Long-term leucine supplementation aggravates prolonged strenuous exercise-induced cardiovascular changes in trained rats
Abstract
New Findings
What is the central question of this study?
Can long-term leucine supplementation prevent prolonged strenuous endurance exercise induced cardiac injury?
What is the main finding and its importance?
Prolonged endurance exercise does not seem to exceed cardiac energetic capacity, hence it does not represent an energy threat to this organ, at least in trained subjects. However, it may induce, in susceptible individuals, a state of cardiac electrical instability, which has been associated with ventricular arrhythmias and sudden cardiac death. This situation might be worsened when combined with leucine supplementation, which leads to increased blood pressure and cardiac injury. Leucine supplementation failed to prevent cardiac fatigue symptoms and may aggravate prolonged strenuous exercise-induced cardiovascular disturbances in trained rats.
Observational studies have raised concerns that prolonged strenuous exercise training may be associated with increased risk of cardiac arrhythmia and even primary cardiac arrest or sudden death. It has been demonstrated that leucine can reduce prolonged exercise-induced muscle damage and accelerate the recovery process. The aim of this study was to investigate the effects of prolonged strenuous endurance exercise on cardiovascular parameters and biomarkers of cardiac injury in trained adult male rats and assess the use of leucine as an auxiliary substance to prevent the likely cardiac adverse effects caused by strenuous exercise. Twenty-four male Wistar rats were randomly allocated to receive a balanced control diet (18% protein) or a leucine-rich diet (15% protein plus 3% leucine) for 6 weeks. The rats were submitted to 1 h of exercise, 5 days per week for 6 weeks. Three days after the training period, the rats were submitted to swimming exercise until exhaustion, and cardiac parameters were assessed. Exercising until exhaustion significantly increased cardiac biomarker levels, cytokines and glycogen content inhibited protein synthesis signalling and led to cardiac electrical disturbances. When combined with exercise, leucine supplementation led to greater increases in the aforementioned parameters and also a significant increase in blood pressure and protein degradation signalling. We report, for the first time, that leucine supplementation not only fails to prevent cardiac fatigue symptoms, but may also aggravate prolonged strenuous exercise-induced cardiovascular disturbances in trained rats. Furthermore, we find that exercising until exhaustion can cause cardiac electrical disturbances and damage cardiac myocytes.
Introduction
It is well established that exercise training induces a variety of cardiovascular adaptations that lead to enhanced sporting performances and health benefits. A meta-analysis quantifying the dose–response relationship between physical activity and risk of coronary heart disease stated that even low- to moderate-intensity leisure-time exercise could induce cardioprotection (Sattelmair et al. 2011). Despite such positive outcomes, numerous observational studies have raised concerns that prolonged strenuous exercise training may be associated with increased risk of cardiac arrhythmia and even primary cardiac arrest or sudden death (George et al. 2008; Benito et al. 2011; O'Keefe et al. 2012).
Given that prolonged exercise has been reported to result in skeletal muscle fatigue and damage, as well as reduced performance, it seems reasonable to expect the same response, namely cardiac fatigue, from cardiac muscle (Dawson et al. 2003). Concerns over the clinical consequences of individual acute bouts of prolonged exercise are often dismissed because the changes reported are small and transitory; however, there has been speculation in recent years that prolonged exercise, whether in a single exposure or in a lifetime of activity, may have some negative consequences for cardiovascular performance or health (George et al. 2008). These prolonged strenuous exercise-induced cardiac disturbances can be attributed to physiological and metabolic factors. Physiological factors include changes in the membrane permeability of cardiomyocytes, left ventricular dysfunction, elevations in cardiac-specific biomarkers and metabolic factors from energy substrate depletion, and cardiac electrical abnormalities (Sahlén et al. 2009; Bhella & Levine, 2010).
It has been demonstrated that branched chain amino acids, particularly leucine, can reduce prolonged exercise-induced muscle damage and accelerate the recovery process (Greer et al. 2007). Furthermore, leucine seems to be the most potent amino acid regarding the effects on protein synthesis and degradation and not only provides substrates for gluconeogenesis, but can also supply the tricarboxylic acid cycle with different anaplerotic substrates (Li et al. 2011). Earlier findings (Crowe et al. 2006) showed that 6 weeks of dietary leucine supplementation significantly improved endurance performance and upper body power in outrigger canoeists. Although the effects of leucine on skeletal muscle, i.e. prevention of muscle damage and fatigue, are well established, it is not known whether the same effects occur with cardiac muscles.
The aim of this study was to investigate the effects of prolonged strenuous endurance exercise on cardiovascular parameters and biomarkers of cardiac injury in trained adult male rats and assess the use of leucine as an auxiliary substance to prevent the likely cardiac adverse effects caused by strenuous exercise. Given that the current evidence is promising, we hypothesized that prolonged strenuous exercise would induce cardiac disturbances (electrical, structural and biochemical) and that leucine supplementation would prevent, or at least mitigate, those outcomes.
Methods
Ethical approval
All the experimental procedures used were in accordance with the Ethics Committee on Animal Experimentation of Unicamp (CEEA/IB/UNICAMP, protocol 2888-1). Animals were killed while under general anaesthesia [90 mg (kg body weight)−1 ketamine (Ketalar®, Pfizer, New York, NY, USA), i.p. and 45 mg (kg body weight)−1, xylazine, (Rompun®, Bayer, Leverkusen, Germany), i.p.], and all the procedures (ECG, blood pressure measurement and gastrocnemius harvest) were performed under general anaesthesia. The level of anaesthesia was assessed by the position of the eye and the degree of depression in the eye's protective reflexes and tail reflex. Two rats received two doses of anaesthetic agents, with the second dose being approximately 10–20% of the initial dose, when the duration of the procedure exceeded 25 min. The animals were killed by cardiac puncture (i.e. exsanguination), and death was confirmed by heart harvest.
Animals and diets
Twenty-four male Wistar rats (12 weeks old, weighing 351 ± 28.87 g) were obtained from the animal facilities of the University of Campinas (São Paulo, Brazil) and divided equally into four groups. They were housed in collective cages at 22–24°C on a 12 h–12 h light–dark cycle, with free access to tap water and food. The semi-purified isocaloric diets were a normal protein diet (C), containing 18% protein (Reeves et al. 1993); or a leucine-enriched diet (L), containing 15% protein plus 3% l-leucine. Approximately 70% carbohydrate (sucrose, dextrin and starch), 7% fat (soybean oil) and 5% fibre (purified microcellulose) were added to the diets. Vitamin and mineral mix, as well as cystine and choline, supplemented the diets. The control diet had 1.6% l-leucine, and the leucine-rich diet contained 4.6% l-leucine, according to a previous study from our group (Cruz & Gomes-Marcondes, 2014). A leucine-supplemented diet has led to a significant increase in the plasma leucine concentration in fetuses from tumour-bearing pregnant mice (Viana & Gomes-Marcondes, 2013) and adults rats (data not shown). Two groups were fed the control diet [sedentary control (C) and trained (T)], and two more groups were fed the leucine-rich diet [control, leucine supplemented (CL) and trained, leucine supplemented (TL)].
Training protocol
The T and TL groups were submitted to a swimming protocol adapted from Barbosa Dos Santos et al. (2013), 5 days per week for 6 weeks, in a water tank (90 cm × 70 cm × 70 cm with water temperature 31 ± 1°C). All of the rats were adapted to the water during the first week of the experiment. The adaptation process consisted of keeping the animals in shallow water, initially for 20 min, and progressively increasing by 10 min day−1 and 10 cm water day−1 for 5 days. Exercise sessions began with 60 min day−1 in the second experimental week, carrying constant loads (added to the tail) of 20 g (∼6% of initial body weight). Initially, the rats carried the load for the first 10 min of these 60 min, increased by 10 min each week, until it reached 60 min of loaded swimming training in the sixth and final week of experiment. Three days after the exercise-training period, rats were submitted to swimming exercise carrying the same load until exhaustion. The time to exhaustion was determined from the beginning of swimming to the point at which rats failed to return to the water surface within 10 s. Animals were killed under anaesthesia between 3 and 4 h after this final exercise bout, in order to achieve peak cytokine concentrations after the exhaustive swimming test (Suzuki et al. 2002; Louis et al. 2007).
Electrocardiogram
An ECG recording was made 1 day before the beginning of training protocol and 1 h after the exhaustive test, or the experimental period for control groups. Anaesthetized rats were maintained in the supine position with spontaneous breathing for ECG recording. The ECG was recorded with three needle electrodes placed subcutaneously on the right and left chest and the left foot. The electrodes were connected to the computerized four-channel ECG MLS360/7 ECG Analysis Module (AD Instruments, NSW, Australia), and there were six minutes of ECG recording (Barbosa Dos Santos et al. 2013).
Blood pressure
To determine arterial systolic and diastolic blood pressures, under general anaesthesia, a cannula was inserted into the left femoral artery and connected to a BP-1-Analog single-channel transducer signal conditioner (World Precision Instruments, USA; Kang et al. 2006). The BP cannula was implanted only once (BP measurement was performed only at the end of the experimental period), immediately before the animal was killed.
Glycogen content
Hearts were quickly removed, frozen immediately in liquid nitrogen and stored at −80°C until further analysis. Cardiac glycogen content was estimated colorimetrically based on the method described by Lo et al. (1970). The absorbance was read on a plate CHAMELEON V Multilabel Microplate Reader (Hidex, Turku, Finland) at 620 nm.
Citrate synthase activity
For citrate synthase analyses, ∼30 mg of cardiac muscle was homogenized in ice-cold extraction buffer (175 mm KCl and 2 mm EDTA, pH 7.4) and centrifuged at 16,000g for 20 min at 4°C. An aliquot of supernatant was combined with a reaction mixture containing 0.1 m Tris, pH 8.3, 1 mm Dinitrothiobis (DTNB) and 3 mm acetyl-CoA. The reaction was initiated by adding 10 mm oxaloacetic acid to the extract. The absorbance was measured spectrophotometrically at 412 nm in 30 s intervals for 5 min using a Dynex MRX plate reader controlled through personal computer software (Revelation; Dynatech Laboratories, Houston, Texas, USA), as previously described (Srere, 1969). All samples were tested for linearity up to 5 min of reaction, and values were normalized by protein concentration (Bradford, 1976).
Western blot
Heart samples (40 μg) were homogenized, and the protein concentration was measured using a colorimetric method (Bradford, 1976). The proteins were revealed using primary antibodies against α-tubulin (#T5168, 1:20,000 dilution; Sigma-Aldrich, St. Louis, MO, USA), phospho-mTORSer2448 (#2971, 1:1000; Cell Signaling, Danvers, MA, USA), phospho-AktThr308 (#4056, 1:1000; Cell Signaling), phospho-AMPK-αThr172 (#2535, 1:1000; Cell Signaling) and proteasome subunits 20S and 19S (#BML-PW8195 and #BML-PW9265, 1: 1.000; Enzo, Farmingdale, New York, USA) and secondary anti-mouse, anti-rabbit and anti-goat antibodies (1:10,000; Cell Signaling). After reaction with a chemiluminescent reagent (Thermo Fisher Scientific, Waltham, MA, USA), the band volume was captured using Alliance Captura 2.7 (UVItec, Cambridge, UK) and quantified using UVI band −1D (UVItec) gel analysis software.
Specific cardiac biomarkers
Blood samples were taken from the heart by ventricular puncture. Serum was separated by centrifugation at 1000g for 10 min at 4°C and stored at −80°C. The analysis of serum cardiac-specific markers [troponin I (cTnI) and troponin T (cTnT)] and serum inflammatory markers [tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] was determined using beads coupled with capture antibodies specific for each protein of interest as specified by the manufacturer, Millipore® (Merck Millipore Corporation, Darmstadt, Germany). The analysis was carried out on Xponent software used with the Luminex® 200 (Luminex Corporation, Austin, TX, USA) equipment, following the manufacturer's technical procedures.
Statistical analysis
The data are expressed as the means ± SD. The data were analysed statistically by ANOVA followed by Tukey's test to establish differences between groups. We used Prism software (Graphpad Software Inc., San Diego, CA, USA). The results were considered significant when P < 0.05.
Results
Cardiac functional parameters
Relative heart weight increased in the T and TL groups when compared with the C and CL groups (Fig. 1). Although collectively there was no significant difference in ECG parameters, individually there were some clinically relevant changes (Fig. 2A and B), which are discussed later. Both arterial systolic and diastolic blood pressures were determined ∼2 h after the last exercise bout. Systolic, diastolic and mean arterial pressures were significantly higher in TL group when compared with all the other experimental groups (Fig. 3).
Cardiac metabolic parameters
The cardiac glycogen content was assessed in order to reveal the metabolic stress (i.e. energetic demand) of exercising until exhaustion and was significantly elevated in trained groups (T and TL) compared with sedentary groups (C and CL). This enhancement was even higher in the TL group when compared with the T group (Fig. 1B).
AMP kinase α (AMPK) was also assessed to measure metabolic stress, because AMPK reflects the energetic status of the cell. AMPK phosphorylation (p-AMPK) did not differ significantly between groups (Fig. 4D).
Citrate synthase activity is the most important biomarker for mitochondrial density in skeletal muscle and biochemical marker of the skeletal muscle oxidative adaptation to a training intervention. Citrate synthase activity did not differ significantly between groups (Fig. 1C).
Cardiac structural parameters: protein synthesis and degradation pathways
In order to evaluate treatment-induced cardiac protein synthesis, we assessed the activation of two main key proteins in the synthesis pathway, namely, Akt and mTOR. Activation of Akt was inhibited in trained groups (T and TL) compared with the control group. Among trained groups, leucine supplementation increased Akt phosphorylation when compared with exercise only (T group; Fig. 4B). Compared with the control group, mTOR activation was significantly inhibited only in the T group (Fig. 4C).
The ubiquitin–proteasome pathway is the most important pathway for protein degradation in cardiac muscle during exercise. To analyse the effect of exercising until exhaustion on the cardiac protein degradation pathway and the modulatory effect of leucine supplementation, we evaluated some key proteins of this process, namely proteasome subunits 19S and 20S (Fig. 5B and C, respectively). Both proteins were elevated only in the TL group when compared with all the other groups.
Cardiac cell damage and systemic inflammation
To determine cardiomyocyte integrity, we assessed the serum concentrations of specific cardiac biomarkers, namely troponin T and troponin I (Fig. 6A and 6B, respectively). Both were significantly elevated in trained groups (T and TL) compared with the sedentary groups (C and CL). This enhancement was even greater in the TL group when compared with the T group.
Discussion
It is generally accepted that leucine supplementation can mitigate endurance exercise-induced skeletal muscle damage and fatigue, accentuate muscle protein synthesis and improve recovery and muscle performance (Rowlands et al. 2015). However, it is unclear whether leucine supplementation would lead to these outcomes in cardiac muscle and whether it could prevent cardiac fatigue. Here we report, for the first time, that leucine supplementation not only fails to prevent symptoms of cardiac fatigue, but may also aggravate prolonged strenuous exercise-induced cardiovascular disturbances in trained rats. Furthermore, we find that exercising until exhaustion can cause cardiac electrical disturbances and cardiac myocyte damage.
Prolonged strenuous exercise presents a unique haemodynamic and metabolic challenge to cardiac muscle and may lead to transient impairment of cardiac function, so-called cardiac fatigue (Sahlén et al. 2009). The primary novel finding of the present study is that leucine supplementation, when combined with prolonged endurance exercise, can induce high blood pressure. This finding was highly unexpected, because endurance exercise has previously been reported to reduce blood pressure in both hypertensive and normotensive human subjects and rats (Whelton et al. 2002; Halliwill et al. 2013). However, leucine has recently been related to a hypertensive response through an mTOR-induced hypothalamic sympathetic stimulation pathway (Harlan et al. 2013). Although a leucine dose-related increase in arterial pressure was found in that study, the leucine was administered intracerebroventricularly. Moreover, previous studies reported higher circulating leucine in hypertensive subjects (Newgard et al. 2009) and correlated plasma leucine concentrations with cardiovascular events (Shah et al. 2010). It is not clear why this haemodynamic response appeared in the present study only when leucine supplementation was combined with prolonged exercise; nevertheless, it is worth mentioning that our exercise protocol can be considered as high intensity (imposed tail weight) and high volume, which could lead to overreaching syndrome and, consequently, to autonomic cardiac dysfunction (Baumert et al. 2006).
Another novel finding of our study was that leucine supplementation combined with chronic high-intensity endurance training can significantly increase myocardial injury when compared with training alone. The arterial hypertensive response found only in the trained, leucine-supplemented group might explain the significant increase in cTnI and cTnT found in this group, because it is well established that hypertension can induce cardiac cell damage and endothelial dysfunction and, ultimately, lead to strokes and cardiovascular events (Vasan et al. 2001). Thus, an arterial hypertensive condition associated with exercising until exhaustion can have an additive effect on cardiac cell damage. Although some might argue that increases in cardiac troponins are only mild and transitory, reflecting a physiological troponin release from the free cytosolic pool rather than damaged contractile elements (Scharhag et al. 2008), this was definitely not the case in the TL group because the cTnT concentration was three times higher than in the T group, and clinically relevant cardiac electrical disturbance was found in some ECG parameters.
Taken collectively, there was no significant difference in ECG parameters between experimental groups. It is important to understand, however, that there is a broad normal range in ECG parameters. Hence, some of them should not be taken collectively; otherwise, clinically relevant cardiac electrical disturbance may not be noted. Moreover, the ECG provides indirect evidence of structural cardiac changes and remodelling that affect automaticity, impulse propagation and other mechanisms of arrhythmia. Thus, individual pre- and postexperimental period analysis should be advised in these studies. A number of studies (Shave et al. 2004; Middleton et al. 2007) have shown that these prolonged exercises inducing diastolic and systolic changes are physiological, mainly in trained subjects (Pelliccia et al. 2000), and our results show that this seems to be true in most cases. However, despite the lack of statistical relevance, we found some important individual changes in both the trained and the trained, leucine-supplemented groups. In our study, one out of five rats of the T group presented significant QTc interval prolongation (from 103 to 207 ms, pre- to post-experimental period, respectively), which is suggestive of increased life-threatening ventricular arrhythmias and risk of sudden death (Straus et al. 2006; Sahlén et al. 2009). Moreover, two out of five rats of the TL group also presented QTc interval prolongation (from 90 to 150 ms and from 50 to 90 ms), and one of them presented prolonged Tpeak-end (from 4 to 18 ms) and an inverted T wave (Fig. 2B), which taken together with the troponin concentrations, strongly suggest cardiac injury and myocardial ischaemia (Sahlén et al. 2009). The transient characteristic of ECG parameter changes reported in previous studies suggests that the impact of prolonged endurance training upon cardiac electrical disturbances is not harmful. Our individual data, however, suggest otherwise. It is likely that some subjects are more susceptible to these prolonged strenuous exercise-induced cardiac disturbances than others, as previously proposed (Sahlén et al. 2009; Aro et al. 2012). Thus, in susceptible individuals (such as those with underlying cardiac disease or with a particular genetic predisposition) these modest and mostly transient cardiac electrical instabilities could, in fact, lead to a significant risk of cardiac events. Our results demonstrated that prolonged endurance exercise combined with leucine supplementation may induce a state of electrical instability, which has been associated with an increased propensity for ventricular arrhythmias and cardiac sudden death.
As expected, the exercise protocols led to an increase in the relative heart weight. This can be explained by increased glycogen content and glycogen molecule-bonded water (Philp et al. 2012). Furthermore, although not assessed, it is reasonable to expect that exercise-induced myofibrillar hypertrophy may have occurred (Rowlands et al. 2015). Endurance exercise led to significant glycogen content enhancement and, when combined with leucine supplementation, this enhancement was even greater.
Furthermore, the endurance exercise training did not increase cardiac citrate synthase activity. Although it is generally recognized that skeletal muscle citrate synthase activity is elevated by endurance exercise training, this seems not to be the case in cardiac muscle. It has been shown (Siu et al. 2003) that 8 weeks of endurance treadmill training did not increase cardiac citrate synthase activity. It was suggested that the myocardium has sufficient pre-existing oxidative capacity to supply the energy required during exercise. Interestingly, prolonged endurance exercise had no effect on the cardiac p-AMPK level. However, these results, as well as all cell signalling pathways, are highly time point dependent. As far as we know, very few studies have examined the cardiac p-AMPK level after endurance exercise, and one of these (Ogura et al. 2011) showed that p-AMPK peaked immediately after 30 min of endurance exercise and returned to the basal level after only 30 min. It is likely that we have failed to coincide with peak exercise-induced phosphorylation of cardiac AMPK-mediated signalling. Additionally, as the glycogen level did not decrease significantly (when compared with the control groups) after exercising until exhaustion, it is expected that AMPK activation will remain unaltered. Taken together, these results suggest that even high systemic metabolic stress induced by prolonged endurance exercise does not exceed cardiac capacity and does not represent an energetic threat for this organ, at least in trained subjects.
Protein synthesis signalling was significantly inhibited after the endurance exercise, and leucine supplementation prevented it. Both Akt and mTOR phosphorylation were significantly reduced in the T group but not in the TL group. In the present study, exercise was carried out until exhaustion, with a mean exercise duration of 3 h 22 min (±10 min). It has been demonstrated previously that cardiac p-mTOR was also diminished during 1 h after 30 min of endurance training (Ogura et al. 2011). Furthermore, a recent study has also demonstrated increased mTOR phosphorylation after endurance exercise and leucine supplementation (Rowlands et al. 2015). Although it has been viewed only in skeletal muscle, it is likely that the cardiac mTOR response to endurance exercise is similar.
Surprisingly, protein degradation signalling was significantly increased only in the trained, leucine-supplemented group (TL group). The reason for this result is not entirely clear, but it seems reasonable to assume that it is a response to hypertension-induced pressure overload (mechanical stress), because it has been demonstrated that the cardiac proteasome system is activated during pressure overload (Cacciapuoti, 2014).
In conclusion, and in contrast to our original hypothesis, leucine supplementation failed to prevent cardiac fatigue symptoms, and may also aggravate prolonged strenuous exercise-induced cardiovascular disturbances in trained rats. The major exercise-induced cardiac disturbances do not appear to be metabolic/energetic but electrical and structural. Prolonged endurance exercise does not seem to exceed cardiac energetic capacity, hence it does not represent an energy threat to this organ, at least in trained subjects. However, prolonged endurance exercise may induce, in susceptible individuals, a state of cardiac electrical instability, which has been associated with ventricular arrhythmias and cardiac sudden death. This situation may be worsened when combined with leucine supplementation, which led to increased blood pressure and cardiac injury. Additional studies are needed to elucidate fully which factors (genetic and/or metabolic) lead to this electrical susceptibility.
References
Additional information
Conflict of interest
None declared.
Author contributions
Conception or design of the work: G.B.S. and M.A.A. Acquisition, analysis or interpretation of data for the work; and drafting the work or revising it critically for important intellectual content: G.B.S., A.G.O., L.A.F.R., M.C.G.-M. and M.A.A. All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.
Funding
We are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for a fellowship granted to G.B.S., and to Ajinomoto Interamericana Indústria e Comércio Ltda (Brazil) for supplying the leucine.
