Experiments were performed on 28 male Wistar rats weighing 220–270g, obtained from the Animal Breeding Unit at the Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Porto Alegre, RS, Brazil. The animals were allocated in groups of three to a cage with free access to water and pellet rodent chow diet and were maintained under standard conditions of temperature (22°C). All procedures were performed in accordance with the Guideline for the Care and Use of Laboratory Animals (NIH Publication n° 85-23, revised 1996). All procedures outlined in this study were approved by the by the UFCSPA Ethics and Research Committee (protocol no. 39/11).

To induce myocardial infarction (MI), rats were anaesthetized with xylazine (12mg/kg IP) and ketamine (90mg/kg IP), intubated and artificially ventilated (SamWay VR 15) with a breathing rate of 60breaths/min and oxygen inspired fraction of 100%. After thoracotomy, coronary artery ligation (CAL) was performed to induce MI and, subsequently HF.12 During the first 48h, the animals were treated for post-operative pain with subcutaneous buprenorphine (0.15mg/kg) and given a single dose of penicillin (20,000U IP). After surgery, the rats remained six weeks in home cage for evolution of MI. This period is necessary to develop HF state.17 It was reported that in six weeks following MI the renal sympathetic nerve activity and left ventricular end-diastolic pressure (LVEDP) increase, and baroreflex control decreases,17 all of these changes are associated with chronic HF. Rats were randomly allocated into four experimental groups: sedentary sham-operated (Sed-Sham, n=7), trained sham-operated (Tr-Sham, n=7), sedentary HF (Sed-HF, n=7) and trained HF rats (Tr-HF, n=7).

One week before the aerobic exercise training protocol the adaptation period was started to familiarize the rats with running on the treadmill with low speed (10m/min) and short duration (10min per session). Rats in the training groups (Sham and HF) performed an aerobic ExT program for 8 weeks (5×/week)16,18 with speed at 16m/min for 60min/day.19 This intensity was maintained at a constant level throughout the experiment. Previous study demonstrated that this exercise protocol improved the aerobic capacity and citrate synthase activity in post-myocardial infarction rats.20 Only rats that ran steadily with little or no prompting were used in the study. The total duration of the experiment was 14 weeks (5 resting in home cage, 1 in adaptation to treadmill and 8 of aerobic ExT protocol).

After a rest period of 48h from the last training session, the cardiac hemodynamic evaluation was performed. The animals were anesthetized with xylazine (12mg/kg IP) and ketamine (90mg/kg IP), and a small incision in the anterior cervical region was performed for the insertion of a polyethylene catheter (PE-50) into the right carotid artery. The arterial pressure (AP) was recorded first during a 5-min period by connecting the arterial cannula to a pressure transducer (Miniature Pulse Transducer RP-155, Narco Biosystems, Houston, TX, USA), coupled to a pressure amplifier (General Purpose Amplifier 4, Model 2, Stemtech, Hudson, WI, USA). Then, the catheter was positioned inside the left ventricle (LV), and the pulse wave was monitored using the typical graphic registration of ventricular pressure and recorded for 5min. Pressure analog signals were digitalized by a data acquisition system (AT/Codas, Dataq Instruments, Akron, OH, USA) at a sampling rate of 2000Hz. These data were used to determine LV maximum change in pressure over time (+dP/dtmax) and LV minimum change in pressure over time (−dP/dtmax), and LVEDP.21 The last parameter was determined manually by detecting the point of inflection to the end of diastole via analysis of the ventricular pressure wave. The total left ventricle area and myocardial infarction scar were manually drawn on scanned images. ImageJ 1.47 software used planimetry to determine the size of the infarct. All photographs were analyzed independently by two blinded investigators. The heart weight-to-body weight ratio (HW/BW) and right ventricular to body weight ratio (RV/BW) values were determined. Lungs were dehydrated (80°C) for 48h and then weighed again to evaluate the water percentage. Cardiac hemodynamic evaluations of left ventricular diastolic function as well as morphometric cardiac were used to characterize HF. Only rats with elevated LVEDP≥16mmHg and myocardial infarction area ≥ 29% were included in the present study.16,21

After cardiac hemodynamic evaluation, the animals were euthanized with an overdose of anesthetic (thiopental 80mg/kg IP), the superficial white gastrocnemius muscle were collected, weighed, immediately frozen in liquid nitrogen, and stored at −80°C. The samples of fast-glycolytic muscles were obtained by dissection of gastrocnemius muscles, which were quickly excised from the left hindlimbs of the animals and dissected according to the muscle color (corresponding to myoglobin concentration) into a white portion derived from the superficial part of both lateral and medial head.

The gastrocnemius muscles were chosen for cytokine assays because of their metabolic characteristics and tend to be more affected in disease conditions.10 The gastrocnemius samples were homogenized in potassium phosphate buffer (KPi, pH 7.4) containing 4.08g/L KH2PO4, 8.9g/L KCl, 8.71μg/mL phenylmethylsulfonyl fluoride (PSMF), 0.1μg/mL aprotinin, 0.1μg/mL leupeptin and 0.1μg/mL pepstatin proteases inhibitors using a hand-held homogenizer. The homogenates were centrifuged at 12,000×g (Mikro 220 R, Hettich Zentrifugen, Tuttlingen, Germany) for 60min at 4°C. The supernatant was removed and TNF-α, IL-6 and IL-10 levels were determined by multiplex bead array using Milliplex™ MAP rat cytokine kits (RCYTO-80K) (Millipore, Billerica, MA, USA). All samples were run in duplicates and the average value is reported as pg/mL.

The data are presented as the mean±SD. The data were tested for normal distribution using the Kolmogorov–Smirnov test. Comparisons were made between groups using two-way ANOVA (exercise training and HF as the main factors) followed by the Newman–Keuls post hoc test. When Gaussian normality failed, a Kruskal–Wallis test on ranks and Dunn's test were performed. Unpaired student t test compared infarcted areas between the Sed-HF and Tr-HF groups. Between-group differences are reported as the mean difference (MD) and 95% confidence interval (95% CI). A p value<0.05 was considered statistically significant.

The perioperative mortality in the HF-groups was 35%. The infarct sizes of the LV were similar in two infarcted groups, (MD −3.1; 95% CI −9 to 3; p=0.4). At the end of study, between-group analyses showed that HW/BW and RV/BW ratio, and pulmonary congestion were higher in the Sed-HF group when compared with Sed-Sham group (p<0.01). ExT reduced pulmonary congestion (p=0.03 for group), cardiac hypertrophy (HW/BW, p=0.03 for interaction effect) and RV hypertrophy (RV/BW, p=0.01 for interaction effect) when compared with Sed-HF group, respectively. After 14 weeks post-MI, the Sed-HF group showed a significant reduction between-group analyses in the muscle weight and ratio of muscle weight to body weight of the gastrocnemius muscle when compared with Sed-Sham group (p=0.01), suggesting muscle atrophy. However, when the exercise-trained HF group was compared with the Sed-HF group, there was an increase in the weight (p=0.02) and ratio of weight to body weight of the gastrocnemius muscle (p=0.01). There was no significant difference in the body weight among all the groups. All of these data are summarized in Table 1.

Table 2 shows the hemodynamic characteristics in sham and HF rats. As expected, LVEDP was elevated in both HF groups compared with the sham groups (p<0.001 for group effect). However, when the trained HF group was compared with the Sed-HF group, there was an improvement in diastolic function (p=0.01 for training effect). This result suggests that 8-week ExT have a beneficial hemodynamic effect in HF rats. The negative derivative of LV pressure (−dP/dtmax) was significantly reduced in the HF groups compared with the sham groups (p<0.05 for group effect). No differences were observed in mean arterial pressure (MAP) or positive derivative of LV pressure (+dP/dtmax). All hemodynamic variables were assessed while the rats were under anesthesia.

Muscular levels of TNF-α (Fig. 1A) were elevated in the Sed-HF group when compared with the Sed-Sham group (MD 1.3; 95% CI −0.04 to 2.5; p<0.05 for group effect). ExT significantly reduced by 59% TNF-α level in Tr-HF group when compared with the Sed-HF group (MD −1.7; 95% CI −2.9 to −0.3; p=0.02 for interaction effect). We observed an increase in IL-10 (Fig. 1B) in trained HF group when compared with the Sed-HF (MD 15; 95% CI 11–26; p=0.01) and Tr-Sham group (MD 9; 95% CI −6 to 27; p=0.01 for group and training effects), respectively. However, no differences were found in the IL-6 (Fig. 1C) among groups. The gastrocnemius muscle IL-10/TNF-α ratio (Fig. 1D) was significantly higher in the Tr-HF group than in the Sed-HF and Sham groups (p<0.01 for training and interaction effects). Cytokine analyses were performed only with muscle samples feasible.

Figure 1.

Mean data showing the effects of exercise training in the gastrocnemius muscle of anti- and pro-inflammatory cytokines. (A) Tumor necrosis factor-alpha (TNF-α); * p<0.05 vs. Sed-HF; # p<0.05 vs. Sed-Sham. (B) Interleukin-10 (IL-10); * p<0.05 vs. Sed-Sham and Sed-HF. (C) interleukin-6 (IL-6). (D) IL-10/TNF-α ratio. * p<0.01 vs. all groups. Values are the means±SD, (n=3–6 per groups).

(0.14MB).