| There is emerging evidence that aldosterone is involved in thepathogenesis of cardiac hypertrophy and heart failure. The use of aldosteronereceptor antagonists for the treatment of myocardial failure and selected casesof hypertension, in combination with the current therapy, has been tested inrecent clinical trials. The Randomized Aldactone Evaluation Study (RALES)study has demonstrated that spironolactone, an aldosterone antagonist, addedto an angiotensin-converting enzyme (ACE) inhibitor in patients with severecongestive heart failure, reduces overall mortality. The EPHESUS study hasshown that eplerenone, added to ACEI or angiotensin AT1receptor blocker(ARB) decreases cardiovascular events and death in patients with leftventricular dysfunction following myocardial infarction. Therefore, clinicalpractice guidelines for heart failure management have designated aldosteronereceptor antagonists as first-line therapies for patients with heart failure andreduced left ventricular ejection fraction (LVEF). However, aldosteronereceptor antagonists carry an increased risk of complications, especiallyhyperkalemia may cause serious arrhythmic events. Accordingly, in clinicalpractice, the benefits of aldosterone receptor antagonists may be reduced andthey are used significantly less frequently compared with the otherguideline-recommended medications.In recent years, low dose of aldosterone receptor antagonists has beenused in increasing number of experimental studies. It is generally acceptedthat “low-dose” refers to a non-pressure-lowering dose, although the doses ofaldosterone receptor antagonists vary with different reports. It was found thatthe low-dose spironolactone (1mg/kg) had no effect on systolic blood pressurebut improved diastolic dysfunction comparable to a conventional dose (30mg/kg) in a transgenic rat model of RAAS over-expression. A subpressordose of eplerenone markedly ameliorated vascular endothelial dysfunction,cardiac inflammation and fibrosis in salt-sensitive hypertensive rats to asimilar degree as an antihypertensive dose of amlodipine. These resultssuggest that the low-dose of aldosterone receptor antagonists exert organprotective effect independent of hemodynamic changes. The clinical study hasshown that low-dose of spironolactone significantly reduce the risk ofhyperkalemia. Thus, we hypothesize that low doses of aldosterone antagonists,added to ACEI or ARB therapy potentiates the cardioprotective effects, andwith little potential of hyperkalemia.To verify our hypothesis, this study was designed to evaluate theeffectiveness of "low-dose" and conventional-dose aldosterone antagonistspironolactone in combination with ARB irbesartan on cardiac hypertrophicremodeling induced by abdominal aorta coarctation (PartialAbdominalAortic Constriction, PAAC) in rats. Plasma K level was measured to assesshyperkalemia risk. The effects of low-dose spironolactone in combinationwith irbesartan on myocardial oxidative stress were investigated in order tounderstand the pharmacological mechanisms underlying the potentiationeffects of the two-drug combination. To further explore the mechanism ofaldosterone induced myocardial hypertrophy, the possibility thatNF-E2-related factor (nuclear factor erythroid2-related factor2, Nrf2) wasinvolved in the regulation of cardiac hypertrophy induced by exogenousaldosterone was tested by using Nrf2knockout mice. The results will providesome new insights into the clinical application of aldosterone antagonists totreat cardiac hypertrophy and heart failure.Section1The effects of low-dose spironolactone added to irbesartan onmyocardial remodelling induced by pressure overload inratsObjective: To evaluate the effectiveness of "low-dose" andconventional-dose aldosterone antagonist spironolactone in combination withARB irbesartan on cardiac hypertrophic remodeling induced by PAAC in rats. Plasma K level was measured to assess hyperkalemia risk.Methods: Male SD rats were chosen, the rats were prepared the model ofmyocardial hypertrophy by PAAC. At4weeks after surgery, cardiachypertrophy was confirmed by echocardiographic measurements. Then, PAACrats were divided into4groups: model, irbesartan (Ir,15mg/kg daily),conventional dose of spironolactone (20mg/kg daily) added toirbesartan(CSp+Ir) and low dose spironolactone (1mg/kg daily) added toirbesartan(LSp+Ir). At the end of8weeks, Left ventricular mass index(LVW/BW) was assessed by morphology measurement. The thicknesses ofleft ventricle walls and cardiac function were measured by transthoracicechocardiography. Left ventricular pressure and function were assessed byhemodynamic examination. Cardiomyocyte hypertrophy and collagenaccumulation in cardiac tissue were measured by HE and Masson stainingrespectively. The mRNA expression of ANP, β-MHC, Procollagen I andTGF-β were measured with RT-PCR.Results:1Effects on cardiac hypertrophy:1) Echocardiography showed that allrats subjected to PAAC developed cardiac hypertrophy at4weeks aftersurgery. Left ventricular posterior wall thickness (LVPW), interventricularseptum thickness (IVs) were increased, while left ventricular end diastolicdimension (LVd) was decreased in PAAC compared with age-matched shamrats. Which indicated that PAAC in this experimental condition result in hearthypertrophy and the model is successful. There was no significant differenceamong the experimental groups before therapy.2) At the end of drugtreatment, body weight was similar among the experimental groups. The ratioof LV weight to body weight (LVW/BW), an index of LV hypertrophy, was34%greater in model rats than in sham rats, and the overload-induced increasein this parameter was not significantly reduced by monotherapy with Ir, but itwas comparably attenuated by low-dose and conventional Sp added to Ir.3)Echocardiography revealed that LVPW, IVs and LVd were greater in modelrats than in sham rats at8weeks. Monotherapy with Ir didn’t affect these parameters. Combination therapy led to significant decrease in theseparameters compared with monotherapy with Ir, whereas there was nodifference in treatment with low-dose and conventional Sp added to Ir.4)Histological analysis revealed that hemodynamic overload had increased thecross-sectional area of cardiac myocytes in model rats by45%compared withthat apparent in sham rats. The extent of load induced cardiomyocytehypertrophy was reduced by treatment with low-dose and conventional Spadded to Ir (p<0.01). Whereas there was no difference in treatment withlow-dose and conventional Sp added to Ir.5) Hemodynamic overload resultedin upregulation of the expression of ANP and β-MHC, molecular markers ofhypertrophy, in the heart of model rats, and this effect was inhibited bylow-dose and conventional Sp added to Ir. Whereas there was no difference intreatment with low-dose and conventional Sp added to Ir. The results indicatedthat low dose Sp added to Ir can reduce PAAC induced cardiac hypertrophy,furthermore, the effect of low dose Sp is comparable with conventional Sp.2Effects on cardiac fibrosis: Marked interstitial fibrosis was also detectedin the left ventricle of model rats as compared with matched Sham; thisincrease in cardiac fibrosis was also significantly reduced by low-dose andconventional Sp added to Ir. There was no difference in treatment withlow-dose and conventional Sp added to Ir. The rise in collagen type I andTGF-β1gene expression in the myocardium from model rats was attenuatedby low-dose and conventional Sp added to Ir. Whereas there was no differencein treatment with low-dose and conventional Sp added to Ir. The resultsindicated that low dose Sp added to Ir can reduce PAAC induced cardiacfibrosis, and the effect of low dose Sp is comparable with conventional Sp.3Effects on cardiac Function: Echocardiography revealed left ventricularejection fraction(LVEF), left ventricular fractional shortening (LVSF) werenot different among the experimental groups (p>0.05), which indicatedsystolic function remained unaltered. Haemodynamic values obtained at theend of the treatment period. PAAC increase systolic blood pressure(SBP) anddiastolic blood pressure(DBP). Ir alone had no significant effect on SBP and DBP (p>0.05), but SBP and DBP was significantly reduced by low-dose andconventional Sp added to Ir. There was diastolic dysfunction in model rats ascompared with Sham as illustrated by increased LVEDP and decreasedDP/dtmin/LVSP (each p <0.05), but dP/dtmaxwere not different from Sham,which indicated systolic function remained unaltered in CMC-PAACrats,which is consistent with the result of echocardiography. Both low-doseand conventional Sp added to Ir reduced LVEDP and increased DP/dt min/LVSP(each p <0.05). The results indicated that low dose Sp added to Ir can reducePAAC induced diastolic dysfunction, and the effect of low dose Sp iscomparable with conventional Sp.4Effects on the concentration of plasma K+and Na+: There was nodifference in the plasma concentration of Na+among all groups of rats.However, conventional dose of Sp added to Ir significantly elevated potassiumlevel in plasma, whereas, low-dose Sp did not affect the plasma K+concentration. The results indicated that low dose Sp added to Ir do not affectthe plasma K+concentration, which is was superior to conventional Sp.Conclusion: Low doses of aldosterone antagonists spironolactone,added to ARB therapy potentiates the cardioprotective effects, and with littlepotential of hyperkalemia.Section2The effects of combined low dose of spironolactone andirbesartan on myocardial oxidative stress induced bypressure overload in ratsObjective: To examine the effects of low dose of spironolactone,irbesartan and their combination on myocardial oxidative stress, and explorethe probable mechanism of the additive beneficial effects on cardiachypertrophy and diastolic dysfunction.Methods: The levels of malondialdehyde (MDA) were measured asindirect oxidative marker. NADPH oxidase activity of left ventricular tissuehomogenate was measured by lucigenin chemiluminescence. SOD activitywas evaluated by nitro-blue tetrazolium methold. Western blot was used toevaluate the protein expression of both NADPH oxidase subunits including NOX2，NOX4and SOD subunits including SOD-1and SOD-2.Results:1Effects on MDA level: We evaluate the level of oxidative stress bydeterminating malondialdehyde (Maleic Dialdehyde, MDA) content.Compared with the sham operation group, model group of myocardial MDAcontent increased significantly (p<0.01), which demonstrate thatPAAC induced myocardial oxidative stress injury. Ir monotherapy, but notlow dose Sp, significantly reduced myocardial MDA level (p<0.05). Thecombination of Ir and low dose Sp further reduce the myocardialMDA level (p<0.05). These results suggest that Ir can significantly improvethe oxidative stress in myocardium, but Ir and low dose spironolactonecombined with better results.2Effects on cardiac NADPH oxidase activity: NADPH oxidase is oneof the main enzyme to generating active oxygen in myocardialtissue, we observed the changes of NADPH oxidase activity in myocardium.Compared with the sham operation group, model group of myocardialNADPH oxidase activity was significantly enhanced (p<0.01).LSp group, Ir+LSp group, Ir decreased the activity of NADPH oxidase, butthere is significant difference only in Ir+LSp group(p<0.05). Compared withLSp group, combined treatment further reduced the activity of NADPHoxidase (p<0.05) in myocardium.3Effects on cardiac NADPH oxidase subunits expression: In order tofurther illustrate the change mechanism of NADPH oxidase activity, weused immunoblotting to examine myocardial NADPH oxidasesubunit NOX2, NOX4protein expression. PAAC significantly increased theexpression of myocardial NADPH oxidase isoforms of NOX4protein(p<0.01), but no significant changes in NOX2isoforms. Monotherapy with Iror low dose Sp tended to reduce the expression of NOX4protein, but thedifference did not reach statistical significance. However, the combination ofIr and low dose Sp significantly attenuated the increases in the expression ofNOX4protein (p<0.05). These results suggest a better inhibitory effect of Ir combined with low dose of Sp on the expression of NOX4protein.4Effects on cardiac SOD activity：Compared with the sham operationgroup, model group of myocardial SOD activity decreased significantly(p<0.05)，and it tended to be reduced by monotherapy with Ir or low dose Sp,but the difference did not reach statistical significance. However, thecombination of Ir and low dose Sp significantly attenuated the decreases inSOD activity (p<0.05).5Effects on cardiac SOD subunits expression: In order to explainthe mechanism of SOD change, we used immunoblotting to examinemyocardial SOD subtype: SOD-1, SOD-2. Compared with the shamoperation group, model group of myocardial SOD-2proteinexpression increased significantly (p<0.01), but SOD-1had noobvious change. Monotherapy with Ir or low dose Sp tended to increase theexpression of SOD-2protein, but the difference did not reach statisticalsignificance. However, the combination of Ir and low dose Sp significantlyattenuated the decreases in the expression of SOD-2.Conclusions:1Abdominal aortic constriction caused the increase of MDA content inrat myocardial tissue, enhanced NADPH oxidase activity and the expressionof subunit NOX4, and decreased the activity of SOD and the expression ofSOD-2subunit, suggesting that oxidative stress induced by NADPH oxidaseand SOD is involved in the process of myocardial hypertrophy and diastolicdysfunction in PAAC rats.2Low dose Sp combined with irbesartan can reduce PAAC inducedmyocardial oxidative stress injury, which may play a role by inhibiting theNADPH oxidase activity and enhancement of SOD activity.Section3Regulation effect of Nrf2transcription factor on aldosteroneinduced cardiac hypertrophy in miceObjective: To explore the role of Nrf2in regulation of myocardialhypertrophy induced by external aldosterone, further explorethe mechanism of aldosterone induced myocardial hypertrophy. Methods: The Nrf2-null mice backcrossed into a ICR background weregifted by academician Li Chunyan of Department of Internal Medicine,Second Affiliated Hospital of Hebei Medical University. The genotypesof the animal were identified from the DNA， mRNA and proteinlevel. Aldosterone-treated wild type(WT) and Nrf2-null mice were implantedwith osmotic mini-pumps for subcutaneous infusion for4wk.(1mg/kg/·day)for4wk. Heart mass index (HW/BW) was assessed by morphologymeasurement. The thicknesses of left ventricle walls and cardiac function weremeasured by transthoracic echocardiography. Blood aldosterone concentrationwere measured using [125I] aldosterone radioimmunoassay. Myocardialcells were digested by trypsin method, and loaded DCFH-DA dye to observethe level of ROS. Western blot was used to evaluate the protein expression ofNrf2. The mRNA expression of Nrf2, HO-1, NQO-1were measured withRT-PCR.Results:1Nrf2knockout mice identification: PCR and agarose electrophoresis oftail DNA show that amplified bands of Nrf2-null mice is in400bp position, and the WT mice is in700bp. we confirmed that the Nrf2ispresent in the WT mouse hearts The protein (Western blot) and RNAexpression (RT-PCR) analysis confirmed disruption of Nrf2in the KO mouseheart.2Effects on plasma aldosterone level: Plasma aldosterone concentrationwere significantly higher than corresponding control group (p<0.05),suggesting that osmotic pump with constant speed can release of aldosteroneinto mice, which significantly increased aldosterone level in the body.3Echocardiography: Echocardiography revealed left ventricular ejectionfraction(LVEF), left ventricular fractional shortening (LVSF) were notdifferent among the experimental groups (p>0.05), which indicated systolicfunction remained unaltered. The Nrf2-null in baseline have a norma lleftVentricular anterior wall thickness(LVID), left ventricular posterior wallthickness(LVPW), LVEF, LVFS. These results indicate that there is no evidence for cardiac dysfunction due to disruption of Nrf2. Aldosteroneinfusion led to an increase in LVID and LVPW, however, the aldosterone–induced increase in LVID and LVPW were exaggerated in Nrf2-null mice.These results suggest that Nrf2deficiency exaggerates aldosterone-induced thechange of echocardiography parameters.4Effects on HW/BW: Aldosterone infusion led to an increase in heartweight to body weight ratio (HW/BW), however, the aldosterone–inducedincrease in HW/BW was exaggerated in Nrf2-null mice. These resultssuggest that Nrf2deficiency exaggerates aldosterone-induced the change ofHW/BW.5Effects on expression of cardiac hypertrophy gene ANP, β-MHC:Aldosterone infusion led to an upregulation of cardiac hypertrophy geneANP, β–MHC. However, the aldosterone-induced molecular markers ofhypertrophy changes were exaggerated in Nrf2-null mice.6Effects on ROS level: In view of the anti-oxidative defence role of Nrf2in the heart, we investigated the role of Nrf2in regulating aldosterone-inducedmyocardial oxidative stress. Aldosterone infusion increased myocardial levelsof H2-DCFDA, a marker of ROS, indicating elevated levels of oxidative stressin the heart. The aldosterone-induced oxidative stress levels in the heart weremarkedly enhanced as a result of Nrf2deficiency.7Effects on expression of Nrf2and antioxidative enzymes, HO-1、NQO-1: The chronic aldosterone infusion-enhanced the mRNA and proteinexpression Nrf2in WT myocardium. In response to the sustained aldosteronestimulation, several Nrf2downstream genes, including HO-1, NQO-1, in WTbut not in Nrf2-null hearts were up-regulated at mRNA level.Conclusion: Our present study demonstrates that Nrf2is a criticalregulator for maintaining structural and functional integrity of the heart in thesetting of sustained aldosterone stimulation. Mechanistically, itis likely thatNrf2protects against maladaptive cardiac remodelling and dysfunction byrepressing ROS production and up-regulating antioxidative enzymes. |
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