International Journal of Cardiology 222 (2016) 569–575 Contents lists available at ScienceDirect International Journal of Cardiology j ourna l homepage: www.e lsev ie r .com/ locate / i j ca rd Effects of early aldosterone antagonism on cardiac remodeling in rats with aortic stenosis-induced pressure overload M.P. Okoshi a,⁎, M.D.M. Cezar b, R.M. Iyomasa a, M.B. Silva a, L.C.O. Costa a, P.F. Martinez c, D.H.S. Campos a, R.L. Damatto a,b, M.F. Minicucci a, A.C. Cicogna a, K. Okoshi a a Department of Internal Medicine, Botucatu Medical School, Sao Paulo State University, UNESP, Brazil b Itapeva Social and Agrarian Sciences College, FAIT, Itapeva, SP, Brazil c Federal University of Mato Grosso do Sul, Campo Grande, Brazil ⁎ Corresponding author at: Departamento de Clinica Me Botucatu, UNESP Rubiao Junior, S/N 18618-970, Botucatu, E-mail address: mpoliti@fmb.unesp.br (M.P. Okoshi). http://dx.doi.org/10.1016/j.ijcard.2016.07.266 0167-5273/© 2016 Elsevier Ireland Ltd. All rights reserved a b s t r a c t a r t i c l e i n f o Article history: Received 20 April 2016 Received in revised form 29 July 2016 Accepted 30 July 2016 Available online 1 August 2016 Aldosterone plays a pivotal role in the pathophysiology of systolic heart failure. However, whether early aldoste- rone antagonism improves cardiac remodeling during persistent pressure overload is unsettled. We evaluated the effects of aldosterone antagonist spironolactone on cardiac remodeling in rats with ascending aortic stenosis (AS). Methods: Three days after inducing AS, weaning rats were randomized to receive spironolactone (AS-SPR, 20 mg/kg/day) or no drug (AS) for 18 weeks, and compared with sham-operated rats. Myocardial function was studied in isolated left ventricular (LV) papillary muscles. Statistical analyses: ANOVA or Kruskal–Wallis tests. Results: Echocardiogram showed that LV diastolic (Sham 8.73 ± 0.57; AS 8.30 ± 1.10; AS-SPR 9.19 ± 1.15 mm) and systolic (Sham 4.57 ± 0.67; AS 3.61 ± 1.49; AS-SPR 4.62 ± 1.48 mm) diameters, left atrial diameter (Sham 5.80±0.44; AS 7.15±1.22; AS-SPR 8.02±1.17mm), and LVmasswere higher inAS-SPR thanAS. Posteriorwall shortening velocity (Sham 38.5 ± 3.8; AS 35.6 ± 5.6; AS-SPR 31.1 ± 3.8 mm/s) was lower in AS-SPR than Sham and AS; E/A ratio was higher in AS-SPR than Sham. Developed tension was lower in AS and AS-SPR than Sham. Time to peak tension was higher in AS-SPR than Sham and AS after post-rest contraction. Right ventricle weight was higher in AS-SPR than AS, suggesting more severe heart failure in AS-SPR than AS. Interstitial collagen frac- tional area andmyocardial hydroxyproline concentrationwere higher in AS than Sham.Metalloproteinase-2 and -9 activity, evaluated by zymography, did not differ between groups. Conclusion: Early spironolactone administration causes further hypertrophy in cardiac chambers, and left ventric- ular dilation and dysfunction in rats with AS-induced chronic pressure overload. © 2016 Elsevier Ireland Ltd. All rights reserved. Keywords: Spironolactone Myocardial fibrosis Cardiac hypertrophy Ventricular function Papillary muscle 1. Introduction Aldosterone is a mineralocorticoid hormone involved in renal so- dium and potassium homeostasis and blood pressure modulation. In- creased systemic and myocardial concentrations of aldosterone are associated with deleterious cardiac effects. Experimental studies have shown that aldosterone directly stimulates myocyte growth and strongly induces myocardial fibrosis [1,2]. The harmful effects of aldo- sterone on the cardiovascular system also include myocyte apoptosis, myocardial oxidative stress and electrical remodeling, vascular injury, endothelial dysfunction, ventricular arrhythmia, and sudden death [3, 4]. After the pioneer studies by Pitt et al. [5–7], aldosterone antagonists started to be evaluated in different cardiac aggression models, which dica, Faculdade deMedicina de SP, Brazil. . showed that they prevent or attenuate left ventricular (LV) structural, functional, and molecular changes [8–11]. In clinical settings, aldoste- rone blockers reduced mortality and hospitalizations in patients with systolic heart failure of any cause [5,7], and in patientswith systolic dys- function aftermyocardial infarction [6]. Therefore, aldosteroneblockade is now recommended for symptomatic systolic heart failure patients [12]. In contrast, recent studies have shown that aldosterone blockade failed to reduce cardiovascularmorbimortality [13] or improvemaximal exercise capacity, symptoms, or quality of life in heart failure patients with preserved ejection fraction [14]. The mechanisms responsible for the lack of response to aldosterone blockers in this situation are not clear. Chronic pressure overload is a major cause of heart failure with pre- served or reduced ejection fraction. During sustained pressure overload, cardiac remodeling is mainly characterized by myocyte hypertrophy and interstitial fibrosis [15,16]. Stable cardiac hypertrophy is usually maintained for a long period and may progress to a decompensated http://crossmark.crossref.org/dialog/?doi=10.1016/j.ijcard.2016.07.266&domain=pdf http://dx.doi.org/10.1016/j.ijcard.2016.07.266 mailto:mpoliti@fmb.unesp.br Journal logo http://dx.doi.org/10.1016/j.ijcard.2016.07.266 Unlabelled image http://www.sciencedirect.com/science/journal/01675273 www.elsevier.com/locate/ijcard 570 M.P. Okoshi et al. / International Journal of Cardiology 222 (2016) 569–575 state with left ventricular dilation and systolic dysfunction [17,18]. The role of aldosterone antagonismon the development of cardiac hypertro- phy during persistent pressure overload is not clear. In spontaneously hypertensive rats with LV hypertrophy and no heart failure features, al- dosterone antagonist spironolactone reduced the frequency of heart failure development, improved myocardial function, and attenuated myocardial fibrosis independent of blood pressure levels [8]. In this study we tested the hypothesis that early aldosterone blockade im- proves cardiac remodeling during chronic pressure overload. Therefore, we evaluated the effects of spironolactone on cardiac structures, ven- tricular and myocardial function, and myocardial fibrosis in rats when administration was initiated three days after ascending aortic stenosis induction. In thismodel, 3–4week-old rats are subjected to a clip place- ment around the ascending aorta [19]. After clip placement, aorta diam- eter is preserved; as rats grow, stenosis progressively develops [20,21]. Therefore, spironolactone was started before LV hypertrophy was established. 2. Materials and methods 2.1. Experimental groups Male Wistar rats weighing 80–100 g were purchased from the Central Animal House at Botucatu Medical School, UNESP. All animals were housed in a temperature controlled room at 23 °C and kept on a 12-hour light/dark cycle. Food and water were supplied ad libitum. All experiments and procedures were approved by the Animal Experimentation Ethics Committee of Botucatu Medical School, UNESP, SP, Brazil. The animals underwent median thoracotomy under anesthesia with intraperitoneal ketamine hydrochloride (50 mg/kg) and xylidine hydrochloride (10 mg/kg). Aortic con- striction was induced by placing a 0.6 mm stainless-steel clip on the ascending aorta via a thoracic incision according to a previously described method [22]. Age-matched sham operated rats were used as controls. Three days after surgery, the rats were randomly assigned into three groups: Sham (n=22), aortic stenosis (AS, n=41) and aortic stenosis treated with spironolactone (AS-SPR, n = 36). Treatment was initiated three days after surgery and kept up for 18 weeks. Spironolactone was added to rat chow at a dosage of 20mg/kg/day. At the end of the experimental period, rats were subjected to transthoracic echocardiogram and euthanized the next day. 2.2. Echocardiographic study Cardiac structures and left ventricular (LV) function were evaluated by transthoracic echocardiogram using a commercially available echocardiograph (General Electric Medi- cal Systems, Vivid S6, Tirat Carmel, Israel) equipped with a 5–11.5 MHz multifrequency transducer as previously described [23–25]. Rats were anesthetized by intramuscular in- jection with a mixture of ketamine (50 mg/kg) and xylazine (0.5 mg/kg). A two- dimensional parasternal short-axis view of the left ventricle (LV) was obtained at the level of the papillary muscles. M-mode tracings were obtained from short-axis views of the LV at or just below the tip of the mitral-valve leaflets, and at the level of the aortic valve and left atrium. M-mode images of the LV were printed on a black-and-white ther- mal printer (SonyUP-890MD) at a sweep speed of 100mm/s. All LV structureswereman- ually measured by the same observer (KO) according to the leading-edge method of the American Society of Echocardiography [26]. Values obtained were the mean of at least five cardiac cycles onM-mode tracings. The following structural variablesweremeasured: LV diastolic and systolic dimensions (LVDD and LVSD, respectively), LV diastolic and sys- tolic posterior wall thickness (LVDPWT and LVSPWT, respectively), LV diastolic and sys- tolic septal wall thickness (LVDSWT and LVSSWT, respectively), aortic diameter (AO), and left atrium (LA) diameter. Left ventricular mass (LVM) was calculated using the for- mula [(LVDD+PWT+SWT)3− (LVDD)3] × 1.04. Relativewall thickness (RWT)was cal- culated as 2 × PWT / LVDD. LV function was assessed by the following parameters: endocardial fractional shortening (EFS), midwall fractional shortening (MWFS), posterior wall shortening velocity (PWSV), early and late diastolic mitral inflow velocities (E and A waves), E/A ratio, E-wave deceleration time (EDT), and isovolumetric relaxation time (IVRT). 2.3. Myocardial functional study Two days after echocardiographic study, myocardial contractile performance was evaluated in isolated LV papillary muscle preparations as previously described [27,28]. Rats were anesthetized with pentobarbital sodium, 50 mg/kg, intraperitoneally, and de- capitated. Hearts were quickly removed and placed in oxygenated Krebs–Henseleit solu- tion at 28 °C. LV anterior or posterior papillary muscle was dissected free, mounted between two spring clips, and placed vertically in a chamber containing Krebs–Henseleit solution at 28 °C and oxygenatedwith amixture of 95%O2 and 5% CO2 (pH 7.38). The com- position of the Krebs–Henseleit solution in mMwas as follows: 118.5 NaCl, 4.69 KCl, 1.25 CaCl2, 1.16MgSO4, 1.18 KH2PO4, 5.50 glucose, and 25.88NaHCO3. The spring clipswere at- tached to a Kyowamodel 120T-20B force transducer and a lever system,which allowed for muscle length adjustment. Preparations were stimulated 12 times/min at a voltage 10% above threshold. After a 60-min period, during which the preparations were permitted to shorten while carrying light loads, muscles were loaded to contract isometrically and stretched to the apices of their length-total tension curves (Lmax). After a 5-min period, during which preparations performed isotonic contractions, muscles were again placed under isometric conditions, and the apex of the length-total tension curve was determined. A 15 minute period of stable isometric contraction was imposed prior to the experimental period. One isometric contraction was then recorded for later analysis. The following parameters were measured from isometric contraction: peak of devel- oped tension (DT, g/mm2), resting tension (RT, g/mm2), maximum rate of tension devel- opment (+dT/dt, g/mm2/s), maximum rate of tension decline (−dT/dt, g/mm2/s), and time to peak of tension (TPT). To evaluate contractile reserve, papillarymusclemechanical performance was evaluated at basal conditions and after the following inotropic stimula- tion: post-rest contraction, extracellular Ca2+ concentration increase, and β-adrenergic agonist isoproterenol addition to the nutrient solution [29]. Papillary muscle cross-sectional area (CSA) was calculated from muscle weight and length by assuming cylindrical uniformity and a specific gravity of 1.0. All force data were normalized for themuscle CSA. After dissecting papillarymuscle, atria and ventricles were separated andweighed. Atria and left and right ventricularweightswere normalized to body weight. 2.4. Morphologic study Transverse LV sectionswere fixed in 10% buffered formalin and embedded in paraffin. Five-micrometer-thick sections were stained with hematoxylin–eosin and collagen- specific stain picrosirius red (Sirius red F3BA in aqueous saturated picric acid) [30]. In each heart, at least 50 myocyte diameters were measured as the shortest distance be- tween borders drawn across the nucleus. On average, 20 microscopic fields were used to quantify interstitial collagen fractional area. Perivascular collagen was excluded from this analysis. Measurements were performed using a Leica microscope (magnification 40×) attached to a video camera and connected to a computer equippedwith image anal- ysis software (Image-Pro Plus 3.0, Media Cybernetics, Silver Spring, MD, USA). 2.5. Myocardial hydroxyproline concentration Myocardial hydroxyproline (HOP) concentration was assessed to estimate tissue col- lagen content. HOP wasmeasured in LV tissue as previously described [31,32]. Briefly, the tissue was dried using a Speedvac Concentrator SC 100 attached to a refrigerated conden- sation trap (TRL 100) and vacuum pump (VP 100, Savant Instruments, Inc., Farmingdale, NY, USA). Dry tissue weight was measured and samples were hydrolyzed overnight at 100 °C with 6 N HCl (1 mL/10 mg dry tissue). A 50 μL aliquot of hydrolysate was trans- ferred to an Eppendorf tube and dried in the Speedvac Concentrator. One milliliter of de- ionized water was added and the sample transferred to a tube with a Teflon screw cap. One milliliter of potassium borate buffer (pH 8.7) was added to maintain constant pH and the sample was oxidized with 0.3 mL of chloramine T solution at room temperature for 20 min. The addition of 1 mL of 3.6 M sodium thiosulfate and thorough mixing for 10 s stopped the oxidative process. The solution was then saturated with 1.5 g KCl. The tubes were heated in boiling water for 20 min. After cooling to room temperature, the aqueous layer was extracted with 2.5 mL of toluene. One and a half milliliters of toluene extract were transferred to a 12 × 75 mm test tube. Then 0.6 mL of Ehrlich's reagent was added and the color allowed to develop for 30 min. Absorbances were read at 565 nm against a reagent blank. Deionized water and 20 μg/mL HOP were used as the blank and standard, respectively. 2.6. Metalloproteinase activity Matrix metalloproteinase (MMP)-2 and -9 activity was determined as previously re- ported [33]. In brief, analysis samples were prepared by dilution in extraction sample bufferwith 50mMTris, pH7.4; 0.2MNaCl; 0.1% TritonX, and 10mMCaCl2. In sample pro- tein was quantified by the Bradford method. Samples with 20 μg of protein were then di- luted in application buffer with 0.5 M Tris, pH 6.8; 100% glycerol, and 0.05% bromophenol blue, and loaded intowells of 8% SDS-polyacrylamide containing 1% gelatin. Electrophore- sis was run in a Bio-Rad apparatus at 80 V for 2 h. Gel was removed, washed twice with 2.5% Triton-X-100, and washed with 50 mM Tris, pH 8.4. Gel was then incubated at 37 °C overnight in activation solution with 50 mM Tris, pH 8.4; 5 mM CaCl2, and ZnCl2. Staining was performed for 2 h with 0.5% coomassie blue and destaining in 30%methanol and 10% acetic acid at room temperature until clear bands over a dark background were observed. The gels were photographed and the intensity of gelatinolytic action (clear bands) was analyzed in UVP, UV, White Darkhon image analyzer. 2.7. Statistical analysis Data are expressed as mean ± standard deviation or median and 25th and 75th per- centiles. Comparisons between groups were performed by one way analysis of variance (ANOVA) followed by Tukey test or Kruskal–Wallis followed by Dunn test. Mortality was assessed by log-rank test (Kaplan Meier). Statistical significance was accepted at the level of p b 0.05. Table 2 Echocardiographic structural cardiac data. Sham (n = 22) AS (n = 20) AS-SPR (n = 21) LVDD (mm) 8.73 ± 0.57 8.30 ± 1.10 9.19 ± 1.15# LVDD/BW (mm/kg) 18.1 ± 1.79 20.1 ± 2.92⁎ 22.2 ± 3.10⁎,# LVSD (mm) 4.57 ± 0.67 3.61 ± 1.49⁎ 4.62 ± 1.48# LVDPWT (mm) 1.56 ± 0.07 2.14 ± 0.28⁎ 2.25 ± 0.27⁎ LVSPWT (mm) 2.80 ± 0.25 3.81 ± 0.35⁎ 3.70 ± 0.32⁎ LVDSWT (mm) 1.59 ± 0.07 2.13 ± 0.28⁎ 2.25 ± 0.26⁎ LVSSWT (mm) 2.59 ± 0.19 3.11 ± 0.34⁎ 3.22 ± 0.40⁎ AO (mm) 3.90 ± 0.28 4.06 ± 0.31 3.99 ± 0.18 LA (mm) 5.80 ± 0.44 7.15 ± 1.22⁎ 8.02 ± 1.17⁎,# LA/AO 1.49 ± 0.11 1.77 ± 0.33⁎ 2.01 ± 0.27⁎,# LA/BW (mm/kg) 12.0 ± 1.15 17.3 ± 3.59⁎ 19.4 ± 2.73⁎,# LVM (g) 1.05 ± 0.13 1.52 ± 0.54⁎ 1.90 ± 0.53⁎,# LVMI (g/kg) 2.17 ± 0.26 3.65 ± 1.24⁎ 4.58 ± 1.35⁎,# RWT 0.18 ± 0.01 0.26 ± 0.03⁎ 0.25 ± 0.04⁎ Data are mean± standard deviation. Sham: control sham-operated rats; AS: aortic steno- sis; AS-SPR: aortic stenosis treated with spironolactone; LVDD and LVSD: left ventricular (LV) diastolic and systolic diameters, respectively; BW: body weight; LVDPWT and LVSPWT: LV diastolic and systolic posterior wall thickness, respectively; LVDSWT and LDSSWT: LV diastolic and systolic septal wall thickness, respectively; AO: aorta diameter; LA: left atrial diameter; LVM: LVmass; LVMI: LVmass index; RWT: relativewall thickness. ANOVA and Tukey. ⁎ p b 0.05 vs Sham. # p b 0.05 vs AS. 571M.P. Okoshi et al. / International Journal of Cardiology 222 (2016) 569–575 3. Results During the experiment, AS and AS-SPR groups hadmortality rates of 51.2% and 41.7%, respectively (p= 0.28). No rat from Sham group died during the experimental period. Body weight (BW) and anatomic pa- rameters are shown in Table 1. Final body weight was lower in AS and AS-SPR than Sham. LV and atria weight, in absolute or normalized to body weight values, and right ventricle weight were higher in AS and AS-SPR than Sham and higher in AS-SPR than AS. In rats, the increase in atria weight and particularly the right ventricle weight are strong predictors of heart failure [34,35]. Therefore, the higher atria and right ventricle weight in AS-SPR suggests that spironolactone induced a more severe degree of heart failure. Aldosterone antagonist doses vary between different experimental studies [36–38]. Inpreviousworks,wehaveobserved that 20mg/kg/day of spironolactone does not reduce arterial blood pressure in normoten- sive (data not shown) or hypertensive rats [8,39]. In addition, lower doses such as 10mg daily of spironolactone had beneficial cardiovascu- lar effects [37]. As the purpose of this studywas to use a dose associated with cardiovascular effects but with no change in blood pressure, the rats received 20 mg/kg/day of spironolactone. After subjecting young rats to ascending stenosis, heart failure and ensuing death usually start to occur 18–22 weeks after surgery [20,22,40]. Therefore, to avoid excessively high mortality rates, we established 18 weeks as the period to treat our rats. Table 2 shows structural cardiac parameters and Fig. 1 shows illus- trative LV M-mode echocardiograms. LV diastolic diameter-to-body weight ratio, LVwalls thickness, left atrial diameter, LVmass, and LV rel- ative thickness were higher in AS and AS-SPR than Sham. LV diastolic (Sham 8.73 ± 0.57; AS 8.30 ± 1.10; AS-SPR 9.19 ± 1.15 mm) and sys- tolic (Sham4.57±0.67; AS 3.61±1.49; AS-SPR 4.62±1.48mm)diam- eters, left atrial diameter (Sham 5.80 ± 0.44; AS 7.15 ± 1.22; AS-SPR 8.02 ± 1.17 mm), and LV mass were higher in AS-SPR than AS. Table 3 shows LV functional parameters. Endocardial fractional shortening was higher in AS than Sham. E wave was higher and E-wave decelera- tion time and isovolumetric relaxation time lower in AS and AS-SPR than Sham. Posterior wall shortening velocity (Sham 38.5 ± 3.8; AS 35.6 ± 5.6; AS-SPR 31.1 ± 3.8 mm/s) was lower in AS-SPR than Sham and AS; E/A ratio was higher in AS-SPR than Sham. Table 4 shows basal papillary muscle data. Developed tension and +dT/dt were lower and resting tension higher in both AS and AS-SPR than Sham. Time to peak tensionwas higher in AS-SPR than Sham. Con- tractile reserve was evaluated after positive inotropic stimulation (Table 5). The +dT/dt was lower in AS and AS-SPR than Sham after all inotropic stimulation. Developed tension did not differ between groups after post-rest contraction and extracellular calcium increase to 2.5mM, andwas lower in AS and AS-SPR than Sham after isoproterenol addition to nutrient solution. Time to peak tension was higher in AS-SPR than Sham after all inotropic stimulation and was higher in AS-SPR than AS Table 1 Anatomic data. Sham (n = 22) AS (n = 20) AS-SPR (n = 21) Initial BW (g) 82.8 ± 7.6 79.8 ± 2.0 79.4 ± 2.3 Final BW (g) 479 ± 56 415 ± 40⁎ 413 ± 36⁎ LVW (g) 0.86 ± 0.11 1.16 ± 0.16⁎ 1.35 ± 0.19⁎,# LVW/BW (g/kg) 1.80 ± 0.11 2.79 ± 0.25⁎ 3.27 ± 0.37⁎,# RVW (g) 0.26 ± 0.04 0.31 ± 0.13 0.40 ± 0.12⁎,# RVW/BW (g/kg) 0.55 ± 0.05 0.75 ± 0.32⁎ 0.96 ± 0.29⁎,# Atria (g) 0.11 ± 0.02 0.18 ± 0.01⁎ 0.27 ± 0.01⁎,# Atria/BW (g/kg) 0.22 ± 0.02 0.43 ± 0.18⁎ 0.64 ± 0.23⁎,# Data aremean± standard deviation. Sham: control sham-operated rats; AS: aortic steno- sis; AS-SPR: aortic stenosis treated with spironolactone; BW: bodyweight; LVW: left ven- tricle weight; RVW: right ventricle weight. ANOVA and Tukey. ⁎ p b 0.05 vs Sham. # p b 0.05 vs AS. after post-rest contraction [Sham 165 (160–170); AS 168 (165–180); AS-SPR 188 (175–195) ms]. LV morphometric and biochemical data are shown in Table 6. Myocyte diameters were higher in AS and AS- SPR than Sham and did not differ between AS and AS-SPR. Interstitial collagen fractional area (Sham 2.29 ± 0.77; AS 5.13 ± 3.37; AS-SPR 3.14 ± 3.25%; Fig. 2) and myocardial hydroxyproline concentration were higher in AS than Sham; in AS-SPR, these parameters had values between those of the Sham and AS groups and did not differ signifi- cantly from either group. Metalloproteinase-2 and -9 activity did not differ between groups. 4. Discussion In this study, we observed that early administration of aldosterone antagonist spironolactone unexpectedly increased cardiac hypertrophy and dilation, and impaired LV function in rats with aortic stenosis- induced chronic LV pressure-overload. Ascending aortic stenosis in rats has been used to promote the grad- ual development of LV pressure overload and hypertrophy, similarly to human chronic pressure overload. In this model, a clip is placed around the ascending aorta in 3–4 week-old rats. Immediately after clip place- ment aorta diameter is preserved; as rats grow, stenosis and LV hyper- trophy progressively develops [20–22]. Rats remain compensated for approximately 18 to 28 weeks [40]. In this study, spironolactone was initiated three days after surgery; therefore, the aldosterone blockade was started before the development of LV hypertrophy. The addition of spironolactone to rat chow did not change food in- take as AS and AS-SPR groups had similar body weights at the end of the experiment. Body weight was approximately 13.5% lower in AS and AS-SPR than Sham, showing the occurrence of cardiac cachexia and the severity of cardiac injury. Cardiac cachexia has been considered an independent predictor ofmortality [41] and has beenwell character- ized in rats with aortic stenosis-induced heart failure [42]. Probably due to the small sample size to evaluate mortality, this parameter did not significantly differ between AS and AS-SPR groups. The echocardiographic study showed that both aortic stenosis groups presented left atrial dilation and LV concentric hypertrophy. The increased E and decreased E-wave deceleration time and isovolumetric relaxation time in AS and AS-SPR are in accordance with the anatomic parameters indicating higher filling pressures [43]. Hyper- trophy in ascending aortic stenosis is usually better adaptive than in Fig. 1. Illustrative left ventricleM-mode echocardiograms. LVDDand LVSD: left ventricular (LV) diastolic and systolic diameters, respectively; PW: LV posterior wall; IVS: interventricular septum; Sham: control sham-operated rats; AS: aortic stenosis; AS-SPR: aortic stenosis treated with spironolactone. Table 3 Echocardiographic left ventricular functional parameters. Sham (n = 22) AS (n = 20) AS-SPR (n = 21) HR (beats/min) 285 ± 30 322 ± 56⁎ 308 ± 26 MWFS (%) 29.5 ± 3.4 32.9 ± 7.2 29.7 ± 5.8 EFS (%) 47.8 ± 5.6 57.8 ± 12.5⁎ 50.7 ± 10.6 PWSV (mm/s) 38.5 ± 3.8 35.6 ± 5.6 31.1 ± 3.8⁎,# E-wave (cm/s) 78.1 ± 7.8 103.7 ± 31.6⁎ 118.3 ± 28.9⁎ A-wave (cm/s) 47.7 ± 10.0 55.9 ± 27.6 47.7 ± 24.1 E/A 1.68 ± 0.28 2.88 ± 2.76 3.40 ± 2.24⁎ EDT (ms) 49.5 ± 6.8 38.4 ± 10.3⁎ 38.7 ± 13.3⁎ IVRT (ms) 28.1 ± 4.0 22.4 ± 4.6⁎ 19.7 ± 6.2⁎ IVRTn 58.6 ± 15.4 51.4 ± 10.2 44.5 ± 13.9⁎ Data are mean± standard deviation. Sham: control sham-operated rats; AS: aortic steno- sis; AS-SPR: aortic stenosis treated with spironolactone; HR: heart rate; MWFS: midwall fractional shortening; EFS: endocardial fractional shortening; PWSV: posterior wall short- ening velocity; E/A: early-to-late diastolic mitral inflow ratio; EDT: E-wave deceleration time; IVRT: isovolumetric relaxation time; IVRTn: isovolumetric relaxation time normal- ized to heart rate. ANOVA and Tukey. ⁎ p b 0.05 vs Sham. # p b 0.05 vs AS. Table 4 Isolated papillary muscle basal data. Sham (n = 13) AS (n = 18) AS-SPR (n = 12) DT (g/mm2) 5.72 ± 1.11 4.51 ± 1.11⁎ 4.83 ± 1.07⁎ RT (g/mm2) 0.60 ± 0.15 0.79 ± 0.21⁎ 0.77 ± 0.26⁎ RT/TT (%) 9.59 ± 2.06 15.3 ± 4.33⁎ 13.8 ± 4.73⁎ TPT (ms) 160 (159–160) 165 (160–170) 180 (168–200)⁎ +dT/dt (g/mm2/s) 65.1 ± 13.6 46.0 ± 12.8⁎ 46.3 ± 12.7⁎ −dT/dt (g/mm2/s) 27.2 ± 5.63 23.4 ± 6.02 26.0 ± 6.68 PM weight (mg) 8.02 ± 1.84 10.04 ± 1.56 9.43 ± 3.11 Lmax (mm) 7.60 ± 1.05 8.18 ± 0.98 8.13 ± 1.06 PM CSA (mm2) 1.05 ± 0.15 1.23 ± 0.19 1.16 ± 0.28 Data aremean± standard deviation ormedian and 25th and 75th percentiles. Sham: con- trol sham-operated rats; AS: aortic stenosis; AS-SPR: aortic stenosis treated with spironolactone; DT: peak of developed tension; RT: resting tension; TT: total tension (RT + DT); TPT: time to peak of tension; +dT/dt: maximum rate of tension development; −dT/dt: maximum rate of tension decline; PM: papillary muscle; Lmax: muscle length at peak of the tension-length curve; CSA: cross-sectional area. ANOVA and Tukey or Kruskal-Wallis and Dunn. ⁎ p b 0.05 vs Sham. 572 M.P. Okoshi et al. / International Journal of Cardiology 222 (2016) 569–575 descending aortic narrowing, as sustained loading occurring early in systole is followed by less concentric hypertrophy [44]. The fact that heart rate differed between AS and Sham groups may suggest that the level of anesthesia was not the same for all rats. However, we believe that the influence of anesthesia on cardiac function was reduced by the great number of animals included in each group. Unexpectedly, we observed that spironolactone increased left atrial and LV dilation, and LV hypertrophy over and above values in the AS group. Despite AS-SPR having larger LV systolic and diastolic diameters than AS, LV hypertrophy remained concentric. Functionally, AS-SPR had worse systolic function than AS, characterized by its reduced PWSV and diastolic function, characterized by a higher E/A ratio. The more highly impaired systolic function in AS-SPR than AS was reinforced by endo- cardial fractional shortening, which was higher in AS than Sham, show- ing better systolic function in AS. In AS-SPR, this parameter had values between those in Sham and AS groups and did not significantly differ from either group, further characterizing the impairment of systolic function with spironolactone. Anatomic data confirmed greater LV hy- pertrophy and also showed greater right ventricular hypertrophy in AS-SPR than AS. In rats with LV pressure overload, right ventricular hy- pertrophy is considered a strongmarker of LV failure and has been char- acterized by a right ventricle weight-to-body weight ratio greater than 0.8 mg/kg [34,35]. Therefore, our data suggest that the AS-SPR group presented a more advanced degree of heart failure than AS. As aldosterone is a potent fibrosis inductor, we quantified myocar- dial fibrosis by histologic and biochemical analysis. The AS had a higher interstitial collagen fractional area and hydroxyproline concentration than Sham, and in AS-SPR these parameters had values between those of the Sham and AS groups, being statistically similar to each group. These data suggest that spironolactone may have attenuated the devel- opment of myocardial fibrosis. Matrix metalloproteinases are proteolytic enzymes responsible for extracellular matrix component degradation. MMP activation can occur in response tomany stimuli including neurohormonal stimulation andmyocardial stretching and precedes ventricular dilation in different Image of Fig. 1 Table 5 Isolated papillary muscle data after positive inotropic stimulation. Sham (n = 13) AS (n = 18) AS-SPR (n = 12) PP60 DT (g/mm2) 6.92 (5.96–7.62) 5.86 (4.59–7.17) 5.17 (4.62–7.44) +dT/dt (g/mm2/s) 81.4 (69.5–94.3) 60.1 (50.4–75.5)⁎ 45.5 (42.6–73.8)⁎ TPT (ms) 165 (160–170) 168 (165–180) 188 (175–195)⁎,# 2.5 mM [Ca2+]0 DT (g/mm2) 6.38 (5.57–7.18) 5.19 (4.31–6.51) 5.10 (4.53–6.83) +dT/dt (g/mm2/s) 75.2 ± 17 60.0 ± 16⁎ 56.3 ± 17⁎ TPT (ms) 150 (144–160) 160 (150–160) 175 (163–183)⁎ 10−6 M Iso DT (g/mm2) 5.83 ± 1.43 4.21 ± 1.27⁎ 3.96 ± 1.19⁎ +dT/dt (g/mm2/s) 83.2 ± 22.7 54.1 ± 18.9⁎ 48.2 ± 17.2⁎ TPT (ms) 120 (120–121) 130 (120–140) 140 (120–140)⁎ Data aremean± standard deviation ormedian and 25th and 75th percentiles. Sham: control sham-operated rats; AS: aortic stenosis; AS-SPR: aortic stenosis treatedwith spironolactone; DT: peak of developed tension; +dT/dt: maximum rate of tension development; TPT: time to peak of tension; PP60: postrest contraction after a 60 s pause; 2.5 mM [Ca2+]0: isometric contraction at 2.5 mM extracellular calcium concentration; 10−6 M Iso: isometric contraction with 10−6 M isoproterenol added to the nutrient solution. ANOVA and Tukey or Kruskal– Wallis and Dunn. ⁎ p b 0.05 vs Sham. # p b 0.05 vs AS. 573M.P. Okoshi et al. / International Journal of Cardiology 222 (2016) 569–575 cardiac injury models [45,46]. In ventricular myocyte culture, aldoste- rone stimulates MMP activity [47], and in patients with ischemic heart failure, spironolactone reduces MMP-9 serum activity [48]. However, in this study we did not observe differences in MMP-2 or−9 activation between groups. As we only evaluatedMMP activation at the end of the experiment, we cannot exclude the influence of increasedMMP activity during cardiac remodeling. Most experimental studies have shown that aldosterone blockade has beneficial effects in different cardiac injury models. In pressure overloaded-induced cardiac remodeling, beneficial effects on different aspects of remodeling were observed when aldosterone blocker was initiated after LV hypertrophy had been established [9,10,49]. As aldo- sterone induces myocyte growth [2], we expected that aldosterone blockade would reduce cardiac hypertrophy. In fact, Pitt et al. [50] showed that aldosterone blocker eplerenone decreased LV hypertrophy in patients with systemic arterial hypertension. On the other hand, in patients with moderate-to-severe aortic stenosis, eplerenone did not change ventricular mass or the period without ventricular dysfunction [51]. Systolic wall stress can be calculated as intraventricular systolic pressure × cavity radius / 2 (LV wall thickness) [52]. When LV pressure is chronically increased, ventricular hypertrophy is initially important for reducing afterload andmaintaining systolic function in the face of el- evated intraventricular systolic pressure [52]. Cardiac hypertrophy is often accompanied by interstitial myocardial fibrosis. However, the role of hypertrophy and myocardial fibrosis during chronic pressure overload remains controversial [15,16]. These processes have been con- sidered to have both beneficial and deleterious effects, according to stimulation characteristics such as overload intensity and duration, and onset severity [15]. In this study, spironolactone caused more maladaptive hypertrophy with more dilation, increased filling pressures, and more pronounced right ventricular hypertrophy, suggesting that spironolactone may be Table 6 Left ventricular morphometric and biochemical parameters. Sham Myocyte diameter (μm; n = 10/group) 12.75 ± 2.06 CFA (%; n = 10/group) 2.29 ± 0.77 HOP (mg/g; n = 10/group) 2.62 ± 0.57 MMP-2 (59/pro MMP-2) 2.76 (1.58–3.03) MMP-2 (64/pro MMP-2) 3.61 (2.20–5.20) MMP-2 ([59 + 64]/pro MMP-2) 5.78 (4.81–7.84) MMP-9 (ativa/pro MMP-9) 0.98 (0.59–1.82) Data aremean± standard deviation ormedian and 25th and 75th percentiles. Sham: control sh CFA: interstitial collagen fractional area; HOP: hydroxyproline; MMP-2 andMMP-9: metallopro Dunn. ⁎ p b 0.05 vs Sham. deleterious during early chronic pressure overload. The essential in- volvement of aldosterone in ventricular grow and development at an early age has not been established. We have not identified any studies evaluating the effects of early aldosteroneblockadeduring chronic pres- sure overload. However, in different experimental models of transgenic or induced myocardial fibrosis inhibition, aortic banding was followed by reduced myocardial fibrosis and increased end-diastolic pressure or LV dilation [53–55]. Thus, our data and these results from literature allow us to hypothesize that, after elevation in LV pressure, the increase inmyocardial interstitial collagen tissue is important in preventing ven- tricular chamber dilation. By inhibiting interstitial fibrosis, spironolactone may have facilitated ventricular dilation to occur early during chronic pressure overload. Ventricular dilation, per se, may be responsible for the impaired LV systolic and diastolic functions observed in the AS-SPR group. The papillary muscle preparations allow us to an- alyze myocardial function and contractility independent of the influ- ence of afterload and preload. Data from papillary muscle functional study reinforces this hypothesis as, except for the increased time to peak of tension, myocardial contractility at basal conditions and after positive inotropic stimulation did not differ between AS-SPR and AS groups. Currently, as several drugs to prevent or reverse myocardial fi- brosis are under investigation, it is important to better understand the role of connective tissue and fibrosis during the early phase of chronic pressure overload. A limitation of this study refers to the fact that cardiac remodeling was only evaluated at the end of the experiment. Additional studies with different evaluation periods and detailed molecular analysis will be helpful in clarifying the physiopathological mechanisms involved in the effects of aldosterone blockade during early pressure overload. Fur- thermore, in this study, aortic stenosis was induced in young rats. As the hypertrophic response to a pressure overloadpresent at birthmaydiffer from the response to one acquired later in life [56], additional research is needed to establish whether our results are applicable to later acquired AS AS-SPR 14.64 ± 2.18⁎ 16.35 ± 2.68⁎ 5.13 ± 3.37⁎ 3.14 ± 3.25 5.27 ± 1.58⁎ 3.29 ± 0.26 1.69 (1.61–2.14) 1.75 (1.59–1.87) 3.80 (3.54–4.05) 3.31 (3.20–4.27) 5.65 (5.26–5.91) 5.16 (4.69–5.93) 1.41 (0.98–2.26) 1.14 (0.76–1.41) am-operated rats; AS: aortic stenosis; AS-SPR: aortic stenosis treatedwith spironolactone; teinase-2 and -9, respectively (n= 7 per group). ANOVA and Tukey or Kruskal–Wallis and Fig. 2. Sirius red-stained myocardial histological sections. A: control sham-operated rats; B: aortic stenosis (AS); C: aortic stenosis treated with spironolactone (AS-SPR). 574 M.P. Okoshi et al. / International Journal of Cardiology 222 (2016) 569–575 aortic stenosis. 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Introduction 2. Materials and methods 2.1. Experimental groups 2.2. Echocardiographic study 2.3. Myocardial functional study 2.4. Morphologic study 2.5. Myocardial hydroxyproline concentration 2.6. Metalloproteinase activity 2.7. Statistical analysis 3. Results 4. Discussion Conflict of interest Ethical statement Grants Acknowledgments References