Effects Of Long-term Combination Therapy With Spironolactone, ACE-I And β-receptor Blocker, On Progression Of Left Ventricular Remodeling In Rats With A Model Of Acute Myocardial Cryoinjury

Animal studies showed that neurohormonal activation occurs immediately after acute myocarial infarction. Long-term exposure to neurohormonal activation finally results in left ventricullar remodeling (LVRM). It is a main predictor for the future cardiac events and prognosis. It is worth emphasizing that the process of LVRM importantly extends to the biology of the cardiac myocyte, the volume of myocyte and nonmyocyte components of the myocardium, and the geometry and architecture of the LV chamber. Therefor, the reversibility of heart failure is determined by whether the changes occured at the level of the myocyte, the myocardium, or the LV chamber are reversible. With the progression of heart failure, it is common that remodeling process after acute myocardial infarction is affected by the activation of neurohormonal, such as renin-angiotensin- aldostero- ne system(RAAS)and sympathetic nervous system.Aldosterone's deleterious effects including myocardial fibrosis,ventriclar wall hypertrophy,congestive heart failure and hypertension are known by degrees and we will have paid more focus on the protection of aldosterone receptor antagonist.Clinical trials and animal studies suggested that ACE-I andβ-receptor blocker could prevent the post-MI cardiac remodeling and myocardium fibrosis which maybe associated with supression of excessive activation of renin-angiotensin-aldosterone system(RAAS)and sympathetic nervous system. Effects of combination therapy were better than use of any alone. Although many clinical trials and animal studies suggested that ACE-I could prevent the post-MI cardiac remodeling,but the phenomenon of"Aldosterone escape"caused by ACE-I during the treatment of heart failure limited the effects of ACE-I.The Randomized Aldactone Evaluate Study (RALES) and Eplerenone Post-Acute Myocardial Infaction Heart Failure Efficacy and Survival Study (EPHESUS indicated that combined with ACE-I, the intervention of Spironolactone ultimately reversed remodeling, improved the nature histroy of heart failure, especially in patients with severe disfounction of heart after AMI. the studies showed aldosterone receports antagnisits had a excellent safety profile.However, whether aldosteorne receptor antagonists have the same efficacy in patients without severe dysfuction after AMI remains to be confirmed. Previously, it is worth emphasizing that we have a lot of clinical experence.It is the foundation of our futher study. Our clinical trials and animal studies showed that coadministation of Spironolactone and ACE-I could suppress left ventricular expansion and myocardial fibrosis, thus improve left ventricular function in the patients without severe dysfunction of heart after AMI. Spironolactone combined wtih Losartan in rats with AMI could decreas ventricular weight and LV weight index,inhibited collagen content and the ratio of type I and III collagen in non-infarct sizes and collagen proliferation,prevente ventricular remodeling,improve LV systolic and diastolic function. The data showed that the efficacy of the combination therapy was superior over any single drug (spironolactone or losartan).Based upon a new method of using spurt, repeated ultra low temperature cryoinjury for 15 seconds in LV free wall(LVFW), we establish an ideal method to make rat model of acute myocardial cryoinjury(AMI)-transmural myo- cardium cryoinjury with fixed and enough cryoinjury size and higher survival rate. We studied the effects of long term coadmnistration of spironolactone, ACE-I andβ-receptor blocker on left ventricular remodelling and cardiocyte apoptosis in the model of the moderate heart failure(cryoinjury size 20-40%) after 14 weeks of cryoinjury in rats and the mechanisms.135 healthy Wistar rats with weight rang from 200-220g, were bought from Hebei Medical University Experimental Animal Center, and were qualified for experiments after medical inspection.This study included four partsas follows: PartⅠ:A pilot study of using spurt,repeated cryoinjury off ventilator to establish a model of acute myocardial infarction in ratsObjective To explore a new method of establishing off ventilator support , spurt, repeated cryoinjury of rat model of acute myocardial infarction.Methods and Results1. Materials andMaterials and methods: Sixtiy wistar rats (males 30) were randomized into experimental groups according to cryoinjury time with 20 rats in each group. They were 15-second group(cryoinjuried for 5 seconds each time, three times in total), 25-second group(5 seconds each time, five times in total) and 40-second group(8 seconds each time, eight times in total). The heart was exposed through a 1.0- to 1.5-cm left lateral thoracotomy. Cryoinjury of the LVFW was performed with the use of an round copper probe 6-mm in diameter cooled to -190℃by immersion in liquid nitrogen for five minutes. It was applied to the LVFW in extenso for 5 seconds and then closed thoracic cavity immediately. When the rats'breathing restored, we repeatedly cryoinjuried the LVFW in situ to ensure a transmural cryoinjury according to the protocol. We observed the rats'survival rate during operation, a post-operation period of 28-day. Also infarct size was shown by heart pathological section stained with Masson's trichrome stain at the twenty-eighth days after operation.2. Results:①Injury and necrosis sizes in all three groups were larger than 20% signified by transmural cryoinjury, completely myocardiolysis and distinct infarct boundary.②The survival rate in 15-second group was higher than that in 40-second group((16/20 versus 5/20, p<0.0167) and the infarct size in 15-second group was smaller than that in 40-second group(28.2775±5.1926)% versus(37.77±10.56)%, p<0.05).③Compared with 15-second group or 40-second group respectively, the survival rate and infarct size in 25-second group was not significantly different (9/20 versus 16/20, p>0.0167; 9/20 versus 5/20, p>0.0167(;31.38±6.50)% versus(28.28±5.19)%, p>0.05(;31.38±6.50)% versus(37.77±10.56)%, p>0.05).④40-second group was with a greater variation than the other two groups in infarct size (standard deviation: 10.56% versus 5.19%, 6.50% in 15, 25-second group, respectively) and its'survival rate was lower. The procedure of 25-second group is more complicated and has a tendency of higher death rate than that in 15-second group.ConclusionBased upon our results, using spurt, repeated ultra low temperature cryoinjury for 15 seconds may be a ideal method to establish the rat model of AMI.PartⅡ: Effects of Long-term Combination therapy with Spironolactone,ACE-I andβ-receptor Blocker, on progression of Left Ventricular Remodeling in Rats with Acute Myocardial cryoinjury.Objective To determine the effects of long time combination of aldactone,ACE-I andβ-receptor blockers on left ventricular remodelling in in Rats with acute myocardial cryoinjury.Methods and Results1. Materials and methods: The rat model of acute myocardial cryoinjury (injury size 20%-40%)was performed with the use of liquid nitrogen in the LV free wall(LVFW)as we described in partⅠ.The cryoinjured 48 rats were randomized into 3 cryoinjury groups (AMI,n=16; AMI+capt.+carv.,n=16; AMI+ combination,n=16)and Sham-operated group was just with lateral thoracotomy and pericardiotomy (Sham-operated, n=12). Immediate administration of Spironolactone(20mg·kg-1·d-1), captopril (25mg·kg-1·d-1), Carvedilol(5 mg·kg-1·d-1) or water according to the experiment design right after palinesthesia. The rats were bred for 14 weeks in individual cages. After 14-week treatment, there were 50 rats were survival (AMI: n=12; AMI+capt.+carv.: n=13; AMI+ combination: n=13; Sham-operated: n=12) and quantitative histology(Masson's trichrome stain), LV founction (Catheterization)were evaluated.2. Results:①There was no significante difference in survival rates between four groups(12/16 in AMI, 13/16 in AMI+capt.+carv., 13/16 in AMI+combination,12/12 in Sham-operated respectively, P=0.348.).There was also no significant difference in 3 cryoinjury groups(12/16 in AMI, 13/16 in AMI+capt.+carv., 13/16 in AMI+combination, P=0.881).②As for cryoinjury size (mean±SD), compared with AMI group there was a singificant reduction in cryoinjury size in two intervention groups -AMI+capt.+carv.or AMI +combination((39.79±6.64)% versus(33.76±6.37)% or(28.01±6.56)% p<0.05) ; Compared with AMI+ capt.+carv., combination therapy group reduced the cryoinjury size even further ((33.76±6.37)% versus (28.01±6.56)%,p<0.05);.③Regarding arterial pressure and heart rate (mean±SD), there was no significante difference in aortic systolic blood pressure (SBP)(p=0.1766),aortic diastolic blood pressure (DBP)(p=0.5529),aortic mean blood pressure(MAP)(p=0.2136)and heart rate(HR) (p=0.1115) have no significant difference in the four groups.④With respect of Left ventricular systolic function:⑴left ventricular systolic pressure(LVSP) (mean±SD): LVSP in AMI+combination was higher than that in the other two cryoinjury groups ( 121.55±12.48mmHg versus 102.63±19.34mmHg or 105.89±14.08mmHg, p < 0.05 ) and had no difference compared with sham-operated group ( 121.55±12.48mmHg versus 132.97±11.72mmHg , p>0.05);⑵dp/dt: there were no differences in the three groups of cryoinjury grpups (AMI: 5696.48±1425.03mmHg/min versus AMI+capt.+carv.: 6358.04±1410.33mmHg/min or AMI+combination: 6500.18±1102.4mmHg/min, p>0.05), but they were all lower than that in the sham-operated group(9235.44±1490.73 mmHg/min, p < 0.05).⑤Left ventricular diastolic function:⑴LVEDP(mean±SD): LVEDP in AMI+ capt. +carv. and AMI + combination were lower than that in AMI(5.24±5.78mmHg or 5.44±8.99mmHg versus 11.95±6.7 mmHg,p<0.05)and were not different compared with sham-operated group (1.33±3.75mmHg, p>0.05);⑵-dp/dt(mean±SD): -dp/dt in AMI+ combination was higher than that in the other two groups(5021.57±1141.26mmHg versus 4005.62±1427.84mmHg or 3760.97±974.64mmHg,p<0.05)but lower than that in sham-operated group (5021.57±1141.26mmHg versus 6062.95±1261.65 mmHg,p<0.05).⑶τ(mean±SD):τin AMI+combination was longer than that in AMI or AMI+ carv.+capt. group(13.46±4.48ms versus 9.00±2.54ms or 10.97±3.42ms,p<0.05), and compared withτin sham-operated, there was no different result between the two groups(13.46±4.48ms versus 13.48±2.98ms,p>0.05).⑥Correlation analysis between heart rate and left ventricular founction in the four groups was proformed: linear regression equation: LVEDP=-10.06 +0.04HR and positive correlation(n=50, r=0.30, p=0.0357). But there were no relationship between heart rate and LVSP(n=50, p=1.1167),-dp/dt(n=50, p=0.1366) and T(n=50,p=0.9710).⑦Correlation analysis between cryoinjury size and left ventricular founction in the four groups: cryoinjury size correlated negatively to LVSP(n=38,r=-0.45, p=0.0044),-dp/d(tn=38,r=-0.49, p=0.0017) and postitively toτ(n=38,r=-0.41, p=0.0113),their linear regression equation are as follows respectivly: LVSP=152.34-1.33SIZE; -dp/dt=6930.1-79.02SIZE;τ=17.55-0.20SIZE. But there were no relationship between cryoinjury size and LVEDP (n=38,p=0.7440).Conclutions1. This findings indicate that immidiate administration of Carvedilol, captopril after acute myocardial cyroinjury for 14w.eeks could reduce cryoinjury size, and when combined with Spironolactone, it can reduce cryoinjury size even further.2. Co-administration of Spironolactone,carvedilol and captopril for 14weeks could significantly reduce cryoinjury size and suppression of heart rate. Cryoinjury size and heart rate were highly correlated with left ventricular systolic and diastolic function.PartⅢ:Effects of long-term combinative therapy with Spironolactone,ACE-I andβ-receptor blocker on cadiocyte apoptosis in rats with a model of acute myocardial cryoinjury.Objective To explore the effects of combination of Spironolactone,ACE-I andβ-receptor blockade for a long time on cadiocyte apoptosis in a model of acute myocardial cryoinjury in Rats.Methods and Results1. Materials and methods: The rat model of acute myocardial cryoinjury was performed with the use of liquid nitrogen in the LV free wall(LVFW)as we have described in partⅠ. The cryoinjured 48 rats were randomedly divided into 3 cryoinjury groups(AMI:n=16; AMI+capt.+carv.:n=16 ; AMI+combination: n=16)and Sham-operated group(Sham-operated: n=12) was just with lateral thoracotomy and pericardiotomy (sham-operated, n=12). Immediate administrati- on of Spironolactone (20mg·kg-1·d-1), captopril (25mg·kg-1·d-1), Carvedilol (5 mg·kg-1·d-1) or water according to the experiment design right after palinesthesia. Rats of the four groups were breed for 14w.eeks in individual cages. After 14 weeks, there were 50 rats were survival(AMI: n=12; AMI+capt.+carv.: n=13; AMI+combination: n=13; Sham-operated: n=12). The cardiomyocyte apoptotic rates in marginal zone near the scar were detected with the method of flow cytometry and ultrastructure of subcellular organelles such as nucleus, mito- chondria and glycogen granules of apoptotic cardiomyocytes were observed by TEM(transmissionelectron microscop).2. Results:①apoptotic rates: apoptotic rate was highly variable in the four groups, which was highest in AMI compared with the other three groups((16.22±3.07)%, p<0.05), and lower in the other three groups respectively: AMI+capt.+carv.( (13.68±1.86)%, p <0.05) AMI+ combination((10.26±2.46)%, p<0.05) and Sham-operated ((5.07±2.27)%), p<0.05).②Correlation analysis between apoptotic rates and cryoinjury size in the AMI three groups: apoptotic rates correlated positively to cryoinjury size ( n=38, r=0.50, p=0.0016)and its linear regression equation is: SIZE=18.44+1.14APOP..③Correlation analysis between cardiomyocyte apoptotic rates and left ventricular founction:⑴cardiomyocyte apoptotic rates correlated negatively to LVSP(n=50, r=-0.60, p=0.0001),-dp/dt(n=50, r=-0.68, p=0.0001)and postivly to LVEDP(n=50,r=0.42, p=0.0024),T(n=50,r=-0.34, p=0.0039). Their linear regression equation are as follows respectivly: LVSP=143.35-2.35APOP.; -dp/dt= 7095.75-211.37APOP.; LVEDP=-1.52+0.66APOP; T=14.58-0.27APOP.④Multiple regression showed: its regression equation: LVSP=160.86-1.2SIZE -0.76APOP.(P=0.0001),41.91% of depreesion of LVSP was associated with myocardium fibrosis and increased cardiomyocyte apoptosis(P SIZE =0.0006, P APOP. =0.0313).⑤Multiple regression analysis was performed: its regression equation: LVEDP =-13.51+0.03HR+0.6 APOP.(P=0.0021), 23.03% of increase of LVEDP was associated with increased cardiomyocyte apoptotic rates(PHR=0.0757, PAPOP.=0.0051).⑥Apoptotic cardiomyocytes showed typical ultrastructure feathers: AMI group and AMI+capt.+carv. group showed as follows: a marked decrease of subcellular organelles such as glycogen granules,marked swollen mitochondria with disrupted cristae,abnormally contracted Myofibrils (Mf) and partially disrupted, Disrupted double-deck perinuclear membrane fusion nuclear membranes. These findings indicated that irreversibly oncotic changes and were going to fuse and disapear; AMI+combination group showed as follows: mitochondria with partially disrupted cristae , cristae membrane fusion and disapearance,a marked decrease of glycogen granules. These findings indicated irreversibly mild pathological change.Conclutions1. In the process of late left ventricular remodeling, cardiomyocyte apoptotic rates in marginal zone near the scar and cryoinjury size were closely related to the ventricular founction and may independently contribute to the progress of heart failure.2. Immediate administration of aldosterone receptor antagonist-Spironol- actone, combined with captopril and Carvedilol could reverse myocardium remodeling and improve the nature histroy of heart failure in the two ways. One is to depress of apoptosis of survival cardiomyocyte in marginal zone near the scar, another is to reduce cryoinjury size. These effects maybe mutually interact.,,,,,,,3. Aldosterone receptor antagonist-Spironolactone might improve the founction of cadiocyte chondriosome via chondriosome signal way and suppress cadiocyte apoptosis through blockading Caspase protein cascade reaction.