Effects And Mechanisms Of Arbs To Inhibit Pressure Overload-induced Cardiac Hypertrophy

Cardiac hypertrophy is not only a physiological adaptively state before heart failure, but also an independent risk factor of major cardiac events. Although there are many factors that can induce cardiac hypertrophy, hypertensive stimulation is a major hypertrophy-inducing factor. AngiotensinⅡ(AngⅡ) and its type 1-receptor (AT1-R) have been known to be greatly involved in the pressure overload-induced cardiac hypertrophy. AT1-R blockers (ARBs) are clinically available as a highly effective and well-tolerated class of drugs for the treatment of hypertension. In addition, a variety of clinical evidences have shown that ARBs provide cardiovascular protection beyond blood pressure (BP)-lowering. Especially, ARBs administration effectively prevents cardiac hypertrophy and improves the cardiovascular outcomes in patients with hypertension. At present, there are several ARBs widely used in clinics such as Losartan, Irbesartan, Telmisartan, Candesartan, Valsartan, Olmesartan, etc. Although some of them have been shown to have more beneficial effects than other types of antihypertensive agents such asβ-adrenergic receptor blockers and calcium channel antagonists, there are also reports indicating that cardiovascular outcomes by ARBs-based treatment do not differ from those by other agents-based treatment. We therefore supposed whether there were differences in cardiac protection among ARBs besides antihypertensive potency.AT1-R is a member of the G protein-coupled receptor (GPCR) superfamily [10] and ARBs are highly selective in binding to the AT1 receptor and blocking diverse effects mediated by AT1 receptor. Structurally, ARBs not only have the common biphenyl-tetrazole ring but also the unique side chains which contribute to drug-specific differences in pharmacokinetic and pharmacodynamic properties and antihypertensive potency. Many in vitro studies including ours have suggested that effects of ARBs on suppression of AT1-R activation differ. However, a rigorous in vivo comparison of different ARBs to regress pressure overload-induced cardiac hypertrophy has not been performed. Moreover, we have recently reported that Candesartan inhibited activation of AT1-R and attenuated cardiac hypertrophy induced by pressure overload in the absence of AngⅡ, however, it is unknown whether this holds true for other ARBs. The efficacies of ARBs on cardiac protection during hypertension are different, which associate with the differences in chemical structures of the drugs. Elucidation of the molecular interaction model for the chemical structure of ARB and AT1-R and the molecular mechanisms of ATl-R activation induced by pressure overload will be of help to discriminate the ARBs according to the efficacies in AT1-R inactivation, to understand the Signaling pathway induced by pressure overload, and to develop a novel ARB with superb efficacy not only in blood pressure-lowering but also in organ protection.PartIDifferent efficacies and mechanisms of ARBs in reversal of pressure-overload cardiac hypertrophy in miceObjectives:This study is designated to compare efficacies and mechanisms of different angiotensin II type 1 (AT1) receptor receptor blockers (ARBs) (including olmesartan, candesartan, losartan, telmisartan and valsartan) in reversal of cardiac hypertrophy induced by pressure overload.Methods:Constricting transverse aorta (TAC) mice were used as a model for pressure-overload cardiac hypertrophy. Wild and angiotensinogen-deficient C57BL/6J mice of 8-10 weeks were each divided into seven groups: sham-operated group (n=5 and n=4), drinking water plus TAC group (n=9 and n=8), olmesartan plus TAC group (n=10 and n=9), candesartan plus TAC group (n=10 and n=8), losartan plus TAC group (n=8 and n=8), telmisartan plus TAC group (n=10 and n=8), and valsartan plus TAC group (n=9 and n=8). After 2 weeks of TAC, mice were subjected to catheterization via the right carotid artery into left ventricle to measure the left ventricular end-systolic pressure (LVESP). Including examination of myocardial fibrosis, four endpoint measurements were made to assess the phenotype of cardiac hypertrophy after 2 weeks of TAC: 1).Two-dimensional guided M-mode echocardiographic assessments were performed with 30MHz high frequency scanhead for the geometry and functions of hearts,2). Heart weight to body weight of ratio (HW/BW) was used to compare the weights of hearts,3). The expression of the genes which are associated with cardiac hypertrophy such as atrial natriuretic peptide (ANP), skeletal a-actin (SAA) and the sarcoplasmic reticulum Ca2+-ATPase (SERCA2) were determined by RT-PCR and confirmed further by qRT-PCR,4).Cardiac hypertrophy was confirmed further by histological analysis on the cross-sectional area (CSA) of cardiac myocytes and mid-transverse sectional area (MSA) in the left ventricle.Results:Only the mice with more than 155mmHg and less than 180mmHg were considered to be successfully TAC-operated and were used for statistic analysis, and the haemodynamics have no significant differences among all the TAC-operated groups. For wild-type mice, all the five ARBs can reverse cardiac hypertrophy and have the same efficacies. However, when angiotensinogen of the mice were knock-out, valsartan and telmisartan can't reverse cardiac hypertrophy, although olmesartan, candesartan and losartan can still.Conclusions:Unlike valsartan and telmisartan that depend on angiotension II, omlesartan, losartan and candesartan do not depend on angiotensin II to inhibit the pressure-overload-induced AT1 receptor activation and therefore act as inverse agonists.Part IIComparison of Olmesartan and Telmisartan to inhibit stress-induced Jak2 pathway in cardiomyocytes of neonatal ratsObjective:To compare the effects of olmesartan and telmisartan on Jak2 pathway induced by stress in cardiomyocytes of neonatal rats(RCM).Methods:To select the better small interfering RNA siRNA segment down-regulating the expression of ATG,4 pairs of chemically synthetical siRNA(siRNA-1, siRNA-2, siRNA-3, siRNA-4) were transfected into RCM isolated by different adherent time method by use of NeoFX, respectively. Negative group and positive group were designed as control. The effects of silence for ATG were determined by Real-time PCR. (2) RCMs were divided into two big groups:wild type group (no siRNA was transfectd), siRNA group (siRNA was transfected). The two big groups were divided into four sub-groups, respectively:control group (no stress), stress group (stress), group treated by olmisartan and group treated by telmisartan. The mRNA level of immediate-early-response genes of c-fos, c-jun and BNP were determined by Real-time PCR and the protein level of ATI,Gaq11, p-ERKs, Jak2, p-Jak2 were detected by western blotting.Results:(1) compared to positive group,4 pairs of chemically synthetical siRNA could down-regulating the expression of ATG, but siRNA-3 segment has the best effect of silence for ATG. (2) As for wild-type group, olmesartan and telmisartan can effectively inhibit stress-induced the expression of c-fos, c-jun and BNP. After mechanical stress, there was a significantly increase for level of Gaq11 in Cytosol of RCM, treatment with olmesartan and telmisartan both significantly inhibited the increment; Mechanical stretch induced association of Jak2 with the AT1 receptor in RCM.Pretreatment of the cells with olmesartan and telmisartan significantly suppressed association of Jak2 with the AT1 receptor; In addition, olmesartan and telmisartan can inhibit the activation of ERKs induced by mechanical stress. For siRNA group in which ATG was down-regulating, compared with telmisartan, olmesartan can significantly inhibited the increment of c-fos and c-jun, level of Gaq11 in cytosol, association of Jak2 with the AT1 receptor, andp-ERKs after stress.Conclusions:mechanical stress activated the AT1-JaK2-ERKs pathway independently of AngII and unlike telmisatan, olmesartan can block the pathway even in the absence of AngII.PartIIIActivation of CaMKII is crucially involved in the stretch-induced activation of AT1 receptorObjective:To explore the effects of CaMKII on the pathway of the activation of AT1 receptor induced by mechanical stress in vitro.Methods:(1) RCM cultured in silica gel dish were each treated with olmesartan and KN-93, the specifical inhibitor of CaMKII. After mechnical stress, p-CaMKII and p-ERKs were detected by WB. (1) HEK293AT1 cultured in silica gel dish randomly divided into four groups accordingly to the plasmids transfected:CaMKIIδB group, CaMKIIδC group, CaMKIIδN group and CaMKIIδM group. The four groups were divided into two groups:not-stress and stress, respectively. After stretching the cells by 20%for 10 min,p-ERKs of all the groups were detected by WB. Results:(1) mechanical stress can signicicantly increase the expression of p-CaMKII without AngII in RCM; Olmesartan inhibited the acivation of CaMKII induced by stress; KN-93 and olmesartan inhibited the acivation of ERKs through suppressing CaMKII and ATI receptor, respetively. (2) compared to corresponding to no-stress group,p-ERKs of the stress groups transfected into the plasmids of CaMKII8C, CaMKII8N, CaMKIIB were increased significantly, butp-ERKs of CaMKIIδM group have no change after stress.Conclusions:Mechanical stress activated the ATl-CaMKII-ERKs pathway without the involvement of AngII and the autoregulatory domain lacated at the middle of CaMKII may play an important role in the pathway. Conclusions1. Unlike valsartan and telmisartan that depend on angiotension II, omlesartan, losartan and candesartan do not depend on angiotensin II to inhibit the pressure-overload-induced AT1 receptor activation and therefore act as inverse agonists.2. Mechanical stress activated the AT1-JaK2-ERK pathway independently of AngII and unlike telmisartan, olmesartan can block the pathway even in the absence of AngII.3. Mechanical stress activated the AT1-CaMKII-ERKs pathway without the involvement of AngII and the autoregulatory domain located at the middle of CaMKII may play an important role in the pathway.Novelty and Value1. We can supply stable and repetitive TAC mice model for the drug screen that regressed cardiac hypertrophy.2. We found JaK2 and CaMKII play an important roles in signalling pathway of cardiac hypertrophy induced by pressure overload. So some active domains of JaK2 and CaMKII may be potential drug targeting for treatment of cardiac hypertrophy.3. We found efficacies and mechanisms of ARBs available in clinical use is different in reversal of pressure-overload cardiac hypertrophy in vivo and in vitro, which will be helpful to discriminate the ARBs according to the efficacies in AT1-R inactivation for the protection of heart.