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Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Laboratory of Experimental Physiology, Porto Alegre, RS, BrazilPost-Graduation Program in Rehabilitation Sciences, (UFCSPA), Porto Alegre, RS, Brazil
Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Laboratory of Experimental Physiology, Porto Alegre, RS, BrazilPost-Graduation Program in Rehabilitation Sciences, (UFCSPA), Porto Alegre, RS, Brazil
Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Laboratory of Experimental Physiology, Porto Alegre, RS, BrazilPost-Graduation Program in Rehabilitation Sciences, (UFCSPA), Porto Alegre, RS, Brazil
Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Laboratory of Experimental Physiology, Porto Alegre, RS, BrazilPost-Graduation Program in Rehabilitation Sciences, (UFCSPA), Porto Alegre, RS, Brazil
Chronic heart failure (CHF) is related with exercise intolerance and impaired nitric oxide (NO) production, which can lead to several functional capacity alterations. Considering the possible superiority of aerobic interval training compared to continuous training and the capacity of l-arginine to restore the NO pathway, the aim of the present study was to investigate whether these treatments are beneficial to exercise capacity, muscle mass preservation and hemodynamic, inflammatory and oxidative stress parameters in CHF rats.
Methods
Thirty-eight male Wistar rats post 6 weeks of myocardial infarction (MI) surgery were randomly assigned into 6 CHF groups: sedentary (SED, n = 6); SED + Arg (n = 7); ACT (n = 8); ACT + Arg (n = 5); AIT (n = 7); AIT + Arg (n = 5). Exercise test capacity (ETC) was performed pre and post 8 weeks of intervention. Supplemented rats received Arg (1 g/kg) by oral gavage (7×/week). Exercise training was performed on a rat treadmill (5×/week). Hemodynamic variables, tissue collection, congestion, inflammatory cytokines, and oxidative parameters were evaluated at the end of protocols.
Results
All trained groups showed a superior exercise capacity compared to SED groups on the post-intervention test (p < 0.0001). Pulmonary congestion was attenuated in AIT and AIT + Arg compared with the SED group (p < 0.05). Left ventricular end-diastolic pressure (LVEDP) was lower in ACT + Arg, AIT, and AIT + Arg groups than SED group (p < 0.05). Association of AIT with Arg supplementation was able to improve hemodynamic responses (left ventricular systolic pressure (LVSP), systolic blood pressure (SBP), +dP/dtmax, and −dP/dtmax (p < 0.05), likewise, decrease muscular and renal lipid peroxidation and tumor necrosis factor (TNF)-α, and increase interleukin (IL)-10/TNF-α plasmatic levels (p < 0.01). Groups that associated aerobic exercise with Arg supplementation (ACT + Arg and AIT + Arg) revealed higher gastrocnemius mass compared to the SED group (p < 0.01).
Conclusions
Both aerobic training protocols were capable to improve aerobic capacity, and the association with Arg supplementation was important to attenuate muscle loss. Moreover, interval training associated with Arg supplementation elicits greater improvements in hemodynamic parameters, contributing to reduction in pulmonary congestion, and demonstrated particular responses in the inflammatory profile and in the antioxidant status.
]. Reduced exercise tolerance is the leading contributor to a poor quality of life in those with CHF. Cardiovascular and ventilatory abnormalities associated with increased activation of neurohumoral systems (i.e., renin-angiotensin-aldosterone and sympathetic systems) are known as the major predictors for early exercise fatigue [
]. In addition, peripheral factors, such as vascular and intrinsic skeletal muscle dysfunctions, inflammation, and increased oxidative stress are apparently correlated with skeletal muscle wasting and exercise intolerance [
]. Abnormalities of the vascular system in CHF can be related to impaired nitric oxide (NO) production. Defects in NO bioavailability might be explained by the low plasmatic levels of its amino acid precursor, l-arginine (Arg) or by reduced endothelial NO synthase expression (eNOS) [
]. Thereby, some studies have been investigating the restoration of Arg by supplementation. Enhanced blood flow through Arg supplementation may be crucial for increasing exercise tolerance or even for increasing nutrient supply to the skeletal muscle [
l-Arginine supplementation causes additional effects on exercise-induced angiogenesis and VEGF expression in the heart and hind-leg muscles of middle-aged rats.
]. Moreover, Arg supplementation, as a treatment in other chronic diseases, has been associated with positive effects on antioxidant enzymes expression and inflammatory responses [
]. Due to this potential role in inflammation, oxidative stress and nutrient supply, l-arginine may also be beneficial to preserve muscle mass.
Further, since exercise intolerance is the main symptom in those with CHF, there are no doubts that exercise training is an indispensable therapeutic approach [
]. Several studies have been conducted in patients with CHF, confirming that physical exercise when performed in stable patients and under professional supervision is a safe tool for reducing hospital admissions and mortality risk. Aerobic exercise training is able to improve functional capacity elicited by molecular changes in multiple organs [
]. Furthermore, moderate-intensity continuous training demonstrates an anti-inflammatory effect in those with CHF. Studies conducted in rats verified reductions in both tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels and also an increase in IL-10 and IL-10/TNF-α ratio levels in plasma or in the skeletal muscle [
Physical exercise improves plasmatic levels of IL-10, left ventricular end-diastolic pressure, and muscle lipid peroxidation in chronic heart failure rats.
Currently, training prescription for cardiac rehabilitation has been discussed, considering intensity, duration, frequency, and caloric expenditure. In this way, aerobic interval training (AIT) has emerged. When the main goal is to achieve high intensities, the training is based in interspersed high-intensity intervals with recovery periods of low to moderate intensities or passive recovery. Consequently, AIT allows heart failure (HF) patients to achieve longer periods in high-intensity zones, compared to aerobic continuous training (ACT) [
]. The AIT protocols have demonstrated to be more effective than ACT for improvements on VO2 peak either in healthy individuals or clinically stable patients with HF [
Effectiveness of high-intensity interval training (HIT) and continuous endurance training for VO2max improvements: a systematic review and meta-analysis of controlled trials.
]. On the other hand, there is limited evidence of AIT on hemodynamic parameters, myocardial hypertrophy, immune function, and redox status in those with CHF [
Effectiveness of high-intensity interval training (HIT) and continuous endurance training for VO2max improvements: a systematic review and meta-analysis of controlled trials.
Effects of high-intensity interval training versus continuous training on physical fitness, cardiovascular function and quality of life in heart failure patients.
Considering the beneficial effects of aerobic exercise training and the time-efficiency of AIT, our aim was to compare both aerobic modalities and to examine the Arg supplementation influence on those therapies outcomes. Thus, the present study was designed to test the effects of 8 weeks of Arg supplementation associated with ACT or AIT on maximal exercise capacity, pulmonary and hepatic congestion, muscle mass, and hemodynamic, inflammatory and oxidative stress parameters in rats with CHF.
2. Methods
2.1 Animals
Experiments were performed on 38 male Wistar rats weighing between 240 and 290 g (~90 days of age) obtained from the Animal Breeding Unit of the Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA-Brazil). Animals were housed in groups of 3 per cage and received water and a standard chow ad libitum in a controlled room maintained at 22 °C under 12:12 hour light-dark cycle. The investigation obeyed law n° 11,794 of 10/08/2008, law n° 6899 of 07/15/2009, and Resolution n° 879 of 02/15/2008 (CFMV). All procedures in this study were approved by UFCSPA Ethics Committee and Institution of Animal Use, under the protocol 14–151.
2.2 Induction of Myocardial Infarction
Rats (n = 90) were anesthetized by inhalation of 2% isoflurane (isoforine, 100 ml-Cristália) 100% oxygen [
], endotracheally intubated, and artificially ventilated (Sam Way VR 15). The animals were kept in anesthetic level III plan Guedel during the surgery [
Analysis of heat loss using inhalation agents in rats subjected to laparotomy and increased intra-abdominal pressure, using digital infrared thermal image.
]. The heart was exposed through the left thoracotomy between the fourth and fifth ribs and then a 6-O mononylon suture (Ethilon, Ethicon, São Paulo, Brazil) was passed into the main left descending coronary artery, at a point between 1 and 2 mm distal to the edge of the left atrium, and the left coronary artery was ligated. The thorax was closed; the skin was sutured, and the pneumothorax was drained by a continuous aspiration system. After the surgeries, the animals were placed in a heated environment for recovery and received an injection of ketoprofen (5.4 mg/kg/ml, i.p.) every 6 h completing 48 h and a single dose of penicillin (20,000 U, i.p.).
2.3 Exercise Test Capacity (ETC)
Five weeks after MI surgery, all rats were submitted to a progressive treadmill running test to measure maximal running capacity. First, animals were submitted to an adaptation period (5 days) and ran 10 to 20 min/day at 10 to 15 m/min. The test protocol was performed at 15° angulation and started at 5 m/min and then the speed was incrementally increased 5 m/min every 3 min until exhaustion. The same test was also performed at the end of the experiments.
2.4 Experimental Design
Animals were allowed 6 weeks for recovery before MI (time necessary to develop the CHF state). Rats were randomized to one of 6 experimental groups: sedentary group (SED, n = 6), sedentary supplemented with Arg group (SED + Arg, n = 7), aerobic continuous training group (ACT, n = 8), aerobic continuous training and supplemented with Arg group (ACT + Arg, n = 5), aerobic interval training group (AIT, n = 7), and aerobic interval training supplemented with Arg group (AIT + Arg, n = 5).
The mortality rate post-surgery of the animals was ~45% and 9 animals were excluded because they lacked the minimum infarcted area to characterize HF (infarct size < 35%) (see Fig. 1).
Supplemented groups were given a daily treatment of Arg (presentation form: powder, 99.9% purity, Sigma-Aldrich, Brazil). The supplementation dose was 1 g/kg/ml per day; solutions were made with distilled water and given by oral gavage to ensure that the desired dose was achieved. Animals were weighed every 15 days to adjust the dose if necessary.
2.6 Training Protocol
After the adaptation period, the animals allocated to the trained groups began the aerobic exercise training. It was performed on a motorized treadmill 5 days/week for 8 weeks. Previous to the specific protocol, both AIT and ACT groups performed 8-minute warm-up periods at 10 m/min. The AIT consisted of 7 periods of 7 min of exercise, consisting of intervals of 3 min at 85% of maximum ETC velocity (~20 m/min) and 4 min of active recovery at 60% of maximum ETC velocity (~14 m/min). In addition, ACT was performed at 60% maximum ETC velocity (~14 m/min) until completing the same distance reached at AIT.
2.7 Cardiac Hemodynamic, Infarct Size, Lung and Liver Edema, Blood and Tissue Collection
At the end of the 8-week training and supplementation protocols, in order to rule out any interference of acute exercise or supplementation, assessments were performed 2 days after the final ETC. Afterward, rats were anesthetized with xylazine (12 mg/kg, i.p.) and ketamine (90 mg/kg, i.p.) for hemodynamic evaluation. A catheter was connected to a pressure transducer (Strain – Gage – Narco Biosystem Miniature Pulse Transducer RP-155, Houston, TX, USA) and coupled to a pressure amplifier (Stoelting, Wood Dale, IL, USA) and was placed into the right carotid artery to measure arterial and ventricular pressures. Arterial pressure was first recorded and then the catheter was positioned inside the left ventricle (LV); both arterial and ventricular pressures were recorded during a 5-minute period. Signals were digitized with a data acquisition system (CODAS – Date Acquisition System). The collected data were used to determine heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), left ventricular systolic pressure (LVSP), maximum positive (+dP/dtmax) and negative (−dP/dtmax) derivatives of left ventricular pressure, and left ventricular end-diastolic pressure (LVEDP).
Arterial blood was collected, stored in 2 ml Eppendorf microtubes containing ethylenediaminetetraacetic acid (EDTA), and subsequently centrifuged (3000 rpm for 10 min at 4 °C). Supernatant plasma was collected and stored at −20 °C. Following the blood collection, tissues (heart, lungs, kidney, liver, and gastrocnemius) were collected and weighed. The heart was removed and the LVs were dissected and weighed. The LV and gastrocnemius weight were adjusted for animal body mass and were expressed in milligrams of tissue per gram of body mass (mg/g BM). The MI area was evaluated by planimetry using Image J 1.47 software. The lungs and liver were dehydrated (80 °C) for 48 h and then weighed again to assess water percentage. The other tissues were frozen at −80 °C.
2.8 Oxidative Stress Evaluation
2.8.1 Tissue Homogenization
Gastrocnemius muscle and kidney samples were homogenized in a KPi buffer (30 mM KCl + KH2PO4), pH 7.4, with a proportion of 9 ml/g of tissue for gastrocnemius and 5 ml/g for the kidneys. Homogenized samples were centrifuged at 3000 rpm for 10 min at 6 °C to remove cell debris, and the supernatant was used as a sample.
2.8.2 Total Protein by Bradford Method
The Bradford method was used to assess the dosage of protein in the tissue [
]. A homogenized sample (10 μl) was diluted in distilled water (190 μl). Sixty microliters of this solution were placed in plastic cuvettes (optical path: 10 mm) containing 2900 μl of Bradford reagent, previously prepared, and 40 μl of distilled water. Absorbance readings were taken at 595 nm, in a Lambda 35 spectrophotometer (PerkinElmer of Brazil, SP, Brazil). The protein standard curve was obtained from known concentrations of bovine serum albumin solutions. Results are expressed in milligrams of protein per milliliter of sample.
2.8.3 Catalase Activity
Catalase activity (CAT) was determined through the decomposition of hydrogen peroxide (H2O2). In a quartz cuvette, 2865 μl of phosphate buffer (50 mM; pH 7.0) and 30 μl of homogenized tissue were added. Then, 105 μl of 0.02 M hydrogen peroxide was added to the solution. Sample absorbance was determined in a Lambda 35 spectrophotometer (PerkinElmer of Brazil, SP, Brazil), at 240 nm per 120 s, and the results are expressed in CAT units per milligram of total protein.
2.8.4 Superoxide Dismutase Activity
Superoxide dismutase (SOD) activity consists of the reaction of inhibition of pyrogallol auto-oxidation by SOD activity. One unit of SOD is defined as the enzyme quantity capable of inhibiting 50% of the reaction. A total of 970 μl of tris buffer (TRIS 50 mM/EDTA 1 mM; pH 8.2), 4 μl of 30 μM catalase and 10 μl of homogenized tissue supernatant was placed into cuvettes. Then, 16 μl of 24 mM pyrogallol in 10 mM HCl was added to the solution. Absorbance measures were determined in a Lambda 35 spectrophotometer (PerkinElmer of Brazil, SP, Brazil), at 420 nm after 60 and 120 s. Results were expressed in SOD units per milligram of total protein.
2.8.5 Malondialdehyde Through Thiobarbituric Acid Reactive Substances Test
To measure lipid peroxidation, malondialdehyde (MDA) concentration was determined by the technique of thiobarbituric acid reactive species (TBARS). To promote the precipitation of proteins, 1 ml of tissue homogenate was added to 1 ml of 10% trichloroacetic acid solution (TCA). After standing on ice for 30 min, samples were centrifuged (3000 rpm for 10 min at 6 °C). Supernatant was collected (~1.8 ml) and 1.8 ml of 0.670% thiobarbituric acid was added (1:1 proportion). Next, the mixture was heated at 100 °C in a water-bath for 1 h. After cooling, 2.5 ml of butanol was added and samples were once again centrifuged (2000 rpm for 5 min at 6 °C), the supernatant was collected and placed in glass cuvettes to determine the absorbance at 532 nm in a Lambda 35 spectrophotometer (PerkinElmer of Brazil, SP, Brazil). The MDA standard curve was obtained from known concentrations of 1,1,3,3 tetramethoxypropane (TMP). Results were expressed in nanomoles per milligram of total proteins.
2.9 Determination of Plasma TNF-α and IL-10 Levels
Plasma TNF-α and IL-10 protein levels were determined by multiplex bead array using Milliplex MAP rat cytokine kits (RCYTO-80 K) (Millipore, Billerica, MA, USA). Cytokines results are reported in picograms per milliliter.
2.10 Statistical Analysis
All data are presented as the mean ± SD. The Kolmogorov-Smirnov test was used to test normal distribution. One-way analysis of variance (ANOVA) and the Student Newman-Keuls post-hoc test were used to compare the groups. The two-way ANOVA followed by the Bonferroni post-hoc test was used to test groups at different times. The accepted significance level was 5% (p < 0.05). Data were evaluated using the software GraphPad Prism 5 for Windows (GraphPad Software, San Diego, CA).
3. Results
3.1 Infarct Size, Body Mass, Heart and Gastrocnemius Mass, Pulmonary and Hepatic Congestion
Infarct size, initial and final body mass, heart and gastrocnemius mass, and congestion measures are shown in Table 1. No statistical differences were observed among groups on the myocardial infarcted area, suggesting a uniform disease condition in all groups. The LVM:BM ratio and initial and final body mass were similar between groups. Groups associated with aerobic exercise and Arg supplementation (ACT + Arg and AIT + Arg) revealed higher gastrocnemius mass compared to the SED group. Lung and liver wet-to-dry ratios were taken to determine the water percentage in tissues, as an indication of congestion. Hepatic congestion did not show statistical differences among groups. By contrast, pulmonary congestion was attenuated in both AIT groups (AIT and AIT + Arg) compared with the SED group.
Table 1Infarct Size, body mass, pulmonary and hepatic congestion, heart and gastrocnemius mass of the 6 studied groups.
Values are mean ± SD. SED: sedentary; ACT: aerobic continuous training; AIT: aerobic interval training; IBM: Initial body mass; FBM: final body mass; IA: infarcted area; PC: pulmonary congestion; HC: hepatic congestion; LVM:BW: left ventricle mass:body mass; GM:BM: gastrocnemius mass: body mass. One-way ANOVA followed by the Student-Newman-Keuls post-hoc test was used for statistical analysis.
At the beginning of the study, all groups presented similar performance on pre ETC (p > 0.05). After 8 weeks of training, AIT and ACT protocols were able to improve maximal exercise capacity when comparing pre and post measurements within each trained group and compared to SED groups on the post-intervention test (Fig. 2.A and .B ) (p < 0.001).
Fig. 2Exercise tolerance test pre and post intervention Values are the means ± SD. SED: sedentary; ACT: aerobic continuous training; AIT: aerobic interval training. Two-way ANOVA followed by Bonferroni post-hoc test was used for the statistical analysis. *P < 0.01 vs. SED and SED + Arg in pre and post moment; ACT, ACT + Arg, AIT, AIT + Arg in pre moment.
Hemodynamic variables are listed in Table 2. There were no statistical differences in resting HR or DBP among groups. The LVEDP was lower in the continuous training group supplemented with Arg (ACT + Arg) and in both interval training groups (AIT and AIT + Arg) than in the SED group. Animals in the AIT + Arg group also presented higher LVSP compared to the sedentary animals (SED and SED + Arg) and higher SBP versus the SED group only. Similar results were observed on positive and negative dP/dt, on which the AIT + Arg group presented superior values when compared to SED, SED + Arg, ACT, and SED, respectively.
Table 2Hemodynamic of the 6 studied groups after 8 weeks of intervention.
Values are mean ± SD. SED: sedentary; ACT: aerobic continuous training; AIT: aerobic interval training; HR: heart rate; LVEDP: left ventricular end-diastolic pressure; LVSP: left ventricular systolic pressure; +dP/dtmax: maximum positive derivative of LV pressure; −dP/dtmax: maximum negative derivative of LV pressure; SBP: systolic blood pressure; DBP: diastolic blood pressure. One-way ANOVA followed by the Student-Newman-Keuls post-hoc test was used for statistical analysis.
3.4 Oxidative Stress and Antioxidant Enzymes Activity
Fig. 3 shows the antioxidant enzymes activity and lipoperoxidation levels in gastrocnemius. The CAT activity in gastrocnemius was similar among groups (Fig. 3.A), while activity of SOD was higher in trained supplemented groups (ACT + Arg and AIT + Arg) compared to other groups (Fig. 3.B). Lower MDA concentrations were observed in supplemented groups when compared to SED and ACT groups (Fig. 3.C).
Fig. 3Oxidative stress in gastrocnemius after 8 weeks of intervention. Concentrations of A) Catalase (CAT) in gastrocnemius; B) Superoxide dismutase (SOD) activity in gastrocnemius; and C) malondialdehyde (MDA) activity in gastrocnemius. Values are the means ± SD. SED: sedentary; ACT: aerobic continuous training; AIT: aerobic interval training. One-way ANOVA followed by Student-Newman-Keuls post-hoc test was used for the statistical analysis. B) * P C) * P.
Fig. 4 shows the antioxidant enzyme activity and lipoperoxidation levels in the kidney. Renal CAT activity was greater in ACT + Arg and AIT + Arg groups compared to SED and also ACT + Arg compared to SED + Arg (Fig. 4.A). No differences were observed among groups in relation to SOD activity in this tissue (Fig. 4.B). Lower MDA concentrations were observed in both trained and supplemented groups (ACT + Arg and AIT + Arg) in relation to all other groups (Fig. 4.C).
Fig. 4Oxidative stress in kidney after 8 weeks of intervention. Concentrations of A) catalase (CAT) in kidney; B) superoxide dismutase (SOD) activity in kidney; and C) malondialdehyde (MDA) activity in kidney. Values are the means ± SD. SED: sedentary; ACT: aerobic continuous training; AIT: aerobic interval training. One-way ANOVA followed by Student-Newman-Keuls post-hoc test was used for the statistical analysis. A) * P C) * P.
Fig. 5 shows the cytokine plasmatic levels after 8 weeks of intervention. Plasma levels of TNF-α were lower in both aerobic interval training groups (AIT and AIT + Arg) compared with all other groups (Fig. 5.A). When IL-10 plasma levels were analyzed there were no significant differences among groups (Fig. 5.B); however, the IL-10/TNF-α ratio was greater in the AIT groups versus all other groups (p < 0.0001) (Fig. 5.C).
Fig. 5Plasmatic levels of anti and pro-inflammatory cytokines after 8 weeks of intervention. Plasmatic concentrations of A) tumor necrosis factor-alpha (TNF-α); B) interleukin-10 (IL-10); and C) IL-10:TNF-α ratio. Values are the means ± SD. SED: sedentary; ACT: aerobic continuous training; AIT: aerobic interval training. One-way ANOVA followed by Student-Newman-Keuls post-hoc test was used for the statistical analysis. *P.
To our knowledge, this is the first study that investigated the effects of Arg supplementation associated with distinct aerobic training protocols (ACT and AIT) in experimental CHF. According to our aims, the major findings of the present study were 1) association of aerobic training and Arg supplementation (ACT + Arg and AIT + Arg groups) improved muscle mass preservation, 2) pulmonary congestion was reduced only in AIT and AIT + Arg groups, 3) both training protocols were able to increase maximal exercise tolerance, and 4) association of AIT with Arg supplementation was able to improve hemodynamic responses (LVSP, SBP, dP/dtmax and −dP/dtmax), likewise, decrease TNF-α and muscular and renal lipid peroxidation, and increase IL-10/TNF-plasmatic levels.
Induction of HF was based on MI through descendent coronary left artery ligation, which is the most frequent experimental model for CHF studies [
]. Our model mimics a severe CHF. An inflammatory profile and higher lipoperoxidation levels were previously demonstrated by this HF model when compared to sham-operated groups [
Physical exercise improves plasmatic levels of IL-10, left ventricular end-diastolic pressure, and muscle lipid peroxidation in chronic heart failure rats.
Physical exercise improves plasmatic levels of IL-10, left ventricular end-diastolic pressure, and muscle lipid peroxidation in chronic heart failure rats.
]. In the present study, there was no difference in the infarcted area size between groups and the average infarcted area was 40%, which is bigger than preceding studies (~35%). Besides, an increased left ventricular mass was present in all groups. Pulmonary and hepatic congestion is a common symptom, although interval training groups showed lower pulmonary congestion compared to the SED group. Gastrocnemius mass was higher in the ACT + Arg and AIT + Arg groups compared to the SED group. In this view, our experimental model probably induces muscle wasting in non-trained animals. Moreover, functional capacity was impaired and the LVEDP was significantly increased (>20 mm Hg) in the SED group.
Exercise tolerance capacity was improved in all trained groups. In contrast to the previous study of our group, AIT was not superior to ACT for improving functional capacity. In the present report, 85% of maximum ETC speed was used in the higher intensity zone, while Nunes et al. used 92% of maximum ETC speed [
]. In that view, exercise intensity possibly has an important role in improving exercise tolerance in CHF. Conversely, a recent meta-regression analysis demonstrated that, in order of importance, the major contributors for exercise capacity are total energy expenditure, session frequency, session duration, and session intensity [
The influence of training characteristics on the effect of aerobic exercise training in patients with chronic heart failure: a meta-regression analysis.
]. Thus, possible explanations for equal improvements in ACT and AIT in the present study are that, in order to correct disparities, both groups had matched session frequency and distance roamed at the end of each training session.
On the other hand, in the present study, AIT showed a superior improvement in the hemodynamic parameters, primarily when associated with Arg supplementation. Association of AIT and Arg was able to improve cardiac relaxation and contractility, as reflected by an increase positive and negative dP/dtmax, as well as normalize LVSP and SBP. Moreover, LVEDP was significantly reduced in both AIT along with ACT + Arg. On AIT groups, hemodynamic responses went along with improvements on pulmonary congestion. Diminished lung water percentage can be explained by lower LVEDP, since higher afterload will possibly culminate in severe pulmonary congestion [
]. The ACT and SED-Arg groups demonstrate a trend to improve LVEDP, although it was not able to show significant responses. Probably, these therapies, when applied only, demonstrate a minor effect on the left ventricular dysfunction. Conversely, when applied together, a synergistic effect may lead to a more pronounced response, as seen in ACT + Arg group. It is also important to highlight that some physiological variables are more likely to show greater responses than others and, although in this study some hemodynamic variables haven't showed statically significant difference, it is not possible to invalidate the general improvements promoted by intervention.
Supplementation of l-arginine on endothelial dysfunction conditions had already demonstrated beneficial effects on hemodynamic parameters. The Arg actions on hemodynamic are primarily related to improvements in blood flow [
Effects of l-arginine supplementation on blood flow, oxidative stress status and exercise responses in young adults with uncomplicated type I diabetes.
]. In our study, Arg supplementation by itself was not able to significantly improve hemodynamic parameters. However, Arg apparently enhanced aerobic training effects, especially when combined with AIT.
The Arg effects may not be exclusively centered on NO production [
]. Angiotensin II (ANG II) modulates sympathetic nerve activity promoting sympatho-excitation. In cardiovascular diseases, an excess of ANG II stimulates removal of mitochondrial ROS to cytosol, leading to more ROS production and, thus, eNOS uncoupling [
]. These data complement the theory for antioxidant actions of Arg and, apart from that, support the synergic effects on hemodynamic parameters among Arg supplementation and aerobic training in our study.
The present results demonstrated that groups with Arg supplementation combined with aerobic exercise, either ACT or AIT, significantly diminished MDA levels. These results are in concordance with a study by Huang et al., which demonstrated Arg was able to blunt lipoperoxidation caused by an exhaustive exercise in both skeletal muscles and kidneys [
]. In our study, gastrocnemius MDA levels were also lower in SED + Arg versus SED and ACT groups. However, SOD and CAT were not increased in the gastrocnemius of the SED + Arg group. Accordingly to this, Arg might have acted as an ROS scavenger, capable of reducing lipid damage itself.
In classical situations of imbalance between ROS formation and ROS degradation, like cardiovascular diseases and exhaustive exercise, the SOD enzyme was decreased in both skeletal muscle and plasma [
]. In concordance with those previous trials, we found augments in gastrocnemius SOD activity in both trained groups after chronic Arg supplementation. No differences were observed in gastrocnemius CAT activity. Conversely, kidney SOD activity was similar among groups, and CAT activity was increased in both trained and supplemented groups (ACT + Arg and AIT + Arg).
Excess of reactive oxygen species (ROS) and reactive nitrogen species (RNS) could intensify the inflammatory process that occurs after muscle damage or in chronic disease conditions, such as HF [
Physical exercise improves plasmatic levels of IL-10, left ventricular end-diastolic pressure, and muscle lipid peroxidation in chronic heart failure rats.
]. Conversely, in the present study, an anti-inflammatory response was observed only in AIT groups; both had lower plasma TNF-α level and increased IL-10/TNF-α ratio in relation to other groups. In accordance to the present report, other studies had shown that continuous exercise was insufficient to modulate inflammatory responses [
]. However, moderate-intensity continuous exercise only attenuated TNF-α, with no significant decreases in relation to the HF sedentary group.
Furthermore, some studies reported that despite any beneficial modulation in the inflammatory biomarkers after continuous aerobic exercise, improvements in cardio-metabolic parameters were observed [
]. It is in accordance with our findings in the ACT + l-Arg, which improved LVEDP, but didn't show positive responses in the inflammatory profile. In fact, a lower TNF-α level is crucial to the CHF clinical state amelioration. However, improvements in the cardio-metabolic parameters are not necessarily dependent of the inflammatory profile only. In CHF, cardiac function may be improved by different mechanisms induced by physical exercise (e.g., myocardial adaptations), and in this case, with a optimizing of Arginine supplementation.
Studies verifying the immune function after high-intensity interval training (HIIT) are still scarce, especially, in the CHF population. However, some studies had shown an anti-inflammatory effect of HIIT on other chronic diseases. Durrer et al. verified a reduction in TLR2 expression in classical an in CD16+ monocytes, which was accompanied by a reduction in the TNF-α after a single bout of HIIT in type 2 Diabetes Mellitus population [
Combined effect of aerobic interval training and selenium nanoparticles on expression of IL-15 and IL-10/TNF—a ratio in skeletal muscle of 4T1 breast cancer mice with cachexia.
]. The mechanisms underlying HIIT anti-inflammatory effects still not clear, although a recent study proposed that it could reduce oxidation and inflammation by up-regulating specific mRNA expression [
High intensity interval training favourably affects angiotensinogen mRNA expression and markers of cardiorenal health in a rat model of early-stage chronic kidney disease.
Gastrocnemius mass was higher in trained groups associated with Arg supplementation. It has already been reported that Arg has the potential to stimulate anabolism and also to improve immune function [
]. Thereby, aerobic exercise training and Arg supplementation may act synergistically in preserving skeletal muscle mass. Since gastrocnemius MDA levels were reduced in both ACT + Arg and AIT + Arg, those groups might have increased anti-inflammatory cytokines or decreased TNF-α in skeletal muscle, leading to a reduced skeletal muscle atrophy.
Our study has some limitations. Training stimulus was the same during the 8 weeks without ETC during protocols for progressive intensity adjustment. In addition, echocardiography to assess cardiac function or changes in ventricular diameter and histological technique for LV mass evaluation could have improved the accuracy of our results. Finally, future studies with ANG II, NO, IL-6, and eNOS analysis may contribute for more robust conclusions about the mechanisms underlying those CHF therapies.
In conclusion, our data indicated that aerobic physical training is important to improve exercise tolerance in CHF-induced rats, despite intensity. In association with Arg supplementation, aerobic exercise was able to attenuate muscle loss. Moreover, AIT associated with Arg supplementation elicits greater improvements in hemodynamic parameters, contributing to a reduction in pulmonary congestion, and was shown to have particular responses in the inflammatory profile and antioxidant state.
Author Contributions
Barcelos GT and Nunes RB: study design, conduct of the study, molecular biological analysis, data collection and statistical analysis, data interpretation, manuscript writing and critical review. Dal Lago P: study design, statistical analysis, data interpretation, manuscript writing and critical review. Rossato DD, Jaenisch RB, Pinheiro LP, Perini JL: conduct of the study and data collection. Carvalho C: conduct of the study, molecular biological analysis and data collection. Rodhen CR: molecular biological analysis.
Funding
This study was supported by funds from CAPES (00889834/0001-08) and CNPq (33.654.831/0001-36), Brasilia.
Acknowledgments
We are thankful to MSc. Jadson Pereira Alves, MSc. Giuseppe Potrick Stefani for their support during the development of the study, and Bruna Marmett, Camila Scheid for their assistance.
Conflict of Interest
The authors declare that they have no conflict of interest.
l-Arginine supplementation causes additional effects on exercise-induced angiogenesis and VEGF expression in the heart and hind-leg muscles of middle-aged rats.
Physical exercise improves plasmatic levels of IL-10, left ventricular end-diastolic pressure, and muscle lipid peroxidation in chronic heart failure rats.
Effectiveness of high-intensity interval training (HIT) and continuous endurance training for VO2max improvements: a systematic review and meta-analysis of controlled trials.
Effects of high-intensity interval training versus continuous training on physical fitness, cardiovascular function and quality of life in heart failure patients.
Analysis of heat loss using inhalation agents in rats subjected to laparotomy and increased intra-abdominal pressure, using digital infrared thermal image.
The influence of training characteristics on the effect of aerobic exercise training in patients with chronic heart failure: a meta-regression analysis.
Effects of l-arginine supplementation on blood flow, oxidative stress status and exercise responses in young adults with uncomplicated type I diabetes.
Combined effect of aerobic interval training and selenium nanoparticles on expression of IL-15 and IL-10/TNF—a ratio in skeletal muscle of 4T1 breast cancer mice with cachexia.
High intensity interval training favourably affects angiotensinogen mRNA expression and markers of cardiorenal health in a rat model of early-stage chronic kidney disease.
p < 0.05 vs. SED.
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