The investigation complies with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996). The experimental protocol was approved by the Institutional Research Ethics Committee from the Federal University of São Paulo, São Paulo, Brazil (process: 1673020414). Sixty-three female Wistar rats, weighing 180–220 g and aged 8–9 weeks, were assigned to one of the following three groups: No-trained Sham (n = 21); no-trained stenosis (SS, n = 21); and trained stenosis (TS, n = 21). Figure 1 illustrates the experimental design, in which at first step the animals were submitted to stenosis or Sham procedure. A week later, echocardiographic examination was performed in all animals, and stenosis rats were then randomly assigned to SS or TS group. Following 3 days, the animals were submitted to functional fitness test on a motor-driven treadmill. Then, animals were followed for over 8 weeks.

Rats were anaesthetized with ketamine plus xylazine (50 mg/Kg, Dopalen®, Vertbrands, Paulínia, SP, BRA, i.p.) mixture, intubated, and mechanically ventilated (Rodent ventilator, model 683, Harvard Apparatus, Holliston, MA, USA). A left thoracotomy was performed, and the pulmonary artery (PA) was carefully dissected free from the aorta. A silk thread was positioned under the PA, and an 18-gauge needle was placed alongside the PA. A suture was tied tightly around the needle, and the needle was rapidly removed to produce a fixed constricted opening in the lumen equal to the needle diameter. The combination of a fixed banding around the PA and the animal growth resulted in a high RV afterload. The Sham animals underwent the same procedure except PAS (Faber et al., 2006).

The functional fitness was evaluated using a motorized treadmill coupled to a gas analyzer (Panlab, Harvard Bioscience Company, MA, USA). Prior to the physical test, rats were introduced to running as previously described by our group (Amadio et al., 2015). Each rat had to undergo a 2-min warm-up period at 25 cm/s, and the running speed was increased by 9 cm/s every 2 min to reach exhaustion. An oxygen uptake steady state on progressive increases in running speed and a respiratory exchange ratio of ≥1.05 were taken into account to define the VO₂max.

After 48 h of stenosis or Sham surgery, rats were anaesthetized with ketamine and xylazine mixture as reported above, and transthoracic echocardiography was performed to determine pressure gradient using a 12 MHz transducer Sonos-5500 (Hewlett-Packard, Andover, MA, USA). The echocardiography was also carried out 9 weeks later to RV morphofunctional study. Measurements were performed according to the echocardiographic RV guidelines. The 2D modality was used to measure the RV. The right parasternal short-axis view at the level of the papillary muscles was used for determination of the diastolic and systolic RV area. Measurements of the RV outflow tract were obtained from the parasternal short-axis view at the level of the aortic valve during end-diastole (Egemnazarov et al., 2015). We included only animals with pressure gradients between 25–55 mmHg (Manchini et al., 2014). The diastolic (RVDA) and systolic (RVSA) transverse areas of the RV were measured at the basal, middle, and apical view. The final value was the arithmetic mean of the measures of the three views. Systolic function was analyzed by the fractional area change (FAC) as a function for the following equation: FAC = RVDA-RVSA/RVDA × 100. Pulsed Doppler at the tricuspid valve level provided the flow velocity curve to analyze the diastolic function (E/A wave ratio).

Rats were subjected to running training on a motor-driven treadmill (CL4002, Caloi, São Paulo, Brazil) after 7 days of surgical procedures for 8 weeks. Rats ran 6 times a week, and each session lasted up to 60 min. For each session, the treadmill speed was 18 m/min for 30 min and 22 m/min for the remaining 30 min. This research protocol has shown to be effective in alleviating the pathological cardiac remodeling (Serra et al., 2008, 2010).

Twenty-four hours after the last training session or sedentary status the rats were anesthetized with urethane overdose (4.8 g kg kg⁻¹ i.p.) and the hearts were quickly removed. The RV chamber was cut, separated from the rest of the heart and weighted to be used as a myocardial hypertrophy indicator. Afterwards, RV tissue was transversally sectioned at the mid-RV level and fixed in 10% formalin buffered solution. Tissue samples were sectioned in 7 μm thickness and stained with hematoxylin-eosin for cross-sectional cardiomyocyte area analysis at approximately 20 visual fields for each animal. Myocytes with visible nuclei and intact cellular membranes were chosen for evaluation. The analyses were performed at 40× magnification using an Olympus image acquisition system (Waltham, MA, USA) (Soci et al., 2011). The RV fibrosis was evaluated in tissue samples stained with picro-sirius red. The collagen content was assessed at 40× magnification using an Olympus image acquisition system (Egemnazarov et al., 2015). The percentage fibrosis was calculated by dividing the total area of collagen by the total analyzed tissue area multiplied by 100.

Total RNA was extracted from frozen RV with 1 ml of TRIzol reagent (Gibco BRL, MD, USA). RNA quantification was determined using a SpectraMax M5 spectrophotometer system (Molecular Devices, CA, USA). One microgram of RNA was used for cDNA synthesis and Real-Time PCR gene expression analysis. First, contaminating DNA was removed using DNase I (Invitrogen, CA, USA) at a concentration of 1 unit/μg RNA in the presence of 20 mM Tris-HCl, pH 8.4, containing 2 mM MgCl₂ for 15 min at 37°C, followed by incubation at 95°C for 5 min. Then, the reverse transcription was performed with 200 μl reaction in the presence of 50 Mm Tris-HCl, pH 8.3, 3 mM MgCl₂, 10 mM dithiothreitol, 0.5 mM dNTPs, and 50 ng of primers with 200 units of Moloney murine leukemia virus-reverse transcriptase (Invitrogen). The conditions were: 20°C for 10 min; 42°C for 45 min; 95°C for 5 min. One microliter of reverse transcription reaction was used for real-Time PCR at Applied Biosystems 7500 Fast PCR (ABI Prism, Applied Biosystems, CA, USA) using the SYBR Green supermix (ABI Prism). Experiments were performed in triplicates for each target genes: β-MHC (primers forward 5′-AAGTGGGCAGCATCACCTAC-3′ reverse 5′-GCCGGCTCTGTAACTTCCTT-3′ (GenBank: NM_021838); α-MHC (primers forward 5′-TCAGGCTTGGGTCTTGTTAGC GAAGAGAAACTTCCAGGGGCA-3′ reverse 5′-AGGCTC TTTCTGCTGGACA-3′ (GenBank: NM_012611.3). Target gene abundance was quantified as a relative value for internal control β-Actin: forward primer 5′-AGAGGGAAATCGTGCGTGAC-3′ and reverse primer 5′-AGGAAGGAAGGCTGGAAGAGA -3′ (GenBank: NM_031144.3).

The cDNA for miRNA analysis was synthesized from total RNA using specific primers according to the TaqMan microRNA assay. The 15 μl reactions obtained by the TaqMan MicroRNA Reverse Transcription Kit protocol (Applied Biosystems, CA, USA) were incubated in a Thermal Cycler (Applied Biosystems, CA, USA) for 30 min at 16°C, 30 min at 42°C, and 5 min at 85°C. They were then maintained steadily at 4°C. The real-time PCR quantification was performed by using the TaqMan MicroRNA Assay protocol (Applied Biosystems, CA, USA). The 20 μl PCR reaction solution contained 10 μl TaqMan Universal PCR master mix II (2x), 1.33 μl RT product, 7.67 μl nuclease-free water, and 1 μl of primers and probe mix from the TaqMan MicroRNA Assay protocol for microRNA-1 and 21. The reactions were performed at 95°C for 10 min, and then in 40 cycles of 95°C for 15 s and 60°C for 1 min. Samples were normalized by evaluating the U6 gene.

Frozen RV was homogenized using ice-cold lysis buffer and proteinase inhibitor cocktail (Manchini et al., 2014). Lysates corresponding to 30 μg of protein were subjected to 10% SDS-PAGE. Separated proteins were transferred to PVDF membrane (Amersham Biosciences, NJ, USA) and transfer effectiveness was examined with 0.5% Ponceau S. After blocking with 5% non-fat dry milk for 2 h at room temperature, PVDF membranes were probed with Abcam (Cambridge, MA, USA) primary antibodies for rabbit ant-Akt₁ (1:5000), rabbit anti-p-Akt₁ (1:2500), rabbit anti-Caspase3; rabbit anti-Bax (1:1000), rabbit anti-Bcl-2 (1:1000), rabbit anti-Bcl-xL (1:500), rabbit anti-β-MHC (1:5000), rabbit anti-α-MHC (1:5000), rabbit anti-L-type Ca⁺⁺ (1:500), rabbit anti-ryanodine receptor (1:1000), rabbit anti-Serca 2 (1:1000), and rabbit anti-Na⁺/Ca⁺⁺ exchanger (1:100) in overnight incubation. Membranes were then washed five times with PBS and incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit (1:20,000; Zymed, CA, USA). Membranes were again washed five times with blocking buffer and then rinsed twice with PBS. Antibodies binding were detected by chemiluminescence reagents (Amersham Biosciences, NJ, USA), and images were captured using an Amersham Imager 600 system. Quantification of target proteins was normalized for the internal control glyceraldehyde 3-phosphate dehydrogenase.

Data were analyzed using GraphPad Prism software version 5.0 (La Jolla, CA, USA) by analysis normality with Shapiro-Wilk test. One-way ANOVA complemented by Newman–Keuls post-hoc was used to detect differences between groups for cross-section analysis. Two-way repeated ANOVA complemented by Bonferroni post-hoc were applied to paired data. Kruskal-Wallis followed by Dunn's multiple comparison tests were applied to non-normality data. Statistical significance was set at p-value ≤0.05. Data are expressed as mean ± standard error of the mean.

All rats survived the stenosis surgery or sham procedure. During the stenosis period, the rats did not show signs of cyanosis or congestion, as demonstrated by the similar liver (SHAM: 68.1 ± 3.5; SS: 66.1 ± 2.9; TS: 67.1 ± 3.6; %, p = 0.3) and lung (SHAM: 71.4 ± 4.7; SS: 71.7 ± 5.2; TS: 71.6 ± 8.6; %, p = 0.9) weights. We performed the experiments in female rats because it allowed a strict control body weight. It is true that the stenosis degree could result in very dissimilar RV afterload during the study, thereby a bias would be set. As illustrated in Figure 2A, all experimental groups showed a similar average body weight at baseline and at the end of the study. The increase in body weight was associated with significant elevation in pulmonary transvalvular gradient only in stenosis groups (Figure 2B). This finding shows that the construction of the outflow tract of the RV was kept during the observation period, and echocardiography showed that the stenosis degree was similar between SS and TS groups. Moreover, mean valvar gradient values are within a range of mild to moderate stenosis definition (Hirth et al., 2006), which usually receives only clinical monitoring of RV remodeling. This issue emerges as a main way of analyzing the impact of exercise because critical stenosis requires a more invasive intervention (Godart et al., 2014). The Figure 2 also shows the functional fitness data. The VO₂max was not significantly different between groups at the baseline and at the end of the study, but there was a slight decrease in the SHAM and SS groups after 8 weeks (Figure 2C). A single functionality difference was evident only in the TT rats, in which VO₂max was significantly increased with training.

On transthoracic echocardiography (Figure 3A), we have confirmed previous findings from the literature that the RV-pressure overload results in a concentric cardiac remodeling (Faber et al., 2006). From the baseline to end of the study, non-trained stenosis rats (SS group) had a significant reduction in diastolic and systolic RV transverse areas, characterizing a concentric RV remodeling. On the other hand, exercise prevented these concentric changes in the cavity. The echocardiographic analysis also revealed reduced RV systolic performance in the first analyzes for animals submitted to stenosis. An evaluation 8 weeks after operation illustrated that systolic dysfunction was stable in SS group, but a significant improvement was noted in the animals submitted to exercise (TS group). The stenosis rats exhibited an increased ratio of RV weight to body weight and cross-section cardiomyocyte area compared with SHAM rats, in which trained animals showed a lower cross-section cardiomyocyte area compared to SS group (Figure 3B). Our structural analysis corroborates a well-established finding of myocardial fibrosis associated with RV pressure overload (Baicu et al., 2012; Egemnazarov et al., 2015). Thereby, stenosis resulted in a significant increase in collagen content (Figure 3C). Notably, exercise prevents the collagen deposition, in which TS group had values that did not differ significantly from the SHAM group.

We analyzed the Akt₁ and p-Akt₁ expression, as altering its activity provides a potent pro-survival signal (Matsui et al., 2003). As illustrated in Figure 4A, there was a significant upregulation of the activated Akt₁ form only in trained animals. We further investigated whether the increase in Akt₁ activity could be associated with a beneficial role of exercise in cell death. Thus, RV apoptosis was assessed by cleaved Caspase-3 levels, which was significantly increased in PAS animals (Figure 4B). These findings were accomplished by significant changes in upstream components of myocardial mitochondrial-dependent apoptotic signaling pathways. Compared to SHAM group, the protein pro-apoptotic Bax level significantly increased while the anti-apoptotic factors Bcl-2 and Bcl-xL decreased in the SS and TS groups. Intriguingly, exercise restored the cleaved Caspase-3 to a similar level of SHAM animals. These findings were accomplished by a significant increase of anti-apoptotic Bcl-xL. Hence, exercise training reversed to normal the Bcl-xL/Bax ratio in myocardial.

MicroRNAs are emerging as key regulators of gene expression, and their role in cardiac hypertrophy is becoming increasingly apparent (Harada et al., 2014). When looking at the expression of microRNA-1 we found no effect of pulmonary stenosis per se, but there was a significant reduction with exercise (Figure 5A). We observed a strong effect of stenosis on the microRNA-21 expression, in which was down-regulated in the SS group (Figure 5B). On the other hand, a restored microRNA-21 level was reported with exercise.

We assessed how the pathophysiologic modifications in the RV were reflected by gene/protein expression changes of two distinct types of myosin heavy chain (MHC), referred as β-MHC and α-MHC. These analyses were carried out because the relative distribution of β- and α-MHC is altered in cardiac hypertrophy and showed to be directly correlated with the myocardial dysfunction (Nadal-Ginard and Mahdavi, 1989). Consistent with myocardial remodeling, there was increased β-MHC and decreased α-MHC gene expression in stenosis animals (Figure 6A). However, the combination of the stenosis with the exercise resulted in additional expression of the β-MHC and attenuation in downregulation of α-MHC. Thereby, the β/α-MHC ratio was increased in SS group whereas this was attenuated by exercise. The Figure 6B illustrates that β-MHC and α-MHC protein expression was upregulated for both SS and TS groups. Moreover, stenosis-induced overexpression of α-MHC protein was significantly enhanced by exercise. Then, the increased β/α-MHC ratio induced by stenosis was completely abrogated in trained animals.

Quantitative changes in expression of the proteins that modulate calcium handling with correlations to functional alterations were reported in several experimental models of cardiac disease (Wankerl and Schwartz, 1955). Here, pulmonary stenosis induced a significant increase in L-type Ca⁺⁺ channel and as Na⁺/Ca⁺⁺ exchanger expression (Figure 7). Furthermore, SS animals exhibited lower ryanodine receptor content and a no significant reduction of Serca 2. Exercise protocol restored both ryanodine receptor and Serca 2 expression to basal levels. In addition, our results demonstrate an imbalance of Na⁺/Ca⁺⁺ exchanger and Serca 2 expression in SS animals, while TS group has not reported this finding.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.