The Mechanism Of Endoplasmic Reticulum Stress In The Progress Of Myocardial Dysfunction Induced By Hypertension

PartⅠEndoplasmic Reticulum Stress Involved with Myocardial Apoptosis in Spontaneously Hypertensive RatsBackgroundIt is well known that left ventricular hypertrophy commonly occurs in hypertensive patients.However,the mechanisms in the progression from left ventricular hypertrophy to left ventricular diastolic dysfunction resulting from hypertension are not fully known.Emerging data now indicate that apoptosis occurs in the critical organs(heart,brain,or kidney) in hypertension.Therefore,we hypothesized that myocardial apoptosis may contribute the incidence of the left ventricular diastolic dysfunction.Endoplasmic reticulum(ER) is an organelle involved in the intrinsic pathway of apoptosis and it is involved in several important functions such as the folding of secretory and membrane proteins.Various conditions can disturb the functions of the ER and result in ER stress(ERS).When ERS conditions persist,initiation of apoptotic processes are promoted by CHOP/GADD153(growth arrest and DNA damage-inducible gene 153),the Caspase-12-dependent pathway,and activation of the c-Jun NH2-terminal kinase(JNK)-dependent pathway.Recent studies have also demonstrated that the apoptosis of cardiocytes is bound up with ERS.However,the mechanisms by which ERS lead to cell death remain enigmatic,particularly in the progression from left ventricular hypertrophy to left ventricular diastolic dysfunction resulting from hypertension.Valsartan is a kind of angiotensis receptor blocker which not only ameliorate hypertension but also protect target organ uniquely.Based on Val-HeFT,valsartan significantly reduces the combined end point of mortality and morbidity and improves clinical signs and symptoms in patients with heart failure,when added to prescribed therapy.The mechanisms of valsartan's effects are complicated,and whether or not the mechanism related to ERS is also unclear.Objectives1.To definite the function of apoptosis in the progress of myocardial dysfunction induced by hypertension.2.To observe the expression of ERS-relative factors in the myocardium in SHRs and controls.3.To observe the relationship between the interventive effect of valsartan and ERS.Methods1.Establishment of animal model1.1 Establishment of cardiac hypertrophy and animal model of the left ventricular diastolic dysfunctionThirty SHRs(8 weeks old,SPF,male) and thirty WKY rats(8 weeks old,SPF,male) were taken from vital river laboratories.The rats were housed in cages,and had free access to standard rat diet and tap water of the center.They were maintained under conditions of standard lighting(alternating 12h light/dark cycle),temperature (22±0.5℃) and humidity(60±10%) for at least 1 week before the experiments.Then, ten SHRs or ten WKY rats were randomly killed in each group when they were 8 weeks,16 weeks and 32 weeks old.All the rats drank freely and their blood pressure was measured every 2 weeks.1.2 the Interventive Effect of valsartanTwenty SHRs(16weeks old,SPF,male) and ten WKY rats(16weeks old,SPF,male) were taken from vital river laboratories.Under the same raising conditions,they were feed for at least 1 week before the experiments.Twenty SHRs were randomly divided into two groups of ten.The first group of SHRs and the ten WKY rats were fed normally to grow for 32 weeks and the other group was fed valsartan(20mg/kg/d) until they were 32 weeks old.All the rats drank freely and their blood pressure was measured every 2 weeks.Both groups of SHRs were killed after the defined period of growth.2.Measure Blood PressureAll the rats drank freely and their blood pressure was measured every 2 weeks.3.Blood pressure and left ventricular mass index(LVMI)LVMI=left ventricular mass(LVM,mg)/body mass(BM,g).4.Echocardiographic examinationAt the beginning and the end of the study,transthoracic echocardiogram was performed in hypertensive and control animals.Rats were placed supine and the anterior chest wall was shaved.Echocardiograms were performed with a Hewlett-Packard Sonos 7500 sector scanner equipped with a 7.5-MHz phased-array transducer.Conventional images included 2-dimensional,M-mode,and continuous wave and pulsed Doppler images.5.HE stainingHE staining was used to study the pathological changes in this study.6.TUNEL stainingTUNEL staining was used to detect the expression of apoptotic cells.7.ImmunohistochemistryImmunohistochemistry was used to detect the protein expression of GRP78,CHOP, p-JNK,Caspase-12,Caspase-3.8.Western-blot analysisWestern-blot analysis was used to determine the protein expression of GRP78,CHOP, p-JNK,JNK,Caspase-12,Caspase-3.9.real time RT-PCR analysisThe mRNA expressions of GRP78,CHOP,p-JNK,Caspase-12 were determined by real time RT-PCR.Results1.Blood pressure and LVMISystolic blood pressure of SHRs group were kept on at high level.Blood pressure and LVMI differed significantly(P＜0.05) in the animals of SHRs compared with the sex-matched control WKY rats.After treatment of valsartan,the level of blood pressure was obviously decreased(P＜0.05) compared with the sex-matched SHRs.2.Establishment of the left ventricular hypertrophy and left ventricular diastolic dysfunctional model(1).Left ventricular diastolic function variables expressed by the ratio of E-wave(early diastolic filling,early peak velocity) and A-wave(late atrial filling,atrial peak velocity) differed significantly(P＜0.05) in SHRs(16 and 32 weeks) compared with those in WKY rats(16 and 32 weeks).A significant decrease in the E-wave velocity,significant increase in the A-wave velocity,and remarkable decrease in the E/A ratio was found in SHRs(16 weeks) and remarkable increase in the E/A ratio was found in SHRs(32 weeks).Left ventricular systolic function parameters including fractional shortening (%),and ejection fraction(%) of the SHRs were no significant difference compared with the WKY rats(32 weeks).Forthermore,the thickness of interventricular septum and left ventricular posterior wall and LVMI also differed significantly(P＜0.05) compared with those in WKY rats at 16 weeks.These results demonstrated that SHRs had cardiac hypertrophy at 16 weeks of age and had left ventricular diastolic dysfunction at 32 weeks old.(2).In the valsartan group,left ventricular diastolic function was obviously improved (P＜0.05) compared with SHRs group.3.HE stainingWith hematoxylin-eosin staining,we found that the cell size was increased significantly in the hearts of SHRs(16weeks) and cardiac muscle fibers(32weeks of SHRs) were disordered,and many of them were collapsed.In the valsartan group, myocyte hypertrophy was improved obviously.4.TUNEL stainingTo assess whether cardiac myocyte apoptosis in hypertensive heart,the tissue sections were labeled with an in situ TUNEL assay.Apoptosis was observed in both the cardiomyocyte and the endothelium of the hypertensive heart.More apoptotic cardiocytes(P＜0.05) were found from SHRs(16 weeks and 32 weeks) groups. Estimation of cardiac apoptosis revealed a nearly threefold increase in TUNEL-positive nuclei in hypertensive heart.In the valsartan group,the level of apoptosis was significantly(P＜0.05) decreased compared with SHRs group.5.ImmunohistochemistryImmunohistochemistry studies showed that GRP78,CHOP,Caspase-12,Caspase-3 were all abundantly expressed in the myocardium of SHRs.In contrast,WKY rats exhibited modest or weak immunoreactivity for this molecule.We also found that in the myocardium of SHRs,the increasing of GRP78,CHOP,Caspase-12 and Caspase-3 positivecells paralleled with the increasing of apoptotic cells.In the valsartan group, the number of cardiac myocyte apoptosis and the expression of GRP78,CHOP and Caspase-3 were significantly(P＜0.05) decreased.However,a similar level of p-JNK protein was observed in all hypertensive and normal rats.6.Western blot analysis of GRP78,CHOP,p-JNK,JNK,Caspase-12 and Caspase-3The densitometric analysis of bands for GRP78,CHOP,Caspase-12,Caspase-3 but not p-JNK,JNK revealed a significant(P＜0.05,P＜0.05) increase in relative protein content in myocardium from SHRs(16weeks,32weeks) in comparison with those from WKY rats.During the initial stage of hypertension,only the protein levels of GRP78 and CHOP were upregulated(P＜0.05).In the valsartan group,protein level of GRP78, CHOP but not Caspase-12 significantly(P＜0.05) decreased compared with SHRs group.7.real time RT-PCR analysis of GRP78,CHOP,p-JNK and Caspase-12 expressionThe mRNA levels of GRP78,CHOP,Caspase-12,but not p-JNK were found to be significantly(P＜0.01) upregulated in the hypertrophic and diastolic dysfunctional heart. However,during the initial stage of hypertension,only the mRNA levels of GRP78 and CHOP were upregulated.A similar level of p-JNK mRNA was observed in all hypertensive and normal rats.In the valsartan group,mRNA levels of GRP78,CHOP were diversity(P＜0.05) compared with SHRs group but not Caspase-12. Conclusions1.Myocardial apoptosis plays an important role in the progress of myocardial dysfunction induced by hypertension.2.Increase expression of GRP78,CHOP,Caspase-12 but not p-JNK in hypertensive heart paralleled with the increase of apoptotic cells.These results suggest that ERS can contribute to cardiac myocyte apoptosis in the progress of myocardial dysfunction induced by hypertension.3.Valsartan could significantly reduced cardiac myocyte apoptosis by inhibiting the expression of CHOP.