The Mechanism Of Atorvastatin Inhibiting Cardialmyocyte Apoptosis In Rats With Heart Failure After Myocardial Infarction

Cardiamyocyte apoptosis is the major reason for cardiac remodeling during heart failure after myocardial infarction, therefore inhibiting cardiamyocyte apoptosis can reduce cardiac remodeling and improve heart function. At present, the mechanism of apoptosis in heart failure is complicated and unclear. Endoplasmic reticulum(ER) resides in cytoplasm of all eukaryotic cells except for mature erythrocytes. Its unique luminal environment renders its involvement in protein folding. When the environment in ER is broken, a response known as endoplasmic reticulum stress (ERS) or unfolded protein response (UPR) results.RAAS, Angll, oxidative stress and inflammatory responses in heart failure will change the environment of ER and activate ERS response. Excessive and/or persistent ERS response will activate apoptosis signal pathways by transcriptional induction of CCAAT/ enhancer-binding protein (C/EBP) homologous protein (CHOP/GADD 153), the caspase12-dependent pathway and activation of the c-Jun NH2-terminal kinase (JNK)-dependent pathway. Therefore, inhibiting cardiamyocyte apoptosis by inhibiting ERS response can improve heart function.Atorvastatin is a hydroxy methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statins), widely used in coronary atherosclerosis heart disease by lowering low-density lipoprotein cholesterol (LDL-C) in plasma. Nevertheless, data from both in vitro and in vivo studies suggest that statins have benefits beyond this, generally attributed to the modification of the lipid profile, called pleiotropic effects. These pleiotropic effects are cholesterol independent as its role in heart failure arouses more attentions. Studies have suggested that statins may improve heart function in ischemic and non-ischemic heart failure and reduce mortality. A study has illustrated that pravastatin can suppress ERS response, cardiamyocyte apoptosis in heart failure induced by hypertension, and improvement of heart function by pravastatin. However it is unclear whether atorvastatin can suppress apoptosis by affecting ERS response in heart failure after MI. Therefore, we study the effects of atorvastatin on cardialmyocyte apoptosis and the role of ERS response in order to investigate the mechanism by which atorvastatin improves heart function after MI.Methods: Heart failure in female Wistar rat models were created by ligating left artery descending(LAD). After ligating LAD for 4 weeks, the rat models were grouped into Model control group (normal diets, n=16) and Atorvastatin group (normal diets+Atorvastatin lOmg/kg.day, PO, n= 16).And after another 4 weeks, the following indexes were evaluated by hemodynamic:LV end-diastolic pressure (LVEDP), left ventricular systolic pressure(LVSP) and±dp/dt max(mmHg/s). The level of brain natriuretic peptide (BNP) in plasma were examined by ELISA.We assayed the cardiac pathomorphism by HE and Masson staining.To assess the level of myocardium apoptosis in heart failure after MI, the cardiac tissue sections were labeled with an in situ TUNEL assay. The expressions of Bcl-2,Bax and caspase-3 proteins were examined by immunohistochemical analysis and the levels of Bcl-2,Bax and caspase-3 mRNA gene examined by PCR. By Western blot method, GRP78 proteins, representing ERS response and caspase-12 and CHOP proteins reflecting signal pathway of apoptosis induced by ERS response were evaluated.We cultured cardiamyocyte in vitro from female rats after 1d. Cardiamyocyte apoptosis were induced by Ang II coculturing and atorvastatin were added. After 24h, we examined the expressions of Bcl-2 protein by Western blot assay. The ratio of apoptosis were examined by TUNEL and flow cytometry (FCM).The expressions of GRP89,caspase-12 and CHOP proteins were examined by Western blot.Results:Compared with Sham group, in Model control group LVEDP were increased, LVSP and±dp/dt max(mmHg/s) reduced and the levels of BNP in plasma were elevated.In Model group, cardiac volume increased, cardiac can't maintain normal geometric shape. Fibrosis and scar tissues formed in infracted area. However, cardiac volume was smaller in Atorvastatin group than in Model control group and cardiac can keep normal spherical shape. In Model group, An amount of cardiac tissues existed in infarcted zone, an affluence of fibrosis stowed in cardialmyocyte clearance by HE staining was also observed.But in Atorvastatin group cardiamyocyte were in order and there was an little of fiber proliferation. By Masson staining we found that a plenty of collagen and a little cardialmyocyte existed in cardiac tissues in Model group.In Atorvastatin group the number of cardiamyocyte did not reduce and collagen did not increase significantly. In both Model control and Atorvastatin groups, apoptosis was observed in cardiac by TUNEL staining. The index of cardiac apoptosis revealed a significant increase in TUNEL-positive nuclei in Model control group than in Atorvastatin group. The expressions of Bax and caspase-3 proteins and mRNA genes increased and the expressions of Bcl-2 and Bcl-2/Bax proteins and mRNA gene reduced in Model control group.However, atorvastatin can down-regulate the expressions of Bax and caspase-3 proteins and mRNA genes and up-regulate the expressions of Bcl-2 and Bcl-2/Bax proteins and mRNA genes. By Western blot assay, the expressions of GRP78, caspase-12 and CHOP proteins increased significantly in Model control group, but atorvastatin can down-regulate the expressions of GRP78,caspase-12 and CHOP proteins. Furthermore, we found that the expressions of GRP78 protein is positively correlated with cardiamyocyte apoptosis, which illustrated that atorvastatin may improve heart function by suppressing cardialmyocyte apoptosis induced by ERS response in heart failure after MI.We succeeded in culturing cardialmyocyte in vitro from rats after boring Id. In AngⅡgroup, the expressions of Bcl-2 and Bcl-2/Bax proteins were low, the expressions of Bax and caspase-3 proteins increased by Western blot; Cardialmyocyte apoptosis ratios were increase by TUNEL staining and FCM; the expressions of GRP78,caspase-12 and CHOP proteins by Western blot were increased; however, the expressions of Bcl-2 and Bcl-2/Bax increased Bax and caspase-3 reduced, apoptosis ratio reduced by TUNEL and FCM, the expressions of GRP78, caspase-12 and CHOP proteins reduced in AngⅡ+Ator group. Furthermore, we found that the expressions of GRP78 protein is positively correlated with cardialmyocyte apoptosis, which illustrated that atorvastatin may inhibit cardialmyocyte apoptosis by surpressing ERS response.Conclusions:1. We succeeded making heart failure rat models after myocardial infarction by ligating LAD. By hemodynamic, LVEDP were increased, LVSP and≠dp/dt max(mmHg/s) reduced and the level of BNP in plasma were elevated by ELISA, which suggested that heart failure models were made successfully.2. Cardiac remodeling occured in heart failure after MI:cardiac volume increased exceptionally, cardiamyocyte were irregular and a lot of fibrosis formed. However, in Atorvastatin group, cardiac can keep normal spherical shape and cardialmyocyte were in order and little fiber proliferated. This suggested that atorvastatin can suppress cardiac remodeling in rat with heart failure after MI.3. Cardialmyocyte apoptosis contributed to cardiac remodeling in heart failure after MI, however, atorvastatin can suppress cardiamyocyte apoptosis in heart failure.4. The expressions of GRP78, caspase-12 and CHOP proteins increased in heart failure after MI and cultured cardialmyocyte induced by AngⅡ. However, atorvastatin can down-regulate the expressions of GRP78, CHOP and caspase-12 proteins in heart failure after MI and cultured cardialmyocyte induced by AngⅡ. The expressions of GRP78 proteins is positively correlated with cardiamyocyte apoptosis, which illustrated that atorvastatin may improve heart function by surpressing cardialmyocyte apoptosis induced by ERS response in heart failure after MI.5. The expressions of Bcl-2/Bax and caspase-3 proteins attributed to the regulation of cardialmyocyte apoptosis in heart failure after MI and cultured cardialmyocyte induced by AngⅡ.U p-regulation Bcl-2/Bax and down-regulation caspase-3 proteins were attributed to atorvastatin inhibiting to cardialmyocyte apoptosis.