Preventive aerobic training exerts a cardioprotective effect on rats treated with monocrotaline

Summary

Pulmonary arterial hypertension (PAH) is a chronic disease which causes overload to the right ventricle. The effect of preventive training on cardiac remodelling in this condition is still unknown. This study aimed to evaluate the influence of preventive training on hypertrophy, heart function and gene expression of calcium transport proteins in rats with monocrotaline‐induced PAH. Thirty‐two male Wistar rats were randomly divided into four groups: S, sedentary control; T, trained control; SM, sedentary monocrotaline; and TM, trained monocrotaline. The preventive training protocol was performed on a treadmill for 13 weeks, five times/week. The first two weeks were adopted for adaptation to training with gradual increases in speed/time. The speed of the physical training from the third to tenth weeks was gradually increased from 0.9 to 1.1 km/h for 60 min. Next, monocrotaline was applied (60 mg/kg) to induce PAH and lactate threshold analysis performed to determine the training speeds. The training speed of the TM group in the following two weeks was 0.8 km/h for 60 min and the T = 0.9 km/h for 60 min; in the final two weeks, both groups trained at the same speed and duration 0.9 km/h, 60 min. Cardiac function was assessed through echocardiography, ventricular hypertrophy through histomorphometric analysis and gene expression through RT‐qPCR. Right cardiac function assessed through the peak flow velocity was SM = 75.5 cm/s vs. TM = 92.0 cm/s (P = 0.001), and ventricular hypertrophy was SM = 106.4 μm² vs. TM = 77.7 μm² (P = 0.004). There was a decrease in the gene expression of ryanodine S = 1.12 au vs. SM = 0.60 au (P = 0.02) without alterations due to training. Thus, we conclude that prior physical training exerts a cardioprotective effect on the right ventricle in the monocrotaline rat model.

Pulmonary arterial hypertension (PAH) is a chronic disease characterized by a progressive increase in pulmonary vascular resistance and pulmonary artery pressure, which causes overload to the right ventricle (RV), leading to a framework of pathological cardiac remodelling characterized by hypertrophy, ventricular dysfunction, and RV insufficiency (Humbert et al. 2004; Ochiai et al. 2008; Zapata‐Sudo et al. 2012; Mocumbi et al. 2015; Talati & Hemnes 2015). Data on the exact prevalence of PAH are unknown, and the true figure could be underestimated (Mocumbi et al. 2015). The disease presents a poor prognosis with a mean survival of 2.8 years (D'Alonzo et al. 1991; Montani et al. 2013; Mocumbi et al. 2015).

In the development of PAH RV dysfunction precedes heart failure (HF) and this occurs when there are alterations in the relaxation and/or contraction of the heart muscle without promoting fluid retention or exercise intolerance (Cohn et al. 2000; Pacagnelli et al. 2014). This RV dysfunction may be related to the molecular mechanisms involved in calcium transport (Opie 1998; Fernandes et al. 2015). Several proteins such as ryanodine receptor (RyR), phospholamban (PLB) and Ca²⁺ ATPase of the sarcoplasmic reticulum (SERCA2a), regulate calcium homeostasis in the heart muscle and are essential for its proper functioning (Opie 1998; Lima‐Leopoldo et al. 2013). The majority of studies focus on the left ventricle (LV), even though RV function is fundamental to survival in pathophysiological conditions such as PAH (Risgaard et al. 2014; La Gerche & Claessen 2015).

The beneficial effects of chronic aerobic physical exercise on RV remodelling in animals with PAH induced by monocrotaline have been described (Handoko et al. 2009; Colombo et al. 2013); but a literature search did not reveal any studies which assessed the effects that preventive aerobic training exerts on RV remodelling induced by PAH. However, studies suggest that chronic aerobic physical training applied in a preventive manner in acute myocardial infarction promoted improvement in cardiac function and survival (Waard & Duncker 2009; Bozi et al. 2013).

To our knowledge, there are no previous studies evaluating the effects that preventive aerobic training exerts on RV remodelling induced by PAH. The aim of this study was to test the hypothesis that physical aerobic training carried out preventively on PAH induced by monocrotaline in rats would ease RV hypertrophy and improve RV function by altering calcium transport genes.

Materials and methods

Ethical approval

All experimental protocols used in this study were in accordance with the principles of laboratory animal care formulated by the Brazilian College of Animal Experimentation (COBEA) and according to the Guide for the Care and Use of Laboratory Animals published by the National Research Council (Clark et al. 1997). All procedures were approved by the Ethics Committee of the University of Western São Paulo–UNOESTE (numbers: 1838 and 1839).

Animals

To conduct this study 32 male Wistar rats were used, 2 months of age and average weight of 206 ± 16.35 g, from the Central Animal Facility of the University of Western São Paulo–São Paulo. The animals were kept in the Animal Experimentation Laboratory of the same institution, in plastic cages with dimensions of 41 × 34 × 16 cm (three animals/cage) at a temperature of 21 to 23°C and relative humidity of 50–60%, with luminosity cycles of 12 h (light/dark) starting with the light cycle at 7 am. The rats received food in a controlled manner (Alisul^®; Supralab, São Leopoldo/RS, Brazil) and water ad libitum.

The animals were randomly distributed into four experimental groups of eight animals each, denominated as sedentary control group (S), sedentary monocrotaline (SM) and the groups undergoing the training protocol: trained control group (T) and trained monocrotaline group (TM).

Experimental design

To conduct the study, the animals in the T and TM groups were submitted to a aerobic training protocol on a treadmill for 13 weeks with a frequency of 5 days a week. The animals underwent two weeks of adaptation to training followed by eleven weeks of training (Machado et al. 2006). At the beginning of the 11th week, the rats of the C and T groups were subjected to an intraperitoneal injection of saline, while the SM and TM groups received an injection of monocrotaline. Twenty‐four hours after the injection, the animals of the T and TM groups continued aerobic training for another 3 weeks (Colombo et al. 2013). For load adjustment and continuity of training, the animals underwent a test for lactate threshold analysis (Carvalho et al. 2005).

Two weeks after completing the exercise protocol, an echocardiographic evaluation was performed which identified right ventricular dysfunction. After the echocardiographic evaluation, the animals were weighed and then euthanized with an intraperitoneal dose of sodium pentobarbital (50 mg/kg). The heart was removed, dissected and the atria (AT), RV and LV separated and weighed. Anatomical, histological and gene expression of cardiac calcium evaluations was performed.

Details of the aerobic training and experimental design (Figure 1) are described below.

Figure 1.

Scheme of the aerobic training and experimental design. S, sedentary control; SM, sedentary monocrotaline; T, trained control; TM, trained monocrotaline; MCT, monocrotaline.

Training protocol

The animals in the T and TM groups underwent an aerobic training protocol performed on a treadmill adapted for rodents (SEBRAE, Presidente Prudente, SP, Brazil). The training was performed for 13 weeks with a frequency of five times per week, consisting of 10 weeks prior to the application of the monocrotaline (2 weeks of adaptation and 8 weeks of training) and 3 weeks after the injection of monocrotaline (Colombo et al. 2013).

During the adaptation period, the training time and treadmill speed were increased gradually, starting at a speed of 0.6 km/h for 15 min on the first day and ending at a speed of 0.9 km/h for 45 min at the end of the second week (Rodrigues et al. 2007). After the adaptation, the aerobic physical training was performed for an additional 8 weeks with gradual increases in intensity (Machado et al. 2006). A 5‐min warm‐up and cool‐down period was included in every session at a speed of 0.6 km/h.

At the beginning of the 11th week of training, 24 h after the animals of the T (saline) and TM (monocrotaline) groups had received the injections, a test was carried out to assess the velocity of the training corresponding to the lactate threshold. This speed was adjusted after conducting further tests to assess the lactate threshold in the 11th and 12th weeks. The exercise intensity in the 11th week was 60 min at 0.8 km/h for the TM group and 0.9 km/h for the T group. In the final week, both groups underwent training at 0.9 km/h for 60 min (Souza et al. 2014).

Lactate threshold

To determine the lactate threshold and the speed at which the lactate threshold occurred, the animals in the T and TM groups were submitted to an incremental exercise test on a treadmill for experimental models.

The protocol used was adapted from that previously described by Carvalho et al. (2005) and was carried out 24 h after the administration of monocrotaline and at the beginning and end of the 11th, 12th and 13th weeks, to adjust the training speed. The test began with a 2‐min warm‐up at 0.5 km/h, followed by 5 min of rest. After this, the speed was increased to 0.7 km/h for 3 min with gradual increases of 0.2 km/h every 3 min with a 0% slope, until the lactate reached a value of 1 mmol/l compared to the initial value, or until exhaustion (Bech et al. 1990; Svedah & Macintosh 2003). Exhaustion was defined as the moment that the rats could no longer keep running for 3 min. After each load increases, the animals were manually removed from the training for 1 min for blood to be collected. Blood samples were collected from the tail of the animal using an Accutrend Plus lactometer (Roche^®, San Cugat del Vallès, Barcelona, Spain). The device was calibrated according to the manufacturer's specifications. The calculation to stipulate the maximum speed was performed using an arithmetic mean of all speeds from each experimental group to reach lactate threshold or exhaustion (Souza et al. 2014).

The lactate threshold was defined as the running speed which could be maintained without an increase in lactate of 1.0 mmol/l above the blood lactate concentration of the previous speed (Ferreira et al. 2007).

Induction of right ventricular dysfunction

At the beginning of the 11th week, saline (NaCl 0.9%) was administered intraperitoneally to the animals in the S and T groups, to ensure that all animals were subjected to the same degree of stress. The protocol for induction of RV dysfunction was performed in the animals of the SM and TM groups, with an intraperitoneal injection of a single dose of monocrotaline (Sigma Chemical, St Louis, MO, USA) in the proportion of 60 mg/kg in 1 mol/l HCl buffer of pH 7.0 with 1 mol/l of NaOH (Souza‐Rabbo et al. 2008).

Feed control

Monocrotaline has been shown to reduce food intake; to control for this effect, the animals were separated into individual cages to dose the daily feed intake. The rats treated with monocrotaline (SM and TM groups) were allowed to eat freely from a supply of standard rat cubes. In the groups S and T, the rats were diet‐matched to the treated rats by allowing them only the amount of food consumed on the previous day by the treated rats. The feed control was performed after the administration of monocrotaline, as the animals, which received the drug, began the framework of pathological RV remodelling, decreasing their feed intake due to RV dysfunction (Lopes et al. 2008).

Echocardiographic evaluation

Echocardiographic evaluation was performed using a commercially available echocardiograph (General Electric Medical Systems, Vivid S6, Tirat Carmel, Israel) equipped with a 5–11.5 MHz multifrequency probe. Rats were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (0.5 mg/kg). Pulmonary artery flow was obtained by pulsed Doppler, and the time to peak flow velocity [pulmonary velocity acceleration time (PVAT)], pulmonary ejection time (PET) and peak flow velocity (PFV) were measured (Martinez et al. 2011; Eguchi et al. 2014). Pulmonary velocity acceleration time is an indicator of the severity of pulmonary hypertension, and the higher the level of systolic pulmonary artery pressure, the smaller the value of PVAT. Pulmonary ejection time is a parameter related to the systolic function and PAH degree. Peak flow velocity is related to RV systolic function (Dabestani et al. 1987).

Evaluation of anatomical parameters

The heart was removed, dissected into the atria (AT), right ventricles (RV) and left ventricles (LV) and ventricular septum and weighed. The anatomical parameters were normalized by the final body weight (AT/FBW, RV/FBW and LV/FBW) and were used as the hypertrophy index. The lungs and liver were also removed, weighed and stored in an oven for 48 h. Next, they were weighed again to calculate the wet/dry weight ratio which was used to evaluate signs of cardiac failure (Carvalho et al. 2010).

Histology and histomorphometric analysis

Cardiac tissue samples were fixed in 10% buffered formaldehyde solution for 48 h. After fixation, the tissue was embedded in paraffin blocks to obtain coronal histological sections of 4 μm. The histological sections were stained on slides with haematoxylin–eosin solution (HE) to measure the cross‐sectional areas of the cardiomyocytes, using a LEICA microscope (model DM750, Leica Microsystems, Wetzlar, Germany). The microscope sent digital images to a computer equipped with the analysis system of the Leica images Application Suite LAS 4.2.0 (Media Cybernetics, Silver Spring, MD, USA) (Gomes et al. 2009; Oliveira‐Júnior et al. 2010).

The images were obtained using a binocular optical microscope. All images were captured by video camera at 400× magnification (objective 40×). The selection of images to capture and digitization were performed visually. The morphometry of the images obtained and digitalized was accomplished using software appropriate for the purpose. For each of the four slices obtained from the RV of each animal, captures were performed in different fields, chosen according to the area where more cells could be viewed in cross section. Fifty cells were measured per ventricle analysed. The cardiomyocytes selected were transversely sectioned and presented a round shape and visible nucleus in the centre of the cell and were located in the subendocardial layer of the muscular wall of the RV. This precaution was aimed at standardizing the maximum cardiomyocytes in the different groups. The average cross‐sectional areas obtained for each group were used as an indicator of cell size (Oliveira‐Júnior et al. 2013).

Gene expression relative to regulators of the intracellular Ca²⁺

The total RNA was extracted from the RV tissue (fresh tissue) using TRIzol (Life Technologies, CA, USA) and then treated with DNAse according to the manufacturer's instructions. The RNA integrity was assessed through electrophoresis. A High‐Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used for the synthesis of complementary DNA (cDNA) from 1000 ng of total RNA. RT‐PCR was used to quantitatively measure the relative levels of mRNA to RyR (Rn01470303_m1), PLB (Rn01434045_m1) and SERCA2a (Rn00568762_m1). To this end, TaqMan Universal PCR Master Mix was used (Applied Biosystems), according to the manufacturer's instructions, and the detection system used was Applied Biosystems Step One Plus. All samples were analysed in duplicate. The cycling conditions were as follows: enzyme activation at 50°C for 2 min; denaturation at 95°C for 10 min; amplification of the cDNA products for 40 cycles of denaturation at 95°C for 15 s; and annealing/extension at 60°C for 1 min. The gene expression was quantified in relation to the values of the S group and after normalization by a β‐actin internal control (ACTB, Rn00667869_m1), being determined by the 2‐ΔΔCt method as previously described (Livak & Schmittgen 2001; Lima‐Leopoldo et al. 2013).

Statistical analysis

For analysis of normality, the Shapiro–Wilk test was used. Data are expressed as mean ± standard deviation, median and minimum and maximum values. For comparison between groups, the nonparametric Kruskal–Wallis was used followed by Dunn's post hoc test (PVAT; PET; atria; wet/dry liver; phospholamban) or the anova parametric test (one‐way), followed by the Tukey's post hoc test (PFV; RV; LV; wet/dry lung; histological and histomorphometric analysis; ryanodine; SERCA2a). A P value <0.05 was considered significant. The software used was GraphPad Prism^®(GraphPad Software, La Jolla, CA, USA).

Results

Echocardiographic evaluation

The results of the echocardiographic evaluation of the RV showed that the animals treated with monocrotaline showed RV dysfunction characterized by a decrease of 23% in the peak flow velocity of the pulmonary artery (S vs. SM, P = 0.001) and a decrease of 30% in the pulmonary velocity acceleration time (S vs. SM, P = 0.005). The physical training normalized the peak flow velocity of the pulmonary artery in the TM group, with an increase of 28% (SM vs. TM, P = 0.001) (Figure 2).

Figure 2.

Echocardiogram. Values are expressed as mean and standard deviation. (a) Peak flow velocity (PFV). (b) Pulmonary velocity acceleration time (PVAT) and pulmonary ejection time (PET). S, sedentary control; SM, sedentary monocrotaline; T, trained control; TM, trained monocrotaline; cm/s, centimetres per second; ms, millisecond. *P < 0.05.

Evaluation of anatomical parameters

Table 1 and Figure 3 show the anatomical parameters of the S, SM, T and TM groups. At 35 days monocrotaline‐treated rats showed signs of RV dysfunction at postmortem examination including atrial hypertrophy (increase 43%; P = 0.001) and right ventricular hypertrophy (increase 46%; P = 0.0004) without pleural effusion or liver congestion. The animals of the S and T control groups showed no changes.

Table 1.

Anatomical data (weight) of the lung and liver, expressed as mean ± standard deviation, median, minimum and maximum values and P‐value

Variables	Groups
	S	SM	T	TM	P‐value
Lung
Wet/dry (g)	4.81 [3.22–5.05]	4.90 [4.17–5.12]	4.87 [4.07–5.08]	4.57 [2.65–5.26]	0.13
Liver
Wet/dry (g)	3.19 [2.89–0.48]	3.27 [3.21–3.74]	3.23 [2.80–3.83]	3.32 [3.25–3.38]	0.14

S, Sedentary control; SM, sedentary monocrotaline; T, trained control; TM, trained monocrotaline; g, grams. P < 0.05.

Figure 3.

Box plot corresponding to the evaluation of anatomical parameters. (a) Atria; (b) RV (right ventricle); and (c) LV (left ventricle). S, sedentary control; SM, sedentary monocrotaline; T, trained control; TM, trained monocrotaline. *P < 0.05.

Histological and histomorphometric analysis

There was a 41% increase in sectional area of the cardiomyocytes, indicating right ventricular hypertrophy, in the animals of the monocrotaline group (S vs. SM, P = 0.0001); the physical exercise was able to relieve the hypertrophy in these animals, as demonstrated by a 21% decrease in the sectional area of the cardiomyocytes (SM vs. TM, P = 0.0001) (Figure 4).

Figure 4.

Histomorphometric analysis. Values are expressed as mean and standard deviation. S, sedentary control; SM, sedentary monocrotaline; T, trained control; TM, trained monocrotaline; *P < 0.05.

Relative gene expression of regulators of intracellular Ca²⁺

A statistical difference was observed only in the RyR gene, with decrease of 46% (S vs. SM, P = 0.02) (Figure 5).

Figure 5.

Gene expression of cardiac calcium proteins. Data are expressed as mean ± standard deviation. RyR, PLB and Serca2a. All analyses of the genes were normalized by β‐actin. S, sedentary control; SM, sedentary monocrotaline; T, trained control; TM, trained monocrotaline; RyR, ryanodine; PLB, phospholamban, SERCA, Serca2a. P < 0.05, *S vs. M.

Discussion

The main finding of this study indicates that preventive aerobic training exerted a cardioprotective effect on the RV, demonstrated by decreased RV hypertrophy and ventricular functional improvement without altering the gene expression of proteins involved in cardiac calcium transport. To our knowledge, this is the first study to assess the cardioprotective effect of prior aerobic training in rats with PAH.

The experimental model used in this study to induce PAH was through monocrotaline, which is widely used to promote RV dysfunction and HF (Lopes et al. 2008; Souza‐Rabbo et al. 2008; Gomez‐Arroyo et al. 2012; Colombo et al. 2013; Pereira et al. 2013; Alencar et al. 2014). In this experimental model, cardiac remodelling is characterized by RV hypertrophy, functional deterioration and progression to right‐sided HF (Handoko et al. 2009; Maarman et al. 2013).

In the present study, the animals progressed to PAH and RV dysfunction without HF, as demonstrated by the echocardiographic alterations, RV hypertrophy and increased areas of cardiomyocytes without presenting fluid retention, which confirms the effectiveness of the chosen model to promote RV dysfunction. The presence of RV dysfunction and not right‐sided HF could be due to the induction of PAH at a later stage, as other studies induce PAH at an earlier stage (Souza‐Rabbo et al. 2008; Zapata‐Sudo et al. 2012; Colombo et al. 2013). The age, weight, the dose of monocrotaline and the number of days rats are kept after monocrotaline injection are greater determinants of the rats having RV dysfunction without HF. Studies have reported but thus far that a dose of monocrotaline at 60 or 80 mg/kg can lead to the development of progressive HF and can be fatal within 3–6 weeks (Hessel et al. 2006; Handoko et al. 2009; Ruiter et al. 2013).

PAH has been widely studied as it causes functional limitations, loss of exercise capacity and poor quality of life (Zafrir 2013; Mocumbi et al. 2015; Sahni et al. 2015). Advances in drug treatment and the implementation of physical exercise improve the prognosis, quality of life and functional capacity of patients, and in experimental animal models, the beneficial effects of training on RV remodelling are highlighted (Mereles et al. 2006; Handoko et al. 2009; Natali et al. 2015). In the past, the practice of physical exercise by patients with PAH was contraindicated due to the risk of sudden death. However, the application of exercise has been reconsidered in these patients due to accumulating evidence of its beneficial effects (Zafrir 2013) there is no previous information demonstrating how preventive aerobic training acts on this condition.

Few studies have evaluated the influence of preventive exercise on cardiac remodelling and the emphasis has been more on the changes in the LV (Portes & Tucci 2006; Veiga et al. 2011, 2013). Studies addressing preventive training were performed in experimental models in rats with acute myocardial infarction and the results were controversial. Aerobic exercise is a non‐pharmacological and effective way to protect the heart against aggression and insult (Portes & Tucci 2006); however, there are studies that found no benefits to this condition (Veiga et al. 2011, 2013). The preventive training protocols for acute myocardial infarction were performed for an average of 8 weeks, which was the period used in the present study (Freimann et al. 2005; Bozi et al. 2013; Veiga et al. 2013).

Studies that used physical training in rats with PAH, previously installed through the monocrotaline model, demonstrated improvements in survival and pulmonary vascular dysfunction, slowing the progression of PAH, with hemodynamic improvement, but without influencing RV hypertrophy (Souza‐Rabbo et al. 2008; Colombo et al. 2013; Natali et al. 2015).

Our study demonstrated, as an important cardioprotective aspect of preventive aerobic training, improvement in function and ventricular hypertrophy. Cardiac function was assessed through echocardiography, demonstrated by the increase in RV contractility, evidenced by the PFV parameter, without changing the PFV which is an indicator of severity of PAH (Greenberg and Eshaghpour 2001; Rudski et al. 2010). Soon physical training improved RV function without changing the PAH severity, which can be justified by some direct actions of the training in the RV, without decreasing the PAH, mechanisms have not been studied but have been elucidated in previous studies (Ryan & Archer 2014; Talati & Hemnes 2015). Other studies using this model evaluated cardiac function through catheterization (Colombo et al. 2013); however, echocardiography also evaluates cardiac function and is not an invasive method (Alencar et al. 2014; Eguchi et al. 2014).

One of the mechanisms that may be involved in RV dysfunction is calcium transport proteins, which include ryanodine receptor (RyR), phospholamban (PLB) and Ca²⁺ ATPase of sarcoplasmic reticulum (SERCA2a) (Opie 1998; Lima‐Leopoldo et al. 2013). The decline in systolic function in the present study may be related to the gene expression of RyR. RyR is a tetrameric protein which has a molecular weight of 565 kDa being predominant in cardiac muscle (Yano et al. 2009). When the RyR2 channel is open, calcium is released from the sarcoplasmic reticulum into the cytoplasm allowing the cardiac excitation/contraction coupling system (Meissner 1994; Eisner 2014). Alterations in RyR2 promote arrhythmias and HF (Marx et al. 2000; Eisner 2014), and even though preventive aerobic training does not alter this expression, other approaches to improve calcium transport in PAH may be considered. Other molecular mechanisms may be involved in the contractile dysfunction, such as endothelin‐1 (ET‐1), atrial natriuretic peptide (ANP), insulin‐like growth factor (IGF‐1) (Loennechen et al. 2001; Wisloff et al. 2002; Fontoura et al. 2014), neurohumoral factors, the sympathetic nervous system through the adrenergic receptors (α1a, β1 and β2) and the endocrine system via angiotensin II (Ang II) (Dai et al. 2011). Likewise, mediators of fibrosis [collagen I, collagen III and TGF‐β1 (Yan et al. 2011)], circulatory neuromodulatory mechanisms (nitric oxide) (Xiao et al. 2012) and expression of MHC‐αeβ (Fernandes et al. 2011) may also be involved. Another aspect to be considered is that the increase in physical activity has known effects on mitochondrial content, mitochondrial respiration and substrate utilization, which may very well have effects on the RV function of monocrotaline rats (treated or untreated) (Piao et al. 2010; Ryan & Archer 2014; Talati & Hemnes 2015). Further studies are necessary to investigate these molecular mechanisms.

Previous studies have demonstrated that reduction in cardiac hypertrophy is directly associated with thinning of the pulmonary artery and interstitial volume, resulting in the decreased pulmonary vascular resistance and consequent reduction in RV afterload (D'Alonzo et al. 1991; Talati & Hemnes 2015). Such a mechanism could explain the attenuating effect of the prior exercise training on cardiac hypertrophy during PAH. The hypertrophy was evaluated by measuring the area of cardiomyocytes in the RV. Differently from the results of Colombo et al. (2013) who conducted training after the application of monocrotaline, in the present study, aerobic training mitigated the RV hypertrophy. Colombo et al. (2013), however, evaluated the hypertrophy by means of anatomical parameters and did not measure the area of cardiomyocytes. The decrease in ventricular hypertrophy in the present study may be related to molecular changes involved in signalling pathways such as the protein GSK‐3β, already evaluated in this model (Colombo et al. 2013). Other pathways may be related to ventricular hypertrophy: mTOR, FOXO3a, FOXO1, calcineurin, mindin, AKT and peroxisome proliferator‐activated receptors (PPAR delta) (Jucker et al. 2007; Yan et al. 2011; Colombo et al. 2013).

Some limitations should be considered. The animals from TM group kept on training for more than 3 weeks to prevent the effects of detraining they would have to interrupt their training. Another aspect to be considered is that this training even being conducted for 3 weeks after the application of monocrotaline, is preventive because the training was performed prior to heart failure as the animals were in a stage of ventricular dysfunction. Another limitation was the biological implications of the analysis technique of mRNA expression and its impact on signalling pathways which cannot be reliably determined using only analysis of RT‐qPCR. In addition, when assessing mRNA expression in isolation, it is not related to the expression of protein activity, and these are intrinsically linked. Thus, if the RV dysfunction caused by PAH reflects a direct or indirect effect on the change in calcium transport, and if this is related to the modulation of transcriptional or post‐transcriptional factors, we would be unable to distinguish this. Thus, future studies are needed to better understand the molecular mechanisms involved in PAH, prior physical training and its effects on RV.

Conclusion

Preventive aerobic training exerted a cardioprotective effect, decreasing hypertrophy and improving heart function in the rats with monocrotaline‐induced RV dysfunction, without modifying the calcium transport genes.

Conflict of interest

There are no conflict of interest issues regarding any of the authors.

Funding source

This study was supported by Pro‐Reitoria de Pesquisa da Universidade do Oeste Paulista‐UNOESTE, Presidente Prudente, SP and Internal Medicine, UNESP, Botucatu, SP, Brazil.