| Cardiac hypertrophy is an independent risk factor for cardiovascular morbidity and mortality in patients with hypertension. Cardiac hypertrophy initially occurs as a compensatory response to pressure overload, but gradually leads to inadequate remodeling, cardiac diastolic and systolic dysfunction, impaired coronary preservation, cardiac arrhythmia, dysregulated cardiac sympathetic nervous activity, and finally induces congestive heart failure and sudden death. Therefore, investigation of novel treatments to prevent and reverse cardiac hypertrophy is an area of intense activity in treatment of hypertension.The main pathologic characteristics of left ventricular hypertrophy (LVH) include cardiomyocyte hypertrophy, cardiacfibroblast proliferation, interstitial fibrosis and intramyocardial small coronary artery remodeling. Several pathogenic factors was proved to be related with LVH such as pressure overload, activated Renin-angiotensin -aldesterone system (RAS), oxygen stress, inflammatory cytokine upregulation, transforming growth factors (TGF-β1), endothelin (ET-1), atrial natriuretic peptide (ANP) and so on. In addition, as members of tumor necrosis factor super family, RANKL/RANK/OPG system was reported to be a multi-functional cell cytokine system, not only to be involved in the formation of bone but also closely related with the development of embryonic heart, post-infarcted ventricular remodeling and immune-inflammatory cardiomyopathy.The regression of left ventricular hypertrophy (LVH) by antihypertensive treatment is associated with improvement in the prognosis of patients with hypertension. However, previous studies have shown that lowering of blood pressure is an important, but not the sole factor for the reversal of cardiac hypertrophy. Recently, accumulating experimental and clinical evidence has indicated that inhibition of oxidative stress (excessive production of ROS) and inflammatory response may delay the progression of cardiac hypertrophy and heart failure. In addition to its antihypertensive activity, amlodipine, a long-acting dihydropyridine calcium channel blocker (CCB), has been shown to exert a favorable effect on regression of cardiac hypertrophy, via inhibition of oxidative stress. Statins, HMG-COA reductase inhibitors, are potent inhibitors of cholesterol biosynthesis, and exert direct antioxidative and anti-inflammatory effects and improve vascular endothelial function, which may result in significant prevention and treatment of cardiovascular disease (CVD), independently of their lipid-lowering effect. A growing body of evidence supports the notion that statins might prevent cardiac hypertrophy and the development of heart failure, through pleiotropic effects.Accumulating evidence has shown that co-administration of amlodipine and statins has additive beneficial effects on inhibition of atherosclerosis and preservation of NO bioavailability, through inhibition of oxidative stress and proinflammatory cytokine expression. Moreover, in the ASCOT-LLA study, low-dose atorvastatin (10 mg/day) was shown to reduce significantly stroke and some cardiac end points when added to amlodipine in patients with hypertension and average cholesterol levels. These results suggest a possible synergetic or additive beneficial effect of combined amlodipine and atorvastatin on cardio vascular disease (CVD). The beneficial effects on vascular protection have been investigated extensively. However, to the best of our knowledge, whether co-administration of amlodipine and atorvastatin has an additive beneficial effect on advanced cardiac hypertrophy, and the underlying pharmacological mechanisms, remain poorly understood.Spontaneously hypertensive rats (SHR) have been used frequently as an ideal model of genetic hypertension and hypertensive heart disease, which develops heart failure with aging, similar to that in humans. SHR with heart failure mimic hypertension-induced cardiac remodeling and heart failure in humans, caused by cardiac and hemodynamic as well as neurohormonal abnormalities during the transition from compensated cardiac hypertrophy to heart failure. In general, SHR usually has elevated blood pressure at the age of 6~8 week, presented with compensatory cardiac hypertrophy at 16~48 week old and cardiac diastolic dysfunction at 36 week old roughly, and finally accompanied with decompensated cardiac hypertrophy, obvious ventricular dilation, diminished cardiac systolic and diastolic function at the age of 70 week.In the present study, we used 36-week-old SHR as a model of hypertensive cardiac hypertrophy complicated with early-stage diastolic dysfunction, to examine the effects of amlodipine and atorvastatin alone and combination of both drugs on advanced cardiac hypertrophy, cardiac diastolic dysfunction, myocardial oxidative stress, regulation of inflammatory cytokine, balance of MMPs/TIMPs system, and expression of RANKL/RANK/OPG system. Thereby, the purpose of the presented study was to investigate the following: whether addition of atorvastatin exerts further beneficial effects on advanced cardiac hypertrophy and diastolic dysfunction compared with amlodipine monotherapy; the possible underlying pharmacologic mechanism of the additive beneficial effect of combination therapy; and proposing new idea and experimental evidence for combination therapy on hypertensive cardiac hypertrophy.Section 1. The effects of combined amlodipine and atorvastatin on cardiac hypertrophy in SHRObjective: To examine the effects of amlodipine, atorvastatin, and their combination on cardiac hypertrophy, cardiac function, circulatory and myocardial ANG II level in SHR, and explore whether combination therapy has synergestic effects on hypertensive cardiac hypertrophy and improvement of cardiac function.Methods: male Wistar-Kyoto (WKY) rats were chosen as WKY control group with normal pressure, male SHR rats were divided into four groups including vehicle-treated SHR, amlodipine-treated SHR (10 mg/kg/d), atorvastatin-treated SHR (10 mg/kg/d), combination of amlodipine and atorvastatin-treated SHR (both 10 mg/kg/d). Drugs were administered by oral gavage over 12 weeks. Left ventricular mass index (LVMI) was assessed by morphology measurement. The thicknesses of left ventricle walls, left ventricle weight (LVW) and cardiac function were measured by transthoracic echocardiography. Left ventricular pressure and function were assessed by hemodynamic examination. Plasma brain natriuretic peptide (BNP) was measured by ELISA assay. Cardiomyocyte hypertrophy and collagen accumulation in cardiac tissue were measured by HE and Masson staining respectively. The hydroxyproline content of cardiac tissue was examined by biochemistry technique. The mRNA expression of ANP,β-MHC, Procollagen I and TGF-βwere measured with RT-PCR. Plasma and myocardial angiotensin II level were analyzed by radio-immune assay.Results:1 Effects on blood pressure, body weight, heart rate and serum lipid level:Amlodipine alone or combined with atorvastatin decreased SBP significantly to a similar degree (P < 0.05), whereas the same dose of atorvastatin alone had little effect on SBP during 12 weeks treatment in SHR (P > 0.05). Twelve weeks treatment with amlodipine, atorvastatin or their combination did not significantly affect body weight, heart rate or serum lipid level of SHR (P > 0.05).2 Effects on cardiac hypertrophy:In contrast to WKY, SHR were presented with increased LVMI, increased plasma BNP level, as well as cardiomyocyte cross-sectional area and interstitial fibrosis in SHR (each P < 0.05). Treatment with amlodipine or atorvastatin alone significantly decreased LVMI, plasma BNP level, as well as cardiomyocyte cross-sectional area and interstitial fibrosis in SHR (each P < 0.05). Moreover, combined amlodipine and atorvastatin treatment induced significant reversal of LVH and attenuation of plasma BNP level, as well as decreased cardiomyocyte cross-sectional area and interstitial fibrosis in SHR to a greater extent than each agent alone (each P < 0.05).3 Effects on expression of cardiac hypertrophic and profibrotic gene markers:Increased expressions of the LV ANP,β-MHC, collagen I and TGF-β1 mRNA were observed in 48 weeks SHR compared with WKY with same age (each P < 0.05). Amlodipine or atorvastatin alone significantly attenuated the increase of LV ANP,β-MHC, pro-collagen I and TGF-β1 mRNA expression in SHR (each P < 0.05), and combination therapy suppressed the expression even further (each P < 0.05).4 Echocardiography:Echocardiographic examination showed that LVW were higher in 48-week-old SHR than that in WKY with same age (P < 0.05). There was diastolic dysfunction in SHR as compared with WKY as illustrated by prolonged IVRT (P < 0.05), but LV end-diastolic dimension as well as LVEF and LVSF were not different from WKY (P > 0.05), Which indicated systolic function remained unaltered in 48-week-old SHR. Treatment with amlodipine or atorvastatin alone tended to decrease IVRT in SHR, but only combination therapy significantly attenuated IVRT (P < 0.05).5 Hemodynamic examinations:There was diastolic dysfunction in SHR as compared with WKY as illustrated by increased LVEDP, prolongedτ, and decreased dP/dtmin/LVSP (each P < 0.05), but dP/dtmax were not different from WKY, Which indicated systolic function remained unaltered in 48-week-old SHR. Both amlodipine and atorvastatin reduced LVEDP andτ, increased dP/dtmin/LVSP (each P < 0.05). Furthermore, in contrast to amlodipine monotherapy, the combination therapy further reduced LVEDP,τand increased dP/dtmin/LVSP in SHR (each P < 0.05).6 Effects on plasma and myocardial Ang II level:There was no difference in plasma Ang II level between SHR and WKY (P > 0.05), however, myocardial Ang II level was obviously higher in SHR than that in WKY (P < 0.05). After 12-week drug administration, amlodipine increased plasma Ang II level (P < 0.05) but atorvastatin and combination therapy did not alter plasma Ang II level in SHR (P > 0.05). This outcome indicated that atorvastatin inhibited the elevation of plasma angiotensin II level induced by amlodipine. Myocardial AngII concentration of amlodipine group is similar to SHR control group, however, Myocardial AngII concentration of atorvastatin and combination therapy group were lower than that of SHR control group (both P < 0.05).Conclusions: Combined amlodipine and atorvastatin treatment had synergistic effects on improvement of hypertensive cardiac hypertrophy and diastolic dysfunction, which was related with decreased myocardial angiotensin II level.Section 2. The effects of combined amlodipine and atorvastatin on myocardial oxidative stress in SHRObjective: To examine the effects of amlodipine, atorvastatin and their combination on myocardial oxidative stress, and explore the probable mechanism of the additive beneficial effects on hypertensive cardiac hypertrophy and diastolic dysfunction.Methods: The serum lipid level was measured by biochemical assay. The concentration of serum oxLDL was detected with ELISA methold. Cardiac oxidative stresses were detected using two approaches. The oxidative fluorescence dye dihydroethidium (DHE) was used to evaluate in situ ROS generation in the LV; the levels of malondialdehyde (MDA) were measured as indirect oxidative marker. NADPH oxidase activity of LV tissue homogenate was measured by lucigenin chemiluminescence. Cu/Zn SOD activity was evaluated by xanthine oxidase methold. Western blot was used to evaluate the protein expression of both NADPH oxidase subunits including p22phox, p40phox, p47phox, Rac-1 and Cu/Zn SOD.Results:1 The levels of serum lipid and oxLDL: There are no differences of lipid levels between WKY and SHR (P > 0.05). Serum oxLDL concentration of SHR control group was significantly higher than that of the control group (P < 0.05). Serum oxLDL concentration both in amlodipine and atorvastatin administration group were significantly dropped compared with that of same aged WKY rats ( each P < 0.05), and those in the combined drug group were even dropped down further than that of the single drug groups( each P < 0.05).2 Effects on cardiac superoxide and MDA level: the intensity of DHE staining and cardiac MDA level of 48-week-old SHR was significantly enhanced compared with WKY (each P < 0.05). Both amlodipine and atorvastatin reduced intensity of DHE staining and LV MDA level in SHR to a similar degree (each P < 0.05). Furthermore, combination treatment decreased cardiac intensity of DHE staining and MDA level of SHR to a greater extent compared with amlodipine alone (each P < 0.05).3 Effects on cardiac NADPH oxidase and Cu/Zn SOD activity: LV NADPH oxidase activity was significantly higher in SHR control group compared with that in WKY group (P < 0.05). Amlodipine or atorvastatin monotherapy significantly reduced LV NADPH oxidase activity in SHR (each P < 0.05), and activity was further decreased by combination therapy (each P < 0.05). Conversely, Cu/Zn SOD activity was significantly lower in the vehicle SHR group than that in the WKY group (P < 0.05). Amlodipine and atorvastatin, alone and in combination, only slightly but not significantly enhanced Cu/Zn SOD activity (P > 0.05).4 Effects on cardiac NADPH oxidase subunits and Cu/Zn SOD expression: p22phox, p40phox, p47phox and Rac-1 levels in membrane fraction of cardiac tissues in SHR were significantly higher than those in WKY (each P < 0.05). p22phox, p40phox, p47phox and Rac-1 levels in membrane fraction from cardiac tissues were significantly reduced by amlodipine or atorvastatin (each P < 0.05). Combination therapy reduced p22phox, p47phox, and Rac-1 levels rather than p40phox further compared with amlodipine monotherapy (each P < 0.05). Cu/Zn SOD levels in SHR were significantly higher than that in WKY (P < 0.05). Cu/Zn SOD levels in SHR were not altered by amlodipine, atorvastatin, or their combination (each P > 0.05).Conclusion: Serum oxLDL concentration, myocardial DHE intensity and MDA level were significantly higher in SHR than those in WKY. These findings suggested that oxidative stress may take part in genesis and development of hypertensive cardiac hypertrophy and cardiac diastolic dysfunction. Inhibition of cardiac hypertrophy and preservation of cardiac function by amlodipine and atorvastatin might be attributed to their inhibition of NADPH oxidase subunits expression and enzyme activity. The addition of atorvastatin to amlodipine achieved more benefits as compared with each monotherapy on advanced cardiac hypertrophy and early diastolic dysfunction. These additive benefits were likely to be related with prominent attenuation on NADPH oxidase-mediated ROS generation in left ventricle from SHR.Section 3. The effects of combined amlodipine and atorvastatin on myocardial proinflammatory cytokine in SHRObjective: To explore whether activated proinflammatory cytokine network may play some role in the additive beneficial effects of amlodipine, atorvastatin, and their combination on cardiac hypertrophy and cardiac diastolic dysfunction, through examining the effects of each drug and co-administration therapy on circulatory levels of hs-CRP, TNF-α, IL-1β, and the protein expressions of cardiac TNF-α, IL-1β, NF-κBp65, IκBαin SHR.Methods: The levels of serum hs-CRP were detected with ELISA. The serums TNF-α, IL-1βwere detected by radioimmunity assay (RIA). Cardiac inflammatory cell infiltration was observed by HE staining. The protein expressions of TNF-α, IL-1β, subunit P65 of NF-κB and IκBαwere detected with Western blot technique. The translocation of NF-κB p65 from cytoplasm to nucleus was observed using immunnohistochemistry.Results:1 The effects on cardiac inflammatory cell infiltration by amlodipine, atorvastatin and combination therapy: cardiac inflammatory cell infiltration in SHR was higher than that in WKY. Both amlodipine and atorvastatin inhibit local inflammatory cell infiltration in cardiac tissue, and combination therapy inhibits further compared with each monotherapy.2 The effects on serum concentration of hs-CRP, TNF-α, IL-1βby amlidipine, atorvastatin, and their combination: the serum levels of hs-CRP, TNF-α, IL-1βwere significantly higher in SHR control group than that of WKY control group (each P < 0.05). The serum levels of hs-CRP and TNF-αin the amlodipine or atorvastatin administration group were significantly dropped compared with SHR control group (each P< 0.05), and those in the combined drug group were even dropped down further than that of the single drug groups (P < 0.05). Both amlodipine and atorvastatin slightly but not significantly decreased serum IL-1βconcentration (each P > 0.05), and combination therapy significantly decreased serum IL-1βconcentration compared with SHR control group (P < 0.05). 3 The effects on protein expression of TNF-α, IL-1βby amlodipine, atorvastatin, and their combination: Compared with that of same aged WKY, the protein expressions of TNF-α, IL-1βwere proved to be enhanced in SHR control group by western blot (P < 0.05). Furthermore, all the proteins were reduced markedly after amlodipine or atorvastatin intervention (each P < 0.05), and that of the combined drug group was even lower than that of the single drug groups (each P < 0.05).4 The effects on protein expression of NF-κB p65 by amlodipine, atorvastatin, and their combination:Western Blot: Compared with that of same aged WKY, the protein expressions of NF-κB p65 were significantly enhanced in SHR control group (each P < 0.05). Furthermore, the protein expressions of NF-κB p65 were reduced markedly after amlodipine or atorvastatin intervention (each P < 0.05), and that of the combined drug group was even lower than that of the single drug groups (P < 0.05).Immunohistochemistry: The expression of NF-κB p65 was weaker within the nucleus of cardiocmyocyte and cardiac fibroblast cells in WKY group, whereas was significantly enhanced in SHR control group, that of the amlodipine and atorvastatin were obviously reduced compared with that of SHR control group, and that in the combined drug group was even lower than that of the single drug groups.5 The effects on protein expression of IκB-αby amlodipine, atorvastatin, and their combination: Western Blot: The protein expression of IκB-αin SHR group was obviously reduced (P < 0.05), whereas that in amlodipine and atorvastatin groups were only slightly enhanced compared with that in SHR group (each P > 0.05), and that in the combined drug group was significantly higher than that in the SHR group (P < 0.05).Immunohistochemistry: just as that of Western Blot.Conclusions: By way of down regulation for myocardial TNF-α, IL-1β, NF-κBp65 as well as up regulation for IκB-α, amlodipine and atorvastatin may obviously reverse cardiac hypertrophy and improve cardiac diastolic dysfunction. Amlodipine combined with atorvastatin may have additive effect on inhibiting of inflammatory response.Section 4. The effects of combined amlodipine and atorvastatin on the balance of myocardial MMPs/TIMPs system in SHRObjective: To explore the effect of amlodipine, atorvastatin, and their combination on the balance of myocardial MMPs/TIMPs system, by investigating the changes of MMP-2, MMP-9 as well as TIMP-1, TIMP-2 expression in SHR.Methods: Geltin zemography was used to evaluate the activity of MMP-2 and MMP-9. The protein expressions of MMP-2, MMP-9 as well as TIMP-1, TIMP-2 were detected with Western blot. RT-PCR was used to observe the gene expression of MMP-2, MMP-9 as well as TIMP-1, TIMP-2.Results:1 The effects on activity of MMP-2, MMP-9 by amlodipine, atorvastatin and their combination:Gelatin zemography: The activity of MMP-2, MMP-9 was higher in SHR than that in WKY (each P < 0.05). Either amlodipine or atorvastatin lowered the activity of MMP-2 and MMP-9 of SHR (each P < 0.05); furthermore, combination therapy had the best lowering effect (P < 0.05).2 The effects on protein and mRNA expression of MMP-2, MMP-9 by amlodipine, atorvastatin and their combination:Western Blot and RT-PCR: The protein and mRNA expression levels of MMP-2 and MMP-9 in SHR were all obviously increased in contrast to that in WKY (each P < 0.05). The protein and mRNA expression levels of MMP-2 and MMP-9 in the amlodipine or atorvastatin administration groups were obviously reduced compared with that in SHR group (each P < 0.05). The protein and mRNA expression levels of MMP-2 and MMP-9 in the combined amlodipine and atorvastatin group were even lower than that in the single amlodipine or atorvastatin groups (each P < 0.05).3 The effects on protein and mRNA expression of TIMP-1 by amlodipine, atorvastatin and their combination:Western Blot and RT-PCR: The protein and mRNA expression levels of TIMP-1 in SHR were obviously increased in contrast to that of WKY (each P < 0.05). Nevertheless, either amlodipine or atorvastatin as well as combination therapy did not alter myocardial TIMP-1 levels in SHR (each P > 0.05).4 The effects on protein and mRNA expressio of TIMP-2 by amlodipine, atorvastatin and their combination:Western Blot and RT-PCR: The protein and mRNA expression levels of TIMP-2 in SHR were not different from that in WKY (each P > 0.05). Either amlodipine or atorvastatin as well as combination therapy did not alter myocardial TIMP-2 levels in SHR (each P > 0.05).Conclusion: Both amlodipine and atorvastatin decreased the activity, protein and mRNA expression levels of MMP-2, MMP-9 in SHR, and combination therapy had the best lowering effects. Thereby, by way of maintaining the balance of MMP-2/TIMP-2 and MMP-9/TIMP-1, amlodipine and atorvastatin may obviously relieve interstitial fibrosis.Section 5. The role of RANKL/RANK/OPG system in cardiac hypertrophy and the effects of combined amlodipine and atorvastatinObjective: To explore the role of RANKL/RANK/OPG system in cardiac hypertrophy, by investigating the changes of RANKL, RANK, and OPG expression in SHR and WKY. Furthermore, to elucidate the effects of amlodipine, atorvastatin, and their combination on the balance of myocardial RANKL/RANK/OPG system.Methods: The immunohistochemistry was used to evaluate the protein expression and tissue localization of RANKL, RANK, and OPG. The protein expressions of RANKL, RANK, and OPG were detected with Western blot. RT-PCR was used to observe the mRNA expression of RANKL, RANK, and OPG.Results:1 The effects on protein and mRNA expression of RANKL by amlodipine, atorvastatin and their combination:Immunohistochemistry: The small amounts of positive expressions of RANKL were present in cardiomyocytes, cardiac fibroblasts, and vascular endothelial as well as smooth mucle cells in WKY. The positive expressions of RANKL in SHR were even darker and larger in size compared with that in WKY in view of their optic density (P < 0.05). In addition, monocytes adhered to coronary artery wall and some inflammatory cells infiltrated in the interstitial area were also stained with RANKL expression. Both amlodipine and atorvastatin decreased the average integrated optic density obviously compared with that of SHR control group (each P < 0.05), and combination therapy reduced it further (each P < 0.05).Western Blot and RT-PCR: The protein and mRNA expression levels of RANKL in SHR control group were all obviously increased in contrast to that in WKY (each P < 0.05). The protein and mRNA expression levels of RANKL in either amlodipine or atorvastatin alone groups were obviously reduced compared with that in SHR control group (each P < 0.05), and that of the combined amlodipine and atorvastatin group was even lower than that of the amlodipine or atorvastatin alone groups (each P < 0.05).2 The effects on protein and mRNA expression of RANK by amlodipine, atorvastatin and their combination:Immunohistochemistry: The small amounts of positive expressions of RANKL were present in cardiomyocytes, cardiac fibroblasts, and vascular endothelial as well as smooth mucle cells in WKY. The positive expressions of RANKL in SHR were even darker and larger in size compared with that in WKY in view of their optic density (P < 0.05). Both amlodipine and atorvastatin decreased the average integrated optic density obviously compared with that of SHR control group (each P < 0.05), and combination therapy reduced it further (each P < 0.05).Western Blot and RT-PCR: The protein and mRNA expression levels of RANK in SHR control group were all obviously increased in contrast to that of WKY (each P < 0.05). The protein and mRNA expression levels of RANK in either amlodipine or atorvastatin alone groups were obviously reduced compared with that in SHR control group (each P < 0.05), and that in the combined amlodipine and atorvastatin group was even lower than that of atorvastatin alone group (each P < 0.05).3 The effects on protein and mRNA expression of OPG by amlodipine, atorvastatin and their combination:Immunohistochemistry: The positive expressions of OPG were present in cardiomyocytes, cardiac fibroblasts but not in vascular endothelial and smooth mucle cells in WKY. The positive expressions of OPG in SHR were even darker and larger in size compared with that in WKY in view of their optic density (P < 0.05). Amlodipine and atorvastatin as well as combination therapy decreased the average integrated optic density obviously compared with that of SHR control group (each P < 0.05), but there was no difference among three different treatment (P > 0.05).Western Blot and RT-PCR: The protein and mRNA expression levels of OPG in SHR control group were all obviously increased in contrast to that of WKY (each P < 0.05). The protein and mRNA expression levels of OPG in either amlodipine or atorvastatin alone groups were obviously reduced compared with that in SHR control group (each P < 0.05), but there was no difference among three different treatment (P > 0.05).Conclusion: Both mRNA and protein expression RANKL, RANK, and OPG in SHR were significantly enhanced compared with WKY, suggesting that the pathologic changes of ventricular remodeling may be associated with the activiation of RANKL/RANK/OPG system. Both amlodipine and atorvastatin inhibit the activation of RANKL/ RANK/OPG system in myocardium and combination therapy inhibits it to a further degree. |
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