Mechanisms Of Losartan Ameliorates β₁-adrenergic Signaling By Increasing Akt Activity And Attenuates Ventricular Remodeling And Cardiac Function In Aortic Banded Rats With Chronic Heart Failure

Congestive heart failure(CHF) is the end-stage and the common destination of coronary heart disease(CHD), hypertension, myocardial disease, valvular heart disease, and some other cardiovascular diseases, which is becoming the main risk for human health.As we have known that the basic pathology of CHF is myocardial remodeling. Myocardial remodeling was resulted from a series of complex molecular and cellular mechanisms of cardiomyocyte and myocardial interstitial tissue. The activition of Sympathetic nervous system(SNS) and endocrine system were the center of CHF, which was related to the progression of CHF. Theβ1-adrenergic receptor(β1-AR) was down-regulated, and out of coupling with Gs protein(desensitization) caused by the activetion of SNS in CHF. However, we still do not know whether and to what extent these alterations are adaptive/protective or detrimental, or both. Therefore, the regulation ofβ-adrenergic signaling undoubtedly improve the protection of cardiac function induced by ventricular remodeling, so that delayed the progress of CHF.At present, most study about CHF was concentrated in the regulation ofβ-adrenergic signaling withβ-receptor blockers, while the study of the regulation ofβ-adrenergic signaling with AT1 receptor antagonist(ARB) have not been reported. The clinical trials about the treatment of ARB in patients with CHF have been proved it could reduce the mortality and improve cardiac function and the quality of life.This paper aimed to study the regulation ofβ-adrenergic signaling and ventricular remodeling with losartan in chronic heart failure rats induced by aortic banded. And to discover the relationship with PI3K-Akt signaling pathway. Thereby to find the pharmacological mechanisms of ARB for treatment of CHF, and to provide the adequate therapeutic basis.PART I: Mechanisms of losartan attenuates ventricular remodeling and cardiac function in aortic banded rats with chronic heart failure Objective: To investigate the regulation of Losartan(Los) on ventricular remodeling and cardiac functionin chronic heart failure rats induced by aortic banded. Methods:1. Experimental group: Adult male Wistar rats were randomly divided coarctation of abdominal aorta(COA+Vehicle), COA+Los, sham-operated(Sham+Vehicle), and Sham+Los groups, followed by 8 weeks treatment with Losartan (20mg/kg per day, orally) or vehicle (drinking water).2. General condition: To observe the breathing, fur color, activity, body weight of rats in each group everyday, and record body weight weekly to observe the changes in body weight and make a survival analysis.3. Cardiac function: The changes of cardiac function in each group were evaluated by ultrasound cardiograph (UCG) and Powerlab system through the pressure transducers.4. Ventricular mass index:To weigh the mass of left and right ventricular accurately,and calculate left ventricular mass index (LVMI) and right ventricular mass index (RVMI).5. Myocardium angiotensin II (Ang II) and aldosterone (ALD) levels were measured by RIA.6. Myocardium tumor necrosis factor-α(TNF-α) level was measured by ELISA, and interleukin-6 (IL-6) level was measured by RIA.7. Myocardium hydroxyproline(HYP) level was measured by chromometry.8. Myocardium type I and type III collogen level and relative ratio were measured by ELISA.9. Histomorphology observation: Myocardium tissue HE staining; Myocardium Masson trichrome staining; Myocardium ultramicrostructure observation.Results:1. General condition: the condition of COA+Vehicle group was worsen day by day, but the condition of COA+Los group is better than that of COA+Vehicle group.2. UCG: Compared with COA+Vehicle group, morphosis and cardiac function in COA+Los group were obviously ameliorated [LVEDD: COA+Los: (6.74±0.52)mm vs COA+Vehicle: (7.52±0.81)mm, P<0.05; LVEF: COA+Los: (63.28±4.32)% vs COA+Vehicle: (43.27±4.25)%, P<0.05].3. Haemodynamic measurement: Compared with COA+Vehicle group, cardiac function in COA+Los group was obviously ameliorated [LVEDP:COA+Los:(11.47±5.06)mmHg vs COA +Vehicle:(21.18±6.56)mmHg, P<0.01].4. Ventricular mass index: Compared with COA+Vehicle group, LVMI in COA+Los group was obviously ameliorated [COA+Los: (2.41±0.15)mg/g vs COA+Vehicle: (3.12±0.18)mg/g, P<0.05].5. Myocardium Ang II and ALD: Compared with COA+Vehicle group, the levels of myocardium Ang II and ALD in COA+Los group were obviously decreased [Ang II: COA+ Los: (60.15±6.22)pg/mgprot vs COA+Vehicle: (92.31±8.31)pg/mgprot, P<0.05; ALD: COA+ Los: (58.15±4.96)ng/mgprot vs COA+Vehicle: (87.31±8.82)ng/mgprot, P<0.05].6. Myocardium inflammation cytokines: Compared with COA+Vehicle group, the levels of myocardium TNF-αand IL-6 in COA+Los group were obviously reduced [TNF-α: COA+ Los: (579.36±52.62)pg/gprot vs COA+Vehicle: (824.31±80.36)pg/gprot, P<0.05; IL-6: COA+Los: (49.12±4.06)pg/mgprot vs COA+Vehicle: (65.22±5.82) pg/mgprot, P<0.05].7. Myocardium HYP: Compared with COA+Vehicle group, the level of myocardium HYP in COA+Los group was obviously decreased[COA+ Los: (0.31±0.04)μg/mgprot vs COA+Vehicle: (0.52±0.07)μg/mgprot, P<0.05].8. Myocardium collogen: Compared with COA+Vehicle group, the contents of myocardium type I and type III collogen in COA+Los group were obviously ameliorated [type-I: COA+ Los: (67.25±5.49)ng/mgprot vs COA+Vehicle: (105.96±9.29)ng/mgprot, P<0.05; type-III: COA+ Los: (9.25±0.84)ng/mgprot vs COA+Vehicle: (10.87±0.93)ng/mgprot, P>0.05], and the ratio of I/III was also decreased[COA+Los:(7.27±0.71)% vs COA+Vehicle: (9.83±0.82)%, P<0.05].9. Histomorphology observation:9.1 Myocardium tissue HE staining: By the microscope observation, the myocardial tissue in COA+Vehicle group showed extensive hypertrophy with a large and deeply stained nuclear, the myocardial fibers were disordered, and some inflammatory cells infiltrated out of vessel. COA+ Los group diaplayed a reduced degree of hypertrophy, and the arrangement of myocardial fibers was improved. Sham group showed the myocardial fibers were arranged regularly, no obvious inflammation.9.2 Myocardium Masson trichrome staining: Masson trichrome stained sections showed less blue staining collagen deposition in COA+Los group vs COA+Vehicle group,and calculated the collagen volume fraction (CVF). Compared with COA+Vehicle group, CVF in COA+Los group was obviously decreased[COA+Los: (11.18±2.75)% vs COA+Vehicle: (18.53±4.12)%, P<0.05].9.3 Myocardium ultramicrostructure observation: Under the TEM, COA+Vehicle group showed the myofibrils were thicken and disarranged, the intercalated disk were confused and blurred, the number of mitochondria was obviously increased and appeared myelin-like change. The myocardial ultramicrostructure in COA+Los group has significantly improved. The myocardial myofibrils in Sham group present relatively well-distributed with a clearly visible intercalated discs and mitochondria.Conclusion: Los attenuated ventricular remodeling and improve cardiac function in aortic banded rats by blocking angiotensin II to bind AT1-R and reducing angiotensin II, aldosterone and inflammatory cytokine in myocardium. PART II: Losartan regulatesβ1-adrenergic signaling and cardiac function in aortic banded rats with chronic heart failure Objective: To investigate the regulation of Losartan(Los) onβ1-adrenergic receptor(β1-AR) signaling and cardiac function in chronic heart failure rats induced by aortic banded.Methods:1. Experimental group: Adult male Wistar rats were randomly divided coarctation of abdominal aorta(COA+Vehicle), COA+Los, sham-operated(Sham+Vehicle), and Sham+Los groups, followed by 8 weeks treatment with Losartan (20mg/kg per day, orally) or vehicle (drinking water).2. Cardiac function: The changes of cardiac function in each group were evaluated by ultrasound cardiograph (UCG) and Powerlab system through the pressure transducers.3. Observation the change of LVSP,±dp/dtmax and HR after intracardiac injection of 0.1, 1.0, 10, 100pg/g of Isoprenaline(ISO) by Powerlab system through the pressure transducers.4. Plasma norepinephrine(NE) level was measured by ELISA.5. The coexpression ofβ1-AR and p-Akt in myocardial cell by immunofluorescence cytochemistry(IFC).6. The mRNA expression ofβ1-AR,β-adrenergic receptor kinase-1(β-ARK1), PI3K-γand Gi protein were determined by reverse transcriptase polymerase chain reaction(RT-PCR).7. The protein expression ofβ1-AR/β-actin,p-Akt/t-Akt were assessed by Western blot.8. The distribution and expression ofβ1-AR in myocardium, liver and adrenal gland by immunohistochemistry(IHC).9. The activity of adenyl cyclase(AC) in myocardium was examined by stimulating of ISO and Forskolin with different concentration.Results:1. General condition: same to part I.2. UCG: same to part I.3. Haemodynamic measurement: 0.1, 1.0, 10, 100pg/g of ISO concentration-dependently improved the change of LVSP,±dp/dtmax and HR. Compared with Sham+Vehicle group, the change rate of these parameters in COA+Vehicle group was significantly depressed. But the change rate of these parameters in COA+Los group was significantly higher than that in COA+Vehicle group.4. Plasma NE: Compared with COA+Vehicle group, the level of plasma NE in COA+Los group was obviously decreased[COA+Los: (682.24±64.66)pg/ml vs COA+Vehicle: (908.24±75.10)pg/ml, P<0.05]. And plasma NE was negatively correlated with LVEF(r=-0.92, P<0.01) and CI(r=-0.73, P<0.05),but was positively correlated with LVEDP(r=0.76, P<0.05).5. IFC: The coexpression ofβ1-AR and p-Akt of myocardial cell in COA+Vehicle group was weaker than that in Sham+Vehicle group, but the coexpression in COA+ Los group was obviously increased.6. RT-PCR:Compared with Sham+Vehicle group, the mRNA expression ofβ1-AR in COA+Vehicle group was obviously decreased, but the mRNA expression ofβ-ARK1, PI3K-γand Gi protein were increased(P<0.05). While the mRNA expression ofβ1-AR in COA+Los group was obviously increased, and the mRNA expression of PI3K-γand Gi protein were decreased(P<0.05) besidesβ-ARK1(P>0.05).7. Western blot:Compared with Sham+Vehicle group, the protein expression ofβ1-AR and p-Akt in COA+Vehicle group were obviously decreased(P<0.05), but the protein expression ofβ1-AR and p-Akt in COA+Los group were obviously increased (P<0.05).8. IHC:The expression and distribution ofβ1-AR in myocardium, liver and adrenal gland of COA+Vehicle group were obviously reduced(P<0.05), but the expression ofβ1-AR in those tissues of COA+Los group were improved(P<0.05).9. The activity of AC: Myocardium cAMP could be increased by different concentrations of ISO and Forskolin. The net increase of cAMP in COA+Vehicle group was significantly lower than that in Sham+Vehicle group, but the activity of AC was significantly improved(P<0.01) Conclusion: Los could reduce plasma NE and effective regulateβ1-adrenergic signaling in chronic heart failure induced by aortic banded rats. Los could enhance the expression ofβ1-AR(up-regulate) and restore the coupling ofβ1-AR(resensitization) by inhibiting the expression of Gi protein and PI3K-γand then increased the activity of p-Akt, so that increase the activity of AC and the positive inotropic and chronotropic response. Los could improve cardiac function and delay the progress of CHF.