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Archives of cardiovascular diseases
Volume 110, n° 2
pages 106-115 (février 2017)
Doi : 10.1016/j.acvd.2016.05.008
Received : 8 September 2015 ;  accepted : 18 May 2016
Downhill exercise training in monocrotaline-injected rats: Effects on echocardiographic and haemodynamic variables and survival
Entraînement en mode excentrique des rats monocrotaline : effets sur la survie, les paramètres échocardiographiques et hémodynamiques
 

Irina Enache a, b, , Fabrice Favret b, Stéphane Doutreleau a, b, Paola Goette Di Marco a, b, Anne-Laure Charles b, Bernard Geny a, b, Anne Charloux a, b
a Service de physiologie et d’explorations fonctionnelles, pôle de pathologie thoracique, hôpitaux universitaires de Strasbourg, 67091 Strasbourg, France 
b Équipe d’accueil 3072, fédération de médecine translationnelle de Strasbourg, université de Strasbourg, 67000 Strasbourg, France 

Corresponding author at: Service de physiologie et d’explorations fonctionnelles, nouvel hôpital Civil, 1, place de l’Hôpital, BP 426, 67091 Strasbourg, France.
Summary
Background

Eccentric exercise training has been shown to improve muscle force strength without excessive cardiovascular stress. Such an exercise modality deserves to be tested in pulmonary arterial hypertension.

Aim

We aimed to assess the effects of an eccentric training modality on cardiac function and survival in an experimental monocrotaline-induced model of pulmonary arterial hypertension with right ventricular dysfunction.

Methods

Forty rats were randomly assigned to one of four groups: 40mg/kg monocrotaline-injected sedentary rats; 40mg/kg monocrotaline-injected eccentric-trained rats; sedentary control rats; or eccentric-trained control rats. Eccentric exercise training consisted of downhill running on a treadmill with a −15° slope for 30minutes, 5 days a week for 4 weeks. Training tolerance was assessed by echocardiography, right ventricle catheterization and the rats’ maximal eccentric speed.

Results

Survival in monocrotaline-injected eccentric-trained rats was not different from that in monocrotaline-injected sedentary rats. Monocrotaline-injected eccentric-trained rats tolerated this training modality well, and haemodynamic status did not deteriorate further compared with monocrotaline-injected sedentary rats. The eccentric maximal speed decline was less pronounced in trained compared with sedentary pulmonary arterial hypertension rats.

Conclusions

Eccentric exercise training had no detrimental effects on right heart pressure, cardiac function and survival in rats with stable monocrotaline-induced pulmonary hypertension.

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Résumé
Contexte

Un entraînement de type excentrique permet d’améliorer la force musculaire sans stimulation cardiovasculaire excessive. De ce fait, cette modalité d’exercice mérite d’être testée en cas d’hypertension artérielle pulmonaire.

Objectif

Nous avons étudié les effets d’un entraînement de type excentrique sur la survie et la fonction cardiaque d’un modèle d’hypertension artérielle pulmonaire avec dysfonction ventriculaire droite.

Méthodes

Au total, 40 rats ont été randomisés dans quatre groupes : 40mg/kg monocrotaline-sédentaire, 40mg/kg monocrotaline-excentrique, témoin-sédentaire, témoin-excentrique. Les rats ont été entraînés pendant 30min, 5jours sur sept, pendant quatre semaines. Ils couraient sur un tapis incliné à –15° (course en descente). L’effet de l’entraînement a été évalué par l’échocardiographie, le cathétérisme cardiaque droit et la vitesse maximale de course.

Résultats

La survie des rats monocrotaline-excentriques et monocrotaline-sédentaires ne différait pas. L’évolution des paramètres hémodynamiques des rats monocrotaline-excentriques a été similaire à celle des rats monocrotaline-sédentaires. La dégradation de la vitesse maximale de course a été moins marquée dans le groupe monocrotaline entraîné que dans le groupe des rats sédentaires.

Conclusion

L’entraînement de type excentrique n’a pas d’effet délétère sur la fonction cardiaque droite, la pression ventriculaire droite et la survie de rats ayant une hypertension artérielle pulmonaire peu évolutive induite par la monocrotaline.

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Keywords : Pulmonary hypertension, Monocrotaline, Eccentric exercise, Training, Echocardiography, Right ventricular dysfunction

Mots clés : Hypertension pulmonaire, Monocrotaline, Exercice excentrique, Entraînement, Échocardiographie, Dysfonction ventriculaire droite

Abbreviations : CLecc, CLsed, LV, MTecc, MTsed, PAAT, PAH, RV, RV EDD, TAPSE, Vmax , VO2


Background

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodelling, which results in an increase in vascular resistance and, progressively, in right ventricular (RV) hypertrophy. Eventually, RV dysfunction and subsequent failure lead to incapacity of the heart to maintain cardiac output, exercise intolerance, poor quality of life and premature death [1, 2]. Advances in pharmaceutical therapies have facilitated improvements in haemodynamics, exercise capacity and patient survival [3, 4]. However, exertional dyspnoea is often persistent, despite adequate treatment, and results in difficulty in performing day-to-day activities. As peripheral muscular deconditioning is involved in functional limitation in PAH, and may in part explain dyspnoea [5, 6], cardiopulmonary rehabilitation has been recently proposed for these patients. Historically, PAH patients have been discouraged from performing intense exercise because of the risk of sudden cardiac death or deterioration of RV function. However, a few prospective studies in stable treated patients have suggested that exercise training can be performed without a negative impact on cardiac function or any major adverse events [7, 8, 9, 10, 11]. In the last 2015 European Society of Cardiology/ERS guidelines for the diagnosis and treatment of pulmonary hypertension [12], exercise training was recommended by the experts as adjunctive therapy. It is important to diversify training modalities, in order to improve PAH patients’ adherence to physical exercise and to adapt physical activity to patients with low cardiovascular reserve.

Eccentric exercise is classically used to improve muscle strength and power in healthy subjects and patients. In addition, eccentric exercise muscle contraction has the advantages of low metabolic cost and then low cardioventilatory solicitation [13]. For a given level of oxygen consumption (VO2 ), eccentric exercise contractions generate higher muscular forces than concentric contractions, and substantial neural adaptations. As a result, for a given level of VO2 , eccentric exercise training can result in greater increases in muscle strength than concentric training, which in turn translate into an increase in skeletal muscle size and strength [14, 15]. It has been shown that it is possible to increase the size and strength of skeletal muscle with eccentric exercise training at a training intensity that is insufficient to have any structural or functional impact on muscles when trained in a concentric manner [15]. Therefore, patients whose low cardiovascular reserve prevents the maintenance of sufficient training intensity using concentric modalities may still be able to improve muscle strength using eccentric exercise training modalities.

Concentric contractions are characterized by muscle fibres shortening (the quadriceps contracts concentrically in order to climb hills or stairs, or to cycle), and eccentric exercise contractions are characterized by muscle fibres lengthening (the quadriceps contracts eccentrically in order to go down hills or stairs, or when braking). Interestingly, a muscle can store energy during a brief stretch. The post-stretch energy release allows a reduction in the metabolic energy expenditure of eccentric exercise contractions [16]. In this paper, we use the term “eccentric exercise”, but we agree that the use of “negative work”, (as proposed by Padulo et al. [17]), related to work induced by lowering or decelerating, may be more appropriate. Indeed, for a given movement, some muscles are in eccentric exercise mode, whereas antagonists contract concentrically to allow movement precision. In animals, eccentric exercise has been based mainly on downhill running [18, 19] or an isokinetic test device [20].

Eccentric exercise training could be proposed for PAH patients on the condition that it has no deleterious cardiac effect. In this experiment, our main purpose was to assess the effects of eccentric exercise training (downhill running) on haemodynamic and cardiac remodelling variables and survival in an experimental monocrotaline-injected model of PAH. We selected a rat model of “stable” PAH for which concentric training has been shown to be beneficial [21]. The exercise training consisted of 30-minute sessions of downhill running, at 50% of each rat's maximal running speed (Vmax ), for 5 days per week.

Methods
Experimental model

Experiments were performed on 40 adult male Wistar rats (Centre d’Élevage Dépré, Saint Dulchard, France) weighing 224±8g. PAH was induced by a single subcutaneous injection (40mg/kg body weight) of monocrotaline (Sigma Medical, St Louis, MO, USA), to mimic stable PAH [21]. Control rats were injected with the same volume of saline solution. Animals were housed in a neutral temperature environment (22±2°C) on a 12-hour light-dark cycle, and received food and water ad libitum. Rats were weighed twice a week. Procedures were conducted in accordance with the US National Institutes of Health guidelines, and were approved by the Ethics Committee of the University of Strasbourg (Reference number: AL/02/13/07/09).

Experimental design

Animals were randomly assigned to one of two study groups: a monocrotaline-injected group and a control group. In the 2 weeks following injection, all rats were made familiar with the treadmill (Panlab S.L., Barcelona, Spain), and ran for 5minutes, three times a week, at a constant speed of 15cm/s, with no slope. Mild electric stimulation was used to encourage the rats to run. After those 2 weeks, all animals performed a maximal incremental running test on the treadmill with a −15° slope, the speed being increased by 10cm/s every 90seconds to maximum tolerance. A rat was considered to be exhausted when the animal accepted the electric stimulus of the treadmill three consecutive times. The animals were then randomly allocated to an eccentric exercise training group (monocrotaline-injected eccentric-trained [MTecc], n =13 rats; or eccentric-trained control [CLecc], n =7 rats) or a sedentary group (monocrotaline-injected sedentary [MTsed], n =13 rats; or sedentary control [CLsed], n =7 rats) (Figure 1). The eccentric exercise training consisted of downhill running with the treadmill set at a −15° slope for 33minutes, 5 days a week for 4 weeks (3minutes of warm-up at a constant speed of 15cm/s, and 30minutes at a constant speed of 50% of the Vmax reached by each rat). We showed in our laboratory that for eccentric exercise, 50% of the Vmax corresponds to about 65% of maximal VO2 (unpublished data). The maximal eccentric exercise incremental test was repeated after a 2-week training period, and at the end of the training programme. Training speed was adapted after the second maximal test for each rat, to keep with the 50% Vmax goal. The sedentary rats (CLsed and MTsed) performed the three maximal exercise tests, and ran on the downhill treadmill for 5minutes, three times a week, at 15cm/s, with the same downhill slope (−15°), for 4 weeks.



Figure 1


Figure 1. 

Study design. Eccentric (ECC) training started 2 weeks after injection. The delay between the third echocardiograph and sacrifice was 5–7 days. 2w, 3w, 4w, 5w and 6w: 2, 3, 4, 5 and 6 weeks after injection, respectively; CL: control rats; CLecc: eccentric-trained control rats; CLsed: sedentary control rats; Echo: echocardiographic evaluation; Max speed: maximal eccentric speed based on an incremental running test; MT: monocrotaline-injected rats; MTecc: monocrotaline-injected eccentric-trained rats; MTsed: monocrotaline-injected sedentary rats; RVcath: right ventricular catheterization.

Zoom

Haemodynamic evaluation
Echocardiography

Echocardiography (Agilent Technologies, Andover, MA, USA; equipped with a 12MHz linear transducer) was performed every 2 weeks after injection, on anaesthetized but spontaneously breathing rats in a supine position. Anaesthesia was induced by inhaled isoflurane at 3.5% (Aerrane™; Baxter S. A. S., Maurepas, France) in an induction chamber, and maintained at 2% using a facemask. The average duration of anaesthesia was 15–20minutes. Three consecutive measures of all variables were performed online, and then averaged. Measured variables for RV remodelling and function were: RV end-diastolic diameter (RV EDD); RV free wall thickness; right ventricle/left ventricle (RV/LV) ratio; and tricuspid annular plane systolic excursion (TAPSE; used as an index of systolic RV function). Pulsed-wave Doppler pulmonary outflow was recorded in the short-axis parasternal view, and the pulmonary artery acceleration time (PAAT; in ms) was measured. Cardiac output (mL/min) was calculated as TVIAo ×HR×SAo , where TVIAo is the time-velocity integral of the pulsed-wave Doppler aortic outflow, HR is the heart rate and SAo is the aortic surface (calculated as π×[D2/4]; D is the aortic diameter just below the aortic valve in the parasternal view). Stroke volume was the ratio between cardiac output and heart rate.

RV catheterization of closed-chest rats

At the end of the study protocol (24hours after the previous training session), closed-chest catheterization was performed under general anaesthesia, but in spontaneously breathing animals, as previously described [22]. RV pressures were recorded by using a fully filled catheter connected to a pressure transducer and physiological recorder (ML 818 PowerLab 15T; ADInstruments, Oxford, UK). RV systolic pressure was assumed to be equal to pulmonary arterial systolic pressure.

Autopsy

After RV catheterization, anaesthesia was maintained with isoflurane 2.5%, and both ventricles and the lung were isolated and weighed. The ratio of RV weight to LV plus septum weight (RV/[LV+septum]) was used as an index of RV hypertrophy and PAH severity. The lung was weighed immediately after sacrifice to obtain wet lung mass. We dried the lung for 60minutes at 120°C on a heater plate. After 24hours at an ambient temperature and humidity, the lung was weighed.

Statistical analysis

Data are expressed as mean±standard error of the mean. Data were analysed using two-way (effects of monocrotaline injection and training were studied) or three-way (monocrotaline administration, training and time effects were studied) analysis of variance followed by a Neuman-Keuls post-hoc test. These analyses were performed on the rats that completed all measurements (echocardiography, exercise test). A Kaplan-Meier analysis and log-rank test were used to evaluate and compare survival in the four groups. Values of P <0.05 were considered as significant.

Results
Survival at 6 weeks

In the monocrotaline-injected group, 11/13 MTsed rats and 10/13 MTecc rats were alive at the end of the study (non-significant difference). Four rats died during the fourth week after injection (MTsed group, n =2; MTecc group, n =2), and one died during the fifth week (MTecc group). All of the control animals survived (Figure 2).



Figure 2


Figure 2. 

Survival analysis. No significant difference was observed between monocrotaline-injected eccentric-trained rats (MTecc) and monocrotaline-injected sedentary rats (MTsed). CLecc: eccentric-trained control rats; CLsed: sedentary control rats.

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Before training (2 weeks after injection)

No difference in maximal speed was found between control and monocrotaline-injected rats (78.8±3.0cm/s vs 79.2±2.6cm/s; non-significant difference). The first echocardiography showed a small increase in RV wall thickness in monocrotaline-injected rats, indicating the beginning of RV remodelling without signs of cardiac dysfunction (0.72±0.05 in monocrotaline-injected rats vs 0.63±0.05mm in control rats; P <0.001) (Figure 3).



Figure 3


Figure 3. 

Echocardiographic variables. These data were recorded in rats that completed the three echocardiographic evaluations. 2w, 4w and 6w: 2, 4 and 6 weeks after injection, respectively; CLecc: eccentric-trained control rats; CLsed: sedentary control rats; MTecc: monocrotaline-injected eccentric-trained rats; LV: left ventricle; MTsed: monocrotaline-injected sedentary rats; PAAT: pulmonary artery acceleration time; RV: right ventricle; RV EDD: right ventricular end-diastolic diameter; SV: stroke volume; TAPSE: tricuspid annular plane systolic excursion. **P <0.01 for monocrotaline-injected versus control rats. ***P <0.001 for monocrotaline-injected versus control rats. $$P <0.01 for monocrotaline-injected rats at 4wk versus 2wk and 6wk versus 2wk. $$$P <0.001 monocrotaline-injected rats at 4wk versus 2wk and 6wk versus 2wk. #P <0.05 for monocrotaline-injected rats at 6wk versus 4wk. ##P <0.01 for monocrotaline-injected rats at 6wk versus 4wk.

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At mid-training (4 weeks after injection)

During the fourth week after the monocrotaline injection, most of the monocrotaline-injected rats had a critical general appearance, and showed a lack of spontaneous activity, tachypnoea and piloerection. Among the rats that passed away during the whole study period, 2 of 3 MTecc rats and 2 of 2 MTsed rats died during this fourth week. These clinical features were concomitant with a significant decrease in maximal speed measured during the incremental test (Figure 4), and with a deterioration in echocardiography-derived variables (Figure 3). The results shown in Figure 3 were obtained from 14 control rats and 21 monocrotaline-injected rats that completed the three echocardiographic evaluations (at 2, 4 and 6 weeks). Significant increases in RV wall thickness and RV EDD, and decreases in TAPSE, PAAT and stroke volume, were observed in both the MTecc and MTsed rats compared with in control rats (P <0.001). We also noted an increase in LV wall thickness (1.41±0.02mm in monocrotaline-injected rats compared with 1.19±0.03mm in control rats; P <0.01) and septum thickness (1.42±0.03mm in monocrotaline-injected rats compared with 1.25±0.04mm in control rats; P <0.001). In monocrotaline-injected rats, these variables had deteriorated significantly at 4 weeks compared with at 2 weeks (P <0.01). Two weeks of eccentric exercise training had no effect on echocardiography-derived haemodynamic variables in MTecc and CLecc rats compared with MTsed and CLsed rats.



Figure 4


Figure 4. 

Eccentric maximal speed (%) at 2 weeks (2wk), 4 weeks (4wk) and 6 weeks (6wk) after injection. These data were recorded in the rats that completed the three exercise tests. CLecc: eccentric-trained control rats; CLsed: sedentary control rats; MTecc: monocrotaline-injected eccentric-trained rats; MTsed: monocrotaline-injected sedentary rats. ***P <0.001 for monocrotaline-injected versus control rats. £P <0.05 for CLecc versus CLsed. §P <0.05 for MTecc vs MTsed.

Zoom

After training (6 weeks after injection)

The clinical appearance of monocrotaline-injected rats improved slightly during the final 2 weeks. Maximal speed increased by 9% in CLecc rats, from 84±3cm/s to 91±5cm/s (P <0.05), and was unchanged in CLsed rats. Maximal speed decreased by 11.5% in MTecc rats (Figure 5), whereas this variable decreased by 27% (P <0.05) in MTsed rats compared with measurements at 2 weeks.



Figure 5


Figure 5. 

Right ventricular systolic pressure (RVSP) measured during RV catheterization at the end of the study. CLecc: eccentric-trained control rats; CLsed: sedentary control rats; MTecc: monocrotaline-injected eccentric-trained rats; MTsed: monocrotaline-injected sedentary rats. ***P <0.001 for monocrotaline-injected versus control rats.

Zoom

Most of the echocardiography-derived variables (RV/LV ratio, RV EDD, PAAT, stroke volume and TAPSE) improved by the end of the study compared with measurements taken 4 weeks after the monocrotaline injection, in both MTecc and MTsed rats (P <0.05) (Figure 3). However, these variables did not return to baseline values. In monocrotaline-injected rats, the septum and LV free wall thicknesses did not differ from those of control rats. Echocardiography data for MTecc rats did not differ from those for MTsed rats. RV catheterization (Figure 5) confirmed the PAH status of monocrotaline-injected rats, but no training effect was observed (RV systolic pressure was 42±3mmHg in MTsed rats vs 36±4mmHg in MTecc rats; non-significant difference).

Anatomical variables

As expected, the increase in body weight in monocrotaline-injected rats was slower than that in control rats. CLecc rats had a lower body weight than CLsed rats (P <0.05), but no difference was seen between MTecc and MTsed rats (Table 1). At the end of the study, RV weight, in absolute and relative value normalized by body mass, was higher in monocrotaline-injected rats than in control rats, as were the RV/[LV+septum] ratio, lung weight and the dry/wet lung ratio. The RV/body weight and LV/body weight ratios were higher in MTecc rats than in MTsed rats (P <0.05 and P <0.01, respectively). Training had no effect on the RV/[LV+septum] ratio, lung weight normalized by body mass or the wet/dry lung ratio. Taken together, these observations suggest that pulmonary vascular remodelling rather than oedema induces increased lung weight in monocrotaline-injected rats [21], and that eccentric exercise does not induce pulmonary oedema.

Discussion

This is the first study to investigate the effects of an eccentric exercise training modality in this rat model of “stable” PAH. We specifically focused on RV remodelling, haemodynamic changes and the survival of monocrotaline-injected rats. The results showed that in trained PAH rats: this training modality was well tolerated and survival was no different to that in sedentary PAH rats; RV remodelling did not worsen and haemodynamic status did not deteriorate further compared with sedentary PAH rats; the decline in the maximal speed of downhill running was less pronounced in trained than in sedentary PAH rats.

Limiting exercise-induced cardioventilatory solicitation may be of interest in pulmonary hypertension. Indeed, RV remodelling is directly related to exercise intensity, cardiac output, RV afterload and RV wall stress, in both animals and humans [23, 24]. The repetition of intense exercise sessions increases the detrimental pressure overload-associated RV inflammation in the poorly adapted RV [21]. Therefore in the present experimental study, we chose an eccentric exercise modality, the benefits of which are the combination of high muscle work with low energy cost [25] and low cardioventilatory solicitation, in comparison with concentric exercise.

Eccentric exercise training tolerance

We showed that in monocrotaline-injected rats, 6 weeks after injection, mortality induced by eccentric exercise training increased, and was close to the 12–20% reported in two studies using the same dose of monocrotaline [21, 26]. The first echocardiographic evaluation was performed 2 weeks after monocrotaline injection, to verify that indirect signs of PAH had developed, and did, in fact, demonstrate a small but significant increase in RV free wall thickness. Signs of RV dysfunction and adverse remodelling confirmed firmly established PAH at 4 and 6 weeks. Nevertheless, echocardiographic variables did not differ according to training status. Eventually, RV catheterization results were similar in MTecc and MTsed rats at 6 weeks. However, it should be underlined that echocardiographic and catheterization measurements were made at rest and under anaesthesia, and could not be performed during exercise tests. Differences between trained and untrained rats might indeed have been revealed by exercise. As a complement to this study, measurements of pulmonary artery wall thickness and RV catheterization in a subset of animals at 4 weeks may be of interest. Indeed, in a previous study, concentric training was found to be beneficial in “stable” PAH but detrimental in “progressive” PAH. Interestingly, after training, RV hypertrophy has been found to be similar in “stable” and “progressive” PAH, whereas pulmonary vascular remodelling was stronger in “progressive” PAH [21]. The time courses of pulmonary artery and RV hypertrophy may be dissociated and deserve to be studied.

Beneficial effects of eccentric exercise training

In addition to non-deleterious effects, we showed some beneficial consequences of eccentric exercise training. The decrease in Vmax was less marked in MTecc rats than in MTsed rats. Part of the increase in Vmax in monocrotaline-injected rats might be the result of the small improvement in cardiac function observed at 6 weeks compared with at 4 weeks [27]. However, this does not explain the difference between MTsed and MTecc rats. Cardiac function did not differ in MTsed and MTecc rats. Consequently, it is likely that the increase in Vmax is mainly attributable to improved neuromuscular function. This is in line with previous findings showing that maximal VO2 did not change after eccentric exercise training in patients with chronic heart failure, whereas peak work rate and maximal strength of the triceps surae were improved [28]. In addition, in rats, concentric but not eccentric exercise training, performed at similar mechanical power output, increased skeletal muscle oxidative capacities [29]. The mechanisms underlying the neuromuscular effects of eccentric exercise training are not fully understood, but several assumptions have been made. Firstly, weak areas of certain muscle fibres may be eliminated after initial bouts of eccentric exercise, and a more resilient structure may be built [13]. Recently, differences in muscle architectural adaptations to eccentric exercise and concentric contractions have indeed been described [30]. Secondly, improvement in muscle activation and neural drive, including a training-associated better contribution of synergistic muscles, probably plays an important role [31]. Thirdly, storage and recovery of elastic strain energy has been shown to improve in response to eccentric exercise training; this has been attributed to changes in the characteristics of titin, a muscle cell protein, as well as other elastic elements, such as the tendon [32].

Eccentric exercise protocol design

Intense eccentric exercise is known to induce muscle damage and pain [25], and eccentric exercise training programmes have to find a balance between efficiency and occurrence of muscle damage. In healthy rats, concentric training programmes are usually based on a frequency of 5 days/week, and a session duration of 30minutes [33]. This frequency and session duration have also been found to be well tolerated and efficient when used in an eccentric exercise-training programme [34]. As eccentric exercise training has been tested in very few studies, and in healthy rats only, we confirmed in preliminary experiments that this frequency and session duration could apply to PAH rats. We selected a −15° treadmill slope because mixed eccentric exercise and concentric exercise is obtained if the slope is less than −15° [35]. On the other hand, it was impossible for animals to run with the treadmill slope set at more than −15° because of the downward slide on our device. Regarding intensity, we set the downhill running speed at 50% of Vmax , which allowed rats to complete the 30-minute session with signs of exhaustion, but did not jeopardize the next day's session tolerance. In our laboratory we demonstrated in control Wistar rats that running at 50% of Vmax with a −15° slope corresponds to 65% of maximal VO2 (unpublished results). In the present study, the training running speed was set at 50% of each rat's Vmax . To take into account the effects of training and disease progression in monocrotaline-injected rats, the individual training running speed was readjusted to 50% after revaluation of the Vmax at mid-training; this allowed the monocrotaline-injected rats to complete the 30-minute session by the end of the 4-week training period. At the end of each session, most of the diseased rats were exhausted and breathless. Cardiorespiratory solicitation is regarded as moderate at 65% of maximal VO2 . However, even at this moderate exercise intensity, desaturation may occur and limit exercise capacity [36, 37], and deserves to be investigated. In contrast, we feel that a higher intensity and/or longer session duration might have been well tolerated by CLecc rats. However, in order to compare MTecc and CLecc, we used similar exercise modalities in the two groups.

Time course of echocardiographic variables

The protocol design we used led to an unexpected finding. In the monocrotaline-injected rats, the echocardiographic functional and morphological variables showed more pronounced deterioration at 4 weeks than at 6 weeks. In addition, the morphological anomalies of the LV were present at 4 weeks and disappeared at 6 weeks. To our knowledge this is the first time that serial echocardiographs have been performed, allowing this particular time course to be described in 40mg/kg monocrotaline-injected rats. Echocardiographic signs were accompanied by clinical deterioration, Vmax diminution and a significant increase in deaths in the monocrotaline-injected group. These results may be explained by the beginning of the reduction of pulmonary arteriolar abnormalities. Indeed, in a recent study, media muscularization in the smallest arterioles had increased at 4 weeks, but returned to control values 8 and 12 weeks after monocrotaline administration, accompanied by an improvement in RV function [27].

PAH model

Monocrotaline injection results in structural remodelling of pulmonary vessels. Pulmonary artery and then RV-induced alterations are dose related: the injection of a low dose of 30mg/kg monocrotaline provokes compensated RV hypertrophy with no signs of failure, whereas the administration of 60–80mg/kg monocrotaline allows RV hypertrophy to progress until failure, with death usually occurring 25–28 days after injection [38, 39]. Handoko et al. [21] demonstrated that the injection of 40mg/kg monocrotaline into rats induces “stable” PAH with preserved cardiac output over 6–8 weeks. Consequently, we chose to use this model in our experiment. However, according to the time course of echocardiographic variables that we have described, the stability of the 40mg/kg monocrotaline model may be questioned. Indeed, others and we found a temporary clinical and cardiac deterioration 4 weeks after monocrotaline injection [27]. Eventually, it would be of interest to evaluate eccentric exercise training in more progressive PAH, as this particular exercise is characterized by lower cardiac solicitation for a given muscle work, compared with concentric exercise. Finding the monocrotaline dose that allows reasonable mortality rates with slow degradation of RV function may be difficult [39]. The alternative would be to use other PAH models characterized by slow degradation of RV function [40, 41, 42].

Conclusions

To conclude, we have shown that eccentric exercise training has no detrimental effects on right heart pressure and remodelling, cardiac function and the survival of rats with stable monocrotaline-induced PAH. The mechanisms leading to the higher Vmax that we observed in MTecc rather than MTsed rats need to be further investigated, but are probably of neuromuscular origin. An eccentric exercise training modality, aimed primarily at increasing muscle strength, may be complementary to concentric training, or used in PAH patients whose low cardiovascular reserve prevents the maintenance of sufficient concentric training intensity.

Sources of funding

This study was partially supported by a Young Scientist grant from Glaxo-Smith-Kline; the sponsor had no involvement in the study design, the collection, analysis and interpretation of data, the writing of the manuscript or the decision to submit the manuscript for publication.

Disclosure of interest

The authors declare that they have no competing interest.

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