammatory reaction[4].Early changes can provide compensatory protection, such as preventing heart rupture and so on,but it causes decompensated changes of heart in the lasting pathological conditions,leading to cardiac filling or systolic dysfunction,further deteriorate and develope into HF[5].The activation of the reninangiotensin-aldosterone system (RAAS) promotes the occurrence of ventricular remodeling[6].Studies have shown that drugs that inhibit RAAS or sympathetic nervous system activity have a protective effect on the heart and can improve ventricular remodeling[7-9].However,the use of these drugs in the clinic has not improved the outcome of high mortality in the HF.Therefore,new drug treatments still need to be explored.

The first in class of the ARNI-sacubitril/valsartan[10-11],which is a sodium complex that combines sacubitril (neprilysin inhibitor) and valsartan (ARB),by inhibiting the degradation of natriuretic peptides (NP) to play the protective effect on the heart.However,NEPI can also increase the level of angiotensin I and II,so the use of NEPI alone has no obvious significance for the treatment of HF.ARNI through the joint NEPI and ARB to block the harmful effects of the RAAS system,thus promotes vasodilation,natriuresis and reverses ventricular remodeling[12-15],which starts new prospects for HF treatment.The PARADIGM-HF study has showed that ARNI has a significant advantage in the treatment of HF with reduced ejection fraction, which can significantly reduce the hospitalization rate and mortality of HF,at the same time,the risks of hyperkalemia, angioedema, and renal damage during treatment are low[16-18].However, there are few studies about the use of ARNI for HF after AMI. whether ARNI can delay ventricular remodeling after AMI needs to be further determined.In this study,rats myocardial infarction model were established by ligating the left coronary anterior descending artery to observe the effect of ARNI on ventricular remodeling in myocardial infarction rats.

2. Materials and methods

2.1 Experimental materials

2.1.1 Experimental animals

60 healthy,clean,male SD rats weighing about 220g were purchased from the Experimental Animal Center of Shanxi Medical University and fed in the animal room of the Physiological Laboratory of Shanxi Medical University.This study was approved by the Animal Ethics Committee of Shanxi Medical University.

2.1.2 Instruments and reagents

sacubitril/valsartan:Beijing Novartis Pharma AG,100mg/tablet,valsartan:Beijing Novartis Pharma AG,80mg/tablet,HX-101E animal ventilator:Chengdu Taimeng Software AG,Taimeng BL-420F biological function experiment system:Chengdu Taimeng Software AG,GEVIVID7 color doppler echocardiography:American GE AG, Masson reagent: Beijing Solable Technology AG.

2.2 Experimental methods

2.2.1 Experimental groups

Sixty male adult SD rats weighing about 220g were randomly divided into 4 groups:Control group,AMI group,valsartan group,ARNI group,15 rats in each group.The treatment dose of valsartan group and ARNI group was 34 and 68mg/kg/day,respectively,the control group and AMI group were given the same amount of normal saline,to 6 weeks of treatment.

2.2.2 Induction of AMI

Induction of AMI.The animals were weighed,The rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate (3ml/kg),fixed in the supine position on the operating table,removed the rats hair of the neck and chest,iodophor sterilization,connected to a respirator through a tracheotomy.The third and fourth ribs were cut to expose the heart.The left coronary artery was ligated about 2 mm from its origin using a sterile 6-0 atraumatic silk suture.The successful modeling indicated by myocardial color to a paler shade,ventricle hypokinesis and ST elevation of the electrocardiogram.The thorax was then closed.The intubation was removed and the animals were observed after the rats had spontaneous breathing.

2.2.3 Cardiac function monitoring

GEVIVID7 color Doppler echocardiography was used to measure the changes of cardiac function before and 6 weeks after treatment.The LVIDd,LVIDs,and EF were used as evaluation indexes.

2.2.4 Myocardial tissue processing

After 6 weeks of treatment,the experimental rats were sacrificed,the heart tissue was taken out and cut according to the left ventricle,right ventricle and atrium,then weighed.The left ventricular myocardial tissue was cut into thin slices perpendicular to the long axis direction,fixed in paraformaldehyde solution,embedded in paraffin and sectioned.The section was stained with Masson and the pathological damage of myocardial tissue was observed.CVF was measured by Image J.(CVF=Myocardial collagen area/total area of tissue measured in the same visual field × 100%).

2.3 Statistical Analysis

SPSS 23.0 software was used for statistical analysis,results shown are means ± SE, and all data were tested for normality and homogeneity variance.Statistical comparisons between groups were analyzed using one-way ANOVA.When data were equal variances,LSD test was used for pairwise comparison,otherwise Welch's approximate variance analysis was performed,and Tamhane's test was used for pairwise comparison.P ＜0.05 was taken to indicate statistical significance.

3. Results

3.1 Effect of ANRI on cardiac function of rats

Before treatment,the left ventricular volume and EF in the control group were within the normal range.Compared with the control group,LVIDs increased significantly with the reduction of EF in the AMI group,valsartan group and ARNI group(P＜0.05).There was no statistical difference in left ventricular volume and EF in the AMI group,valsartan group and ARNI group.After 6 weeks of treatment, compared with the AMI group,LVIDd and LVIDs both reduced significantly with the increase of EF in the ARNI group (P＜0.05). ( Table 1 )

Table 1 Cardiac function in rats (n=15, ±s)

Table 1 Cardiac function in rats (n=15, ±s)

Vs control group , aP＜0.05 ; Vs AMI group , bP＜0.05 ; Vs valsartan group , cP＜0.05 .

Group Before treatment 6 weeks of treatment LVIDd(mm) LVIDs(mm) EF(%) LVIDd(mm) LVIDs(mm) EF(%)control group 4.92±0.40 2.37±0.34 86.73±5.35 4.88±0.38 2.41±0.34 86.40±3.92 AMI group 5.15±0.22 2.95±0.19a 61.33±2.69a 8.50±0.17a 6.73±0.42a 57.93±3.73a valsartan group 5.15±0.24 3.03±0.24a 61.60±2.61a 8.08±0.61a 6.33±0.66a 63.00±3.32ab ARNI group 5.15±0.24 2.97±0.22a 62.40±4.93a 7.78±0.32ab 5.61±0.36abc 67.73±3.15abc F 2.519 13.776 95.635 369.292 392.055 184.135 P 0.067 ＜0.001 ＜0.001 ＜0.001 ＜0.001 ＜0.001

3.2 Myocardial tissue weighing results

Compared with the control group,the left ventricular weight,right ventricular weight,and atrial weight increased significantly in the AMI group,valsartan group,and ARNI group (P＜0.05);compared with the AMI group,the left ventricular weight,right ventricular weight,and atrial weight were significantly reduced in the valsartan group and ARNI group (P＜0.05);compared with the valsartan group,the left ventricular weight was further reduced in the ARNI group (P＜0.05).( Table 2 )

Table 2 Myocardial tissue weighing results(n=15, ±s )

Table 2 Myocardial tissue weighing results(n=15, ±s )

Vs control group , aP＜0.05 ; Vs AMI group , bP＜0.05 ; Vs valsartan group , cP＜0.05 .

Group Left ventricular weight(mg) right ventricular weight(mg) atrial weight (mg)control group 547.33±15.80 169.33±9.61 110.67±9.61 AMI group 920.00±43.59a 254.67±29.00a 190.67±17.10a valsartan group 862.67±28.15ab 232.00±24.84ab 164.00±12.42ab ARNI group 828.67±21.34abc 216.00±36.41ab 154.67±18.46ab F 902.645 60.617 75.424 P＜0.001 ＜0.001 ＜0.001

3.3 Masson staining of myocardial tissue results

The results of Masson staining of myocardial tissue showed that the cells were well-arranged in the control group.Blue stained collagen fibers were seen around the infarcts and myocardial fibers were obviously broken,arranged disorderly in the AMI group, valsartan group,and ARNI group.( Figure 1 ).Compared with the AMI group, the CVF was significantly reduced in the valsartan group and the ARNI group (P＜0.05);Compared with the valsartan group,the CVF was further reduced in the ARNI group (P＜0.05) .( Table 3 )

Figure 1 Masson staining of myocardial tissue results( × 200 magnification)

Table 3 Myocardial collagen area results

4. Discussion

At present, coronary heart disease is still one of the major deathcausing diseases in the world. Among them, the risk of AMI is greater. Although the development of coronary intervention surgery has improved the mortality rate in the acute stage of AMI, the prognosis of patients after AMI is poor, and the incidence of HF after AMI is about 25%[19].Ventricular remodeling after AMI is an important pathological mechanism leading to cardiac dysfunction and deterioration of cardiac function, which is mainly manifested cardiomyocyte hypertrophy,apoptosis,inflammation changes and fibroblast proliferation,further leads to thickening of the ventricular wall,enlargement of the heart cavity and decrease of ventricular ejection fraction,eventually develops into HF.HF has a 50% mortality rate within 5 years[20],which seriously threatens human health.The study found that the activation of the NP system plays an important beneficial role in HF, but due to "NP resistance", which leads to the accumulated concentration is not enough to maintain the hemodynamic balance of the heart, so it is necessary to increase the plasma level of NP to restore rebalance of cyclic steady state.The excessive activation of RAAS in HF leads to adverse effects of cardiac hypertrophy and oxidative stress[21].ARNI works on NP and RAAS simultaneously,which exerts the beneficial effects of natriuretic peptides while inhibits the adverse effects of RAAS[22,23],becoming a new target for HF treatment.At present,there are few studies about whether ARNI can delay ventricular remodeling after AMI.In this experiment,the AMI model was established first,and the drug intervention treatment was performed after the model was established successfully.The end point of the treatment was to clarify the effect of ARNI on ventricular remodeling in experimental animals after AMI,by evaluating cardiac function and pathological changes.

The results of experimental postoperative rats color doppler echocardiography showed that compared with the control group, the LVIDs increased and with the reduction of EF in the model group, suggesting that the modeling was established successfully. At the end of the treatment, the results of reexamination of cardiac color doppler echocardiography showed that all AMI rats occured ventricular remodeling, that is, the volume of the left ventricle increased.Compared with the AMI group,the LVIDd and LVIDs reduced significantly with the increase of EF in the ARNI group, suggesting that ARNI was treated in the early stage of AMI rats can reduce ventricular dilation and increase ventricular contractility, thereby improve cardiac function.

Cardiac weighing results and Masson staining results of cardiomyocytes showed that AMI rat can cause cardiomyocyte hypertrophy and fibrosis changes. Compared with the AMI group,the left ventricular weight, right ventricular weight, atrial weight, and CVF reduced significantly in the ARNI group and valsartan group.Compared with the valsartan group, the left ventricular weight and CVF reduced further in the ARNI group.Which suggested ARNI and valsartan can inhibit cardiomyocyte hypertrophy and fibrosis after AMI,and the effect of ARNI was more significant.

In summary, ventricular remodeling after AMI is an important cause of the deterioration of cardiac function.Therefore,effectively inhibiting the occurrence of ventricular remodeling has important significance for the prognosis after AMI. The above-mentioned research results showed that ARNI can inhibit the changes of ventricular remodeling after AMI such as cardiomyocyte hypertrophy and myocardial fibrosis,thereby exerting ARNI's role in protecting the heart and improving cardiac function.In this experiment,the selection of drug dosage was directly referred to the literature[24].The difference effect of the drug caused by different drug concentration was not studied, and there was certain limitation.