Effects of moderate and high intensity isocaloric aerobic training upon microvascular reactivity and myocardial oxidative stress in rats.

Systemic and central cardiovascular adaptations may vary in response to chronic exercise performed with different intensities and volumes. This study compared the effects of aerobic training with different intensities but equivalent volume upon microvascular reactivity in cremaster muscle and myocardial biomarkers of oxidative stress in Wistar rats. After peak oxygen uptake (VO2peak) assessment, rats (n = 24) were assigned into three groups: moderate-intensity exercise training (MI); high-intensity exercise training (HI); sedentary control (SC). Treadmill training occurred during 4 weeks, with exercise bouts matched by the energy expenditure (3.0-3.5 Kcal). Microvascular reactivity was assessed in vivo by intravital microscopy in cremaster muscle arterioles, while biomarkers of oxidative stress and eNOS expression were quantified at left ventricle and at aorta, respectively. Similar increasing vs. sedentary control group (SC) occurred in moderate intensity training group (MI) and high-intensity training group (HI) for endothelium-dependent vasodilation (10-4M: MI: 168.7%, HI: 164.6% vs. SC: 146.6%, P = 0.0004). Superoxide dismutase (SOD) (HI: 0.13 U/mg vs. MI: 0.09 U/mg and SC: 0.06 U/mg; P = 0.02), glutathione peroxidase (GPX) (HI: 0.00038 U/mg vs. MI: 0.00034 U/mg and SC: 0.00024 U/mg; P = 0.04), and carbonyl protein content (HI: 0.04 U/mg vs. MI: 0.03 U/mg and SC: 0.01 U/mg; P = 0.003) increased only in HI. No difference across groups was detected for catalase (CAT) (P = 0.12), Thiobarbituric acid reactive substances (TBARS) (P = 0.38) or eNOS expression in aorta (P = 0.44). In conclusion, higher exercise intensity induced greater improvements in myocardium antioxidant defenses, while gains in microvascular reactivity appeared to rely more on exercise volume than intensity.

Introduction

Aerobic training is widely acknowledged as an effective strategy to maintain health and reduce cardiovascular risk [1]. Microvascular endothelial function [2-5] and myocardial antioxidant defenses [6-9] have been extensively investigated within this context, as reflecting early systemic and central cardiovascular changes [2, 6, 10, 11]. In regards to endothelial function, albeit chronic exercise seems to improve vasodilation of microvessels due to shear stress and circulating factors [2, 12], some research suggested that too vigorous training may increase oxidative stress and inflammation, therefore leading to deterioration of endothelial function [13, 14]. On the other hand, myocardium integrity seems to be favored by high-intensity training [15], due to greater production of antioxidants protecting against reactive oxygen species (ROS) [7, 16]. In short, different exercise intensities may elicit dissimilar chronic effects upon systemic and central cardiovascular markers–although endothelial function may be jeopardized by high-intensity training [14, 17], myocardium antioxidant protection could be benefited [15].

Thus, for a better understanding of cardiovascular benefits and risks due to exercise training, attention must be given to the relative role of its intensity and volume. A possible approach to address this question would be to compare the effects of aerobic training performed with different intensities, but similar overall volume as defined by the energy expenditure (EE)–in other words, isocaloric training bouts. Given that improvements in endothelial function may rely on the amount of EE during exercise [17, 18], it is feasible to speculate that isocaloric protocols would be able to induce favorable effects regardless of training intensity [19]. Moreover, this approach would help to avoid bias, since protocols with higher intensities can also be related to greater EE. This confounding factor precludes isolating the specific effects of exercise intensity upon endothelial function or myocardium integrity.

To date no study using animal models investigated the relative effects of exercise intensity and volume on endothelial function of microvessels (systemic cardiovascular marker), and antioxidant protection in myocardium (central cardiovascular marker). Thus, the present study aimed to investigate the effects of aerobic training performed with different intensities but equivalent volume, on microvascular reactivity in striated muscle and biomarkers of oxidative stress in myocardium of Wistar rats.

Methods

Ethical approval

Twenty-four male Wistar rats (Rattus norvegicus, Anilab, RJ, Brazil) were kept under 12:12-hour light-dark cycle in a temperature-controlled environment (22°C) with free access to water and standard rat chow (Nuvilab CR-1, NuvitalTM, Curitiba, PR, Brazil). Experiments were performed according to principles of laboratory animal care (NIH pub. No. 86–23, revised 1996) and the protocol was approved by the Ethical Committee of the University of Rio de Janeiro State (License number: 024/2015).

Study design

After assessment of oxygen uptake at rest (VO2rest) and maximal exercise (VO2peak), the animals (270 g, 12 weeks old) were randomly assigned into three groups: (a) moderate-intensity exercise training (MI; n = 8); (b) high-intensity exercise training (HI; n = 8) and (c) sedentary control (SC; n = 8). Two isocaloric exercise bouts were performed after the maximal exercise testing, in order to match the duration of training sessions according to the overall EE. After this, HI and MI underwent exercise bouts during four weeks on a motorized treadmill. At the end of the training period, cardiorespiratory fitness and microvascular reactivity were assessed in vivo. At the end of each microcirculation experiment, animals profoundly anesthetized were euthanized by exsanguination. The eNOS expression was analyzed from aorta fragments. Left ventricle fragments were collected and immediately frozen in liquid nitrogen for measuring biomarkers of oxidative stress (antioxidants and oxidized biomolecules).

Maximal graded exercise testing

Oxygen uptake at rest (VO2rest) and during maximal exercise (VO2peak) were determined by indirect calorimetry via metabolic cart (OxyletTM, Panlab Harvard Apparatus, Barcelona, Spain). The gas analyzer was coupled to a treadmill in a Plexiglas chamber, connected through a tube to an air pump used to maintain the airflow inside the chamber. The gas analyzer continuously measured relative concentrations of oxygen (O2) and carbon dioxide (CO2) effluent in the chamber. The VO2 was calculated by specific software (MetabolismTM, Panlab Harvard Apparatus, Barcelona, Spain) using equations described elsewhere [20]. Standard conditions of temperature, pressure and humidity (STPD) were kept in all experiments.

The VO2rest was assessed during 30 min and data obtained during the last 5 min were averaged and recorded. Prior to VO2peak assessment, the rats underwent treadmill adaptation sessions during three days, with speed set at 16 cm/s during 10–15 min. The testing protocol consisted of load increments of 8 cm/s every 3 min, until the rats were no longer able to run. Exhaustion was determined when animals remained at the end of the treadmill (electrical shock grid) for 5 seconds and VO2peak corresponded to the highest VO2 obtained during the test [20]. The shock grid delivered very low electrical currents (0.2 mA) and was used only to induce the rats to run.

Training protocol (isocaloric exercise bouts)

The target workload during exercise bouts was calculated using the oxygen uptake reserve (VO2R) method, as previously described [19]: VO2R = (fraction intensity) (VO2peak−VO2rest) + VO2rest, where VO2peak corresponded to the highest VO2 during maximal exercise testing. The relative intensity was defined according to each group; animals assigned to MI and HI exercised at speeds corresponding to 50% and 80% of VO2R, respectively. Running speeds matching the relative intensities were individually calculated, based on VO2 obtained in the maximal exercise testing.

The duration of isocaloric bouts was calculated (predicted) from values of VO2R and then converted to EE, as described elsewhere [20]. In order to confirm that both MI and HI bouts elicited similar EE, the VO2 was measured throughout two exercise bouts (test and retest). When it was necessary, adjustments in predicted duration were made before the second bout, to ensure that the rats would perform the isocaloric exercise sessions with different intensities. Since the EE equivalence was confirmed in the retest, HI and MI groups performed the isocaloric training sessions without indirect calorimetry measurements. The exercise sessions were performed on a motorized treadmill (Insight Scientific EquipmentsTM, São Paulo, SP, Brazil) during four weeks, five times a week, according to individual values of duration and running speed. In order to assess the excess post-exercise oxygen consumption (EPOC), the VO2 was assessed during 30 min following the isocaloric bouts in a randomized subgroup of four animals selected from HI and MI.

Assessment of microvascular reactivity

A standardized surgical procedure to evaluate microvascular reactivity in cremaster muscle was performed [21, 22]. In brief, the rats were anesthetized with ketamine and xylazine ip (65 and 10 mg·kg-1 respectively), and the connective tissue was separated of cremaster. The muscle was then exposed on glass stage surface by pins fixed in edges of tissue. The cremaster was continuously superfused at a rate of 4 ml/min by HEPES-supported HCO3—buffered saline solution [composition in mM: NaCl 110.0, KCl 4.7, CaCl2 2.0, MgSO4 1.2, NaHCO3 18.0, N-2-hydroxyethylpiperazine-N′-2ethanesulfonic acid (HEPES) 15.39 and HEPES Na+-salt 14.61] bubbled with 5% CO2−95% N2. The pH was set at 7.4 and the temperature of superfusion solution was maintained at 37.5°C. The preparation was placed under an intravital microscope (LeicaTM DMLFS, optical magnification ×600, NA 0.65, Wetzlar, Germany) coupled to a closed-circuit TV system, in order to record images of the arterioles. The cremaster preparation was maintained during 30 min at rest, before starting the experimental protocol to evaluate microvascular reactivity, as described elsewhere [22, 23].

Three arterioles (2nd and 3rd order) were analyzed in each cremaster preparation. After 30 min of rest, the images of arterioles were collected at baseline and after topical application of acetylcholine (ACh) and sodium nitroprusside (SNP) (Sigma-AldrichTM, St. Louis, MO, USA) at 10−8, 10−6 and 10−4 M. ACh and SNP were used to evaluate endothelium dependent and independent vasodilation, respectively. Each application was performed during 10 min, with a syringe infusion pump (model 55–2222, Harvard ApparatusTM, Boston, MA, USA), producing a cumulative dose–response curve. Internal diameter of arterioles in each moment was measured by specific software (Image J TM, U.S. NIH, Bethesda, MD, USA). At the end of each experiment, rats were sacrificed by anesthetic overdose followed by exsanguination, by removal of myocardium and aorta.

Assessment of eNOS expression (Western blot analysis)

The thoracic aorta was dissected and protein extracted, as previously described [23]. In brief, the aorta was lysed in 50 mM HEPES (pH 6.4), 1% Triton X-100, 1 mM MgCl2, 10 mM EDTA, 1 mg/ml DNase, 0.5 mg/ml RNase containing the following protease inhibitors: 1 mM benzamidine, 1 mM PMSF, 1 mM leupeptin, and 1 mM soybean trypsin inhibitor (Sigma-AldrichTM, St. Louis, MO, USA). Protein content was measured using PierceTM BCA Protein Assay Kit (Thermo Fisher ScientificTM, MA, USA) and samples containing 50 μg of protein were resolved by electrophoresis (7.5% SDS-PAGE), transferred to PVDF membranes and stained with Ponceau to verify whether the same quantity of protein is present in all lanes. Proteins in PVDF membranes were probed with mouse monoclonal anti-eNOS (1:1000; Becton Dickinson, NJ, USA), incubated overnight at 4°C. A Ponceau Red staining was used as loading control. After extensive washings in TBS-Tween, PVDF membranes were incubated for 2 h at room temperature with horseradish peroxidase-conjugated secondary antibody anti-mouse IgG, diluted 1:5,000 and developed using Amersham ECL Western Blotting Detection Kit system (GE Healthcare Life SciencesTM, Pittsburgh, PA, USA).

Enzymatic assays and assessment of oxidative damage

Left ventricular tissue was dissected and homogenized (about 200 mg of tissue) on ice in PBS buffer 0.1 M (0.1 M NaCl, 0.1 M NaH2PO4.H2O, 0.1 M NaH2PO4.2H2O, 0.1 M KCl, 6 mM EDTA, pH 7.5). Samples were centrifuged at 5,000 rpm for 20 min at 4°C and supernatant was collected. All samples were stored at -80°C for further analysis of enzymatic assays and oxidative damage. SOD, GPX and CAT activity were evaluated in left ventricle homogenate. Results are expressed as U/g of protein. Total protein content was quantified using the BCA assay kit (BioagencyTM, Sao Paulo, SP, Brazil).

Measurement of SOD activity is based on its inhibition by pyrogallol autoxidation and assessed by spectrophotometric readings at 420 nm during 5 min [24, 25]. Catalase activity was assessed by standard methods, as described elsewhere [26]. Briefly, this method is based on the rate of hydrogen peroxide decomposition, following the decay in absorbance at 240 nm during 1 minute. GPx activity was assessed by the rate of NADPH disappearance, measured by spectrophotometry (340 nm, during 3 min reading) [27].

The oxidative damage of proteins was assessed through formation of carbonyl groups based on the reaction with dinitrophenylhydrazine (DNPH) [28]. Carbonyl contents were determined by spectrophotometry at 370 nm. Lipid membrane damage was quantified by the formation of byproducts of lipid peroxidation (malondialdehyde, MDA), which are thiobarbituric acid reactive substances (TBARS). The MDA reacts with thiobarbituric acid resulting in a pinkish substance, which is subsequently analyzed by spectrophotometry [29]. TBARS were determined by reading the absorbance at 532 nm (Fluostar OmegaTM, BMG Labtech, Ortenberg, Germany).

Statistical analysis

Normal distribution was ratified by the Kolmogorov-Smirnov test for data regarding maximal graded tests and isocaloric exercise bouts, which are presented as mean ± SEM. Microvascular reactivity are presented as median (1st − 3rd quartile). Comparisons between pre vs. post-exercise training were performed for VO2 and maximal speed by means of 2-way repeated measures ANOVA, followed by LSD post hoc testing in the event of significant F ratios. Microvascular reactivity and biomarkers of oxidative stress were compared only at post training, using Kruskal-Wallis test followed by Dunn test as post hoc verification. In all cases significant level was set at P ≤ 0.05 and calculations were performed using the Statistica 10.0 software (StatsoftTM, Tulsa, OK, USA).

Results

Table 1 exhibits data of cardiorespiratory fitness assessed by maximal exercise testing, before and after training. At baseline, VO2peak (P = 0.98) and maximal running speed (P = 0.38) were similar across groups. After training, VO2peak decreased in SC (P = 0.007), increased in HI (P = 0.001) and increased twice in HI than MI, although this difference lacked of statistical significance (VO2peak Δ = 4.9 vs. 2.2 ml.kg.-1min.-1; P = 0.12). On the other hand, the increase in maximal speed was significantly greater in HI vs. MI (P = 0.016) and vs. SC (P = 0.02). Despite the differences detected for VO2 after training, gains in body mass were similar across groups during all the experimental period (P = 0.59) (Fig 1), as well as the energy intake (SC: 94.9 ± 2.2 kcal/day; MI: 93.3 ± 4.8 kcal/day; HI: 92.9 ± 4.2 kcal/day; P = 0.58).

Table 1

Data from maximal graded exercise test and isocaloric exercise training protocol.

Maximal Graded Test		SC	HI	MI
VO2 peak (ml.kg.-1min.-1)	Before	78.3 (1.9)	78.5 (2.5)	77.9 (2.7)
	After	74.5 (1.6) A	83.4 (3.2) A	80.1 (2.4)
	Δ	-3.7 (1.3)	4.9 (1.0) B	2.2 (1.1) B
Maximal speed (cm.s-1)	Before	66.0 (2.0)	61.0 (2.1)	65.0 (3.5)
	After	61.0 (2.5)	74.0 (2.9) A B	68.0 (3.7)
	Δ	-5.0 (2.1)	13.0 (3.0) * B	3.0 (2.1) B
Isocaloric bouts (predicted)		SC	HI	MI
Target VO2 (ml.min-1)		-	20.4 ±0.5 *	14.1 ±0.7
Total EE (kcal)		-	5.0	5.0
Duration (min)		-	52.6 ±2.6 *	63.6 ±2.3
Running speed (cm.s-1)		-	44 ±2 *	18 ±1
Isocaloric bouts (1st session)		SC	HI	MI
Target VO2 (ml.min-1)		-	18.5 ±0.7 *	14.5 ±0.5
Total EE (kcal)		-	2.7 ±0.2 * #	4.2 ±0.1 #
Duration (min)		-	31.6 ± 1.4* #	57.4 ±1.5
Running speed (cm.s-1)		-	44 ±2 *	18 ±1
Isocaloric bouts (2nd session)		SC	HI	MI
Target VO2 (ml.min-1)		-	18.5 ±0.5 * #	14.6 ±0.7
Total EE (kcal)		-	3.3 ±0.1 #	3.4 ±0.1 #
Duration (min)		-	38.4 ±1.2 * #	48.3 ±1.8 #
Running speed (cm.s-1)		-	44 ±2 *	18 ±1

Data are showed as mean ± SEM (n = 24).

Δ: Absolute value obtained after training minus before training

A: significant difference vs. before training

B: significant difference vs. SC group

*: significant difference vs. MI group

#: significant difference vs. own predicted values.

Fig 1

Body mass during the experimental period (4 weeks).

Data are expressed as mean ± SEM (n = 24). No significant difference was found between the groups (P = 0.59).

Table 1 also depicts data extracted from the first and second isocaloric bouts (test and retest sessions). As expected, running speeds (P = 0.013) and therefore target VO2 (P = 0.0002) were always higher in HI vs. MI. In the first exercise bout, total EE was significantly different (P = 0.007) and could not be matched between groups, because animals in HI were not able to complete the predicted exercise duration before exhaustion. The second bout was performed after adjustments (by reducing duration for MI group) and differences in total EE between groups were no longer detected (P = 0.61), albeit target VO2 in HI has remained higher than MI (P = 0.007). This is reinforced by the EPOC, which was significantly influenced by exercise intensity. At the beginning of recovery, HI had significantly higher VO2 than MI (HI = 17.58 ± 1.4 mL/min vs. MI = 13.15 ± 0.8 mL/min vs.; P = 0.03) and this pattern was extended until the end of recovery, as shown by the VO2 range (HI = 7.85 ± 0.6 mL/min vs. MI = 4.45 ± 0.8 mL/min; P = 0.01).

Changes in arterioles diameter in relation to basal conditions (considered as 100%) under topical application of ACh and SNP are depicted in Fig 2. Baseline refers to a period before ACh or SNP application and after 30-min accommodation; at this phase, mean diameter was always similar across groups (SC: 71.73 ± 2.9 μm, MI: 74.91 ± 3.0 μm, HI: 70.55 ± 4.9 μm; P = 0.54). Endothelium-dependent vasodilation in response to the highest ACh concentration (10-4M) and to the intermediate concentration (10-6M) was significantly lower in SC than MI (P = 0.0016 and P = 0.003) and HI (P = 0.0012 and P = 0.01). In response to the lowest concentration of ACh (10-8M), HI presented significantly increased vasodilation vs. SC (P = 0.02), but not MI (P = 0.17). No difference among groups was detected regarding endothelium-independent vasodilation induced by SNP at any concentrations (10-8M: P = 0.34; 10-6M: P = 0.49; 10−4: P = 0.28).

Fig 2

Microvascular reactivity in vivo of arterioles in the cremaster muscle at the end of the experimental period (4th week).

Data are expressed as median (1st − 3rd quartile) (n = 24). (a) Endothelium-dependent vasodilation; (b) endothelium-independent vasodilation. *: Significant difference vs. SC (P < 0.05).

Exercise performed with different intensities elicited different adaptations in antioxidant enzymatic activity and oxidative damage at left ventricle homogenates, as shown in Fig 3. CAT activity did not change in any group (P = 0.12). In contrast, SOD and GPX activity were significantly increased in HI (P = 0.04; P = 0.01), but not MI (P = 0.29; P = 0.26). Protein carbonyl content was higher in HI (P = 0.003), suggesting an increase in protein oxidative damage, whereas no change was observed after training in MI (P = 0.07). Malondialdehyde content did not differ among groups (P = 0.38). Fig 4 exhibits results of eNOS expression in aorta. Western Blot analysis could not detect differences between groups (HI = 1.10 ± 0.2 a.u., MI = 1.39 ± 0.2 a.u., SC = 1.00 ± 0.1 a.u.; P = 0.44).

Fig 3

Biomarkers of oxidative stress analyzed on left ventricle samples at the end of the experimental period (4th week).

Data are expressed as mean ± SEM (n = 24). (a) CAT Activity; (b) SOD Activity; (c) GPX Activity; (d) TBARS—MDA content; (e) Protein Carbonyls. *: significant difference vs. SC (P < 0.05); **: significant difference vs. SC (P < 0.01).

Fig 4

Western blot analysis of aortic eNOS at the end of the experimental period (4th week).

Data are expressed as mean ± SEM (n = 24). No significant difference was found between the groups (P = 0.44).

Discussion

This study investigated the effects of high- and moderate-intensity aerobic training with equivalent volume (or ‘dose’ reflected by EE) upon microvascular reactivity in cremaster muscle and biomarkers of oxidative stress in myocardium, considered as central and systemic cardiovascular health markers, respectively. It has been hypothesized that protocols with equivalent training volume would elicit similar outcomes, regardless of differences in training intensity. At least for microcirculation, our findings confirmed this hypothesis, since improvements in endothelium-dependent vasodilation were similar across training groups. On the other hand, only high-intensity training was able to improve SOD, GPX, and protein carbonyl content in myocardium.

Some prior research investigated the effects of different exercise intensities upon isolate vasculature [2, 13, 14] or myocardium [9, 15]. However, we could not find studies assessing the concomitant effects of aerobic training upon those peripheral and central markers. Evidently, this approach seems to be more adequate to investigate whether adaptations induced by different training protocols upon a given marker would extend to others. Moreover, bias related to exercise volume has never been addressed by previous research about effects of exercise intensity upon different cardiovascular markers [14, 30, 31].

Consistently with our initial hypothesis, significant improvements in endothelium-dependent vasodilation were similar across training groups in the highest concentration of ACh (MI = 168.7% vs. HI = 164.6%; P = 0.91), suggesting that effects upon microcirculation would be more related to exercise volume than intensity. This finding concurs with data previously reported, suggesting that potential effects of chronic exercise on vasculature would be rather associated to overall exercise ‘dose’ (as reflected by EE) than isolate relative intensity or duration [17, 18]. Potential mechanisms underlying chronic adaptations in endothelium include increased shear stress and NO bioavailability [2, 32]. Another finding that reinforces the beneficial role of these mechanisms on the endothelium function is the lack of differences in endothelium-independent vasodilation between control and trained groups, also in the highest concentration of SNP (SC = 136.1%, MI = 141.6%, HI = 136.1%; P = 0.28), which by the way is supported by previous studies [33, 34].

Altogether, those findings support the hypothesis that the caloric cost of physical training per se may elicit favorable adaptations in endothelial function. Conversely, our findings did not confirm the premise that microvascular endothelial improvements would be due to greater systemic NO production, since no difference between groups was found for aortic eNOS expression. Several studies indicated that an increase in vascular eNOS would occur in response to prolonged training (>10 weeks) [2]. In the present study, the training duration of 4 weeks was perhaps too short to elicit significant changes in eNOS expression [35].

Some prior research suggested that aerobic training may improve vasoreactivity in arterioles, regardless of concomitant increasing in metabolic activity or blood flow during exercise (i.e. non-exercised muscles) [4, 32, 36]. By choosing the cremaster muscle to assess in vivo vasoreactivity instead of active muscle beds (soleus, gastrocnemius) as performed in most studies [2, 32, 34], we have reinforced the premise that chronic exercise effects upon microcirculation are rather systemic than local [32, 33]. This systemic response seems to be independent of exercise intensity, which gives room for interesting practical applications in regards to aerobic training and cardiovascular health.

Moreover, our findings suggest that favorable microcirculation adaptations to physical training might occur irrespective of predominance of type I fibers in a given muscle; indeed, slow oxidative fibers are often associated with higher vasodilation capacity [37], while the predominance in cremaster muscle is of fast glycolytic fibers [38]. Despite the overall strengths by deciding for cremaster muscle, the assessment of vasoreactivity solely in such muscle represents the main limitation of the present study.

With respect to the effects of training intensity upon myocardium, only high-intensity training improved SOD and GPx activities, while CAT did not respond to any exercise intervention (P = 0.12). These findings were expected and concur with prior studies showing that activity of GPx [9, 15] and SOD [9] increased after vigorous aerobic training, while effects upon CAT remain unclear [15, 39]. The fact that MDA and protein carbonyls differ in many aspects (i.e. repair systems, chemical stability, molecular formation, or sensitivity for detection) [40, 41] can help to explain why MDA levels remained unaltered, while protein carbonyls increased. Furthermore, the increase of protein carbonyls levels in HI evokes persistent doubts in regards to whether augmentation in tissue damage by ROS-generation in the heart would be necessary to stimulate antioxidant protection, or whether this acute effect would be harmful due to constant cell-damaging [13, 14]. While this issue remains unclear, it is well elucidated that SOD plays an important protective role through superoxide dismutation [15] and GPX is capable of reducing a broad range of hydroperoxides, thereby conferring great protection against cell-damage by oxidation [7, 15].

We cannot provide a definitive explanation regarding the fact that higher exercise intensity induced greater improvements in myocardium antioxidant defenses, while gains in microvascular reactivity appeared to rely more on exercise volume than intensity. However, plausible reasons might be suggested. Firstly, it is important to consider that different protocols of exercise can elicit distinct cardiovascular adaptations, either central or peripheral. With respect to the vascular milieu, a minimal amount of aerobic exercise seems to be capable to induce changes in the endothelium via several pathways, involving both physical and chemical stimuli. The accumulated evidence suggests that the endothelium is sensitive to a minimal increase in blood flow and metabolic demands elicited by aerobic exercise, due to increased laminar shear stress, greater nitric oxide (NO) release, reduced endothelin-1 concentration, and lowered reactive oxygen species (ROS) scavenged by antioxidant enzymes [42]. The induction of SOD activity through exercise training leads to ROS detoxification, ultimately reducing the degradation of NO and improving the endothelium dependent vasodilation. In fact, several prior studies reported an improvement in endothelial function after chronic exercise performed with either high or moderate intensity vs. sedentary controls [31, 43].

In which concerns the adaptation to oxidative stress in the myocardium, favorable effects seems to be rather provoked by aerobic exercise performed with high- than low- to moderate intensity [9, 44]. The myocardium has an elevated mitochondrial volume and exhibits one of the highest mass-specific oxygen consumption rates in the body. Moreover, it constantly copes with high rates of oxidant formation and stress [8], which helps to explain the presence of relatively high levels of GSH antioxidants. For this reason, only high cardiac workloads are able to increase the production of superoxide and oxyradicals, thereby activating antioxidant enzymes [44]. Some evidence in rodents and humans confirms that exercise needs to be of sufficient intensity to result in accumulation of ROS and subsequent oxidative stress, which progressively improves the cardioprotective effects mediated by antioxidants [45]. Kemi et al. [46] reinforced the premise that a minimum intensity threshold would be necessary to induce myocardium adaptations, suggesting that besides requiring high intensity training, those effects would also demand several weeks to be fully active. On the other hand, improvements in endothelium-dependent vasodilation would occur earlier and in response to exercise performed with lower intensities.

Some limitations of this study must be acknowledged. Firstly, it is difficult to fully extrapolate the findings of experiments developed with animals to humans. Although biologically similar in many aspects, rats may fail to entirely mimic cardiovascular adaptations observed in humans, since they have different dynamics for the heart rate, oxygen consumption, metabolism, energy cost during exercise, or lipid metabolism [47, 48]. Moreover, antioxidant activity, lipid peroxidation, and protein damage in vasculature have not been assessed in the present study. Those biomarkers could indicate whether exercise volume and intensity might also modulate oxidative stress in the vessels. Additionally, vasoreactivity was only assessed in the cremaster muscle. Although cremaster has been widely used for evaluating systemic vasodilation in vivo, its utilization limits the generalization of the present data. Hence, further studies including a translational approach are warranted to investigate the effects of training intensity upon microcirculation in other body regions.

Conclusion

Overall, our findings corroborate the premise that a minimal increase in daily caloric expenditure promoted by exercise would be enough to maintain the endothelial health, but not to enhance antioxidants in the myocardium. Given that gains in antioxidant enzymes were only detected in the group that performed high-intensity training, it seems that vigorous exercise would be necessary to improve myocardial antioxidant defenses.

Raw data of maximal graded test, isocaloric exercise bouts, and EPOC.

(XLSX)

Click here for additional data file.

Raw data of oxidative stress, microvascular reactivity, and eNOS expression.

(XLSX)

Click here for additional data file.

27 Jun 2019

PONE-D-19-14652

Effects of moderate and high intensity isocaloric aerobic training upon microvascular reactivity and myocardial oxidative stress in rats

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The authors present an interesting work with sufficient body of evidence. The paper is well presented and well documented. I have some major comments, though, that have to be addressed by the authors before the manuscript is accepted for publication.

1. Can the authors hypothesize why protein carbonyls increased in HI training, whereas TBARS remained unaffected? Are they aware of a putative molecular mechanism that could explain these findings given that both biomarkers refer to macromolecule oxidation?

2. Why did the authors choose a rather unusual muscle, namely cremaster, in order to evaluate microvascular reactivity?

3. Do the authors have a plausible explanation regarding the fact that higher exercise

intensity induces greater improvements in myocardium antioxidant defenses, while gains in microvascular reactivity appeared to rely more on exercise volume than intensity? Please refer to other references from the literature.

4. The English language needs improvement throughout the manuscript.

Reviewer #2: Effects of moderate and high intensity isocaloric aerobic training upon microvascular reactivity and myocardial oxidative stress in rats

The question posed by the authors of this study is interesting. Intensity of exercise and adaptations on endothelial function or myocardium antioxidant protection is an area of scientific interest. Also, studies on intensity and volume of exercise on animal models are scarce. The study is generally well written with good presentation of the data needed. I will proceed to a few suggestions to the authors.

Introduction

Line 57: “seems to be”

Methods

Line 130: A title is needed

Results/ tables/ graphs

It is not clear what the overall diet of rats is. There is data to suggest that microvascular reactivity and oxidative stress markers are influenced by diet. It would be good to mention at least the overall energy intake value (kcal/day) and the macronutrient content of the diet. Was it isocaloric and specific among the two groups of intervention? All groups gained the same kg at the end of the study; however, was quality the same? What was the diet of control group?

It is not clear whether measurements shown in graphs are taken on 4 wks. This should be written on the legends.

Line 228: “significantly lower”

Line 230: “significantly increased”

Discussion

Limitations are well pointed. Possibly it would be of interest to see some sentences regarding the speculated implications of the results on human subjects.

**********

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Reviewer #1: No

Reviewer #2: No

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7 Sep 2019

Reviewer #1:

The authors present an interesting work with sufficient body of evidence. The paper is well presented and well documented. I have some major comments, though, that have to be addressed by the authors before the manuscript is accepted for publication.

1. Can the authors hypothesize why protein carbonyls increased in HI training, whereas TBARS remained unaffected? Are they aware of a putative molecular mechanism that could explain these findings given that both biomarkers refer to macromolecule oxidation?

Answer: We thank the reviewer for the opportunity to clarify this question. Some mechanisms might indeed explain the responses observed for TBARS (MDA) and protein carbonyls. One of them is the difference between the repair systems of lipid and protein oxidation. Oxidized proteins are completely degraded by proteases before protein replacement and resynthesis. In the event of high oxidative stress, the proteolytic capacity of cells may not be sufficient enough to counteract the increasing of oxidized protein molecules, which accumulate in the cells as “oxidative junk”. On the other hand, products from lipid peroxidation are easily decomposed into several reactive species (including MDA) which act as cross-linking reagents for protein oxidation (please refer to Life 2000; 50: 279-89). To summarize, MDA is less stable than protein carbonyls and its degradation to participate in protein oxidation is faster (due to a less complex system). All those features might contribute to differences of chemical stability between MDA and protein carbonyls, which may have also influenced the results. In molecular terms, oxidized protein seems to have earlier formation and higher stability than oxidized lipids. In this sense, Dalle-Done et al. (Clin Chim Acta. 2003; 329: 23-38) suggested that cells are able to degrade oxidized proteins within hours (or days), while end products of lipid peroxidation are detoxified in a few minutes (please refer to Antioxid. Redox Signal 2013;18:1208-46; and Clin Chim Acta 2003;329:23-38). Carbonyl groups have therefore earlier formation and circulate for longer time, which leads to a greater accumulation in the cells vs. MDA. This fact might increase the relative sensitivity for the detection of oxidized molecules.

It is worthy to mention that the characteristics of the training protocol may have influenced the results regarding those biomarkers. The levels of MDA, protein carbonyl, and antioxidant enzymes are acknowledged to vary depending on the training period and assessment window (please refer to Comp Biochem Physiol 2006;143:239-45). This means that antioxidant enzymes or oxidized molecules may not equally respond to the training stimuli and at the same time. In our study, it is reasonable to speculate that albeit protein carbonyl appears altered after 4 weeks of high intensity training, it is unclear whether a longer training period would provoke similar responses. It is expected that both MDA and oxidized proteins would be lowered after 8-12 weeks as chronic adaptation to exercise training (please refer to J Appl Physiol 2000; 89: 21-8).

In order to clarify this issue, a sentence related to these putative mechanisms were added to the Discussion section: “The fact that MDA and protein carbonyls differ in many aspects (i.e. repair systems, chemical stability, molecular formation, or sensitivity for detection) can help to explain why MDA levels remained unaltered, while protein carbonyls increased” (page 13, Lines 297-300).

2. Why did the authors choose a rather unusual muscle, namely cremaster, in order to evaluate microvascular reactivity?

Answer: It is an important issue and we agree that our option must be justified. Actually, the effects of chronic exercise upon microvascular reactivity have been assessed in a wide range of skeletal muscles (e.g. soleus, gastrocnemius, gracilis, spinotrapezius) (please refer to Med Sci Sports Exerc 2006; 38: 445-54). Studies examining non-locomotor muscles or muscles with low- to intermediary metabolic demand are scarce. The cremaster is a striated and non-locomotor muscle without any major or direct recruitment during physical activity. Following this reasoning, we have chosen the cremaster to confirm that the effects of exercise training would be systemic rather than a consequence of increased muscle mechanical activity.

Some prior studies have examined non-locomotor muscles to investigated the effects of exercise upon microcirculation, as the cremaster or cheek pouch (please refer to J Appl Physiol 2005;98:2113-8; J Appl Physiol Respir Environ Exerc Physiol 1981; 51: 282-7; and Microvasc Res 2014; 93: 34-41). Overall, these muscles have proven to be adequate tissues for the assessment of microcirculatory changes. Moreover, we highlight the fact that they are easily prepared due to their morphological features (i.e. thin layers, well distributed vascularization, or distinct visualization of microvessels). In short, albeit we agree with the reviewer that the cremaster is an unusual muscle to evaluate the microvascular reactivity, the assessment of its arterioles by intravital microscopy enables to visualize several biological vascular processes (please refer to Methods Mol Biol 2015; 1339: 357-66).

3. Do the authors have a plausible explanation regarding the fact that higher exercise intensity induces greater improvements in myocardium antioxidant defenses, while gains in microvascular reactivity appeared to rely more on exercise volume than intensity? Please refer to other references from the literature.

Answer: Firstly, it is important to consider that different protocols of exercise can elicit distinct cardiovascular adaptations, either central or peripheral. With respect to the vascular milieu, a minimal amount of aerobic exercise seems to be capable to induce changes in the endothelium via several pathways, involving both physical and chemical stimuli. The accumulated evidence suggests that the endothelium is sensitive to a minimal increase in blood flow and metabolic demands elicited by aerobic exercise, due to increased laminar shear stress, greater nitric oxide (NO) release, reduced endothelin-1 concentration, and lowered reactive oxygen species (ROS) scavenged by antioxidant enzymes (please refer to Sports Med 2009; 39: 797-812).

The induction of SOD activity through exercise training leads to ROS detoxification, ultimately reducing the degradation of NO and improving the endothelium dependent vasodilation. In fact, several prior studies reported an improvement in endothelial function after chronic exercise performed with either high or moderate intensity vs. sedentary controls (Cardio Res. 2009; 81, 723–32; Circulation. 2008; 118:346-54; J Appl Physiol. 2016; 121: 279-88). In which concerns the adaptation to oxidative stress in the myocardium, favorable effects seems to be rather provoked by aerobic exercise performed with high- than low- to moderate intensity (please refer to Am J Physiol. 1993; 265(6 Pt 2):H2094-8; Mol Med Rep. 2015; 12: 2374-82). The myocardium has an elevated mitochondrial volume and exhibits one of the highest mass-specific oxygen consumption rates in the body. Moreover, it constantly copes with high rates of oxidant formation and stress (please refer to J Appl Physiol 2000; 89: 21-8), which helps to explain the presence of relatively high levels of GSH antioxidants. For this reason, only high cardiac workloads are able to increase the production of superoxide and oxyradicals, thereby activating antioxidant enzymes (please refer to Mol Med Rep. 2015; 12: 2374-82). Some evidence in rodents and humans confirms that exercise needs to be of sufficient intensity to result in accumulation of ROS and subsequent oxidative stress, which progressively improves the cardioprotective effects mediated by antioxidants (please refer to Adv Clinic Chem 2008; 46: 1-50). Kemi et al. (Cardio Res 2005; 67: 161-72) reinforced the premise that a minimum intensity threshold would be necessary to induce myocardium adaptations to oxidative stress, suggesting that besides requiring high intensity training, those effects would also demand several weeks to be fully active. On the other hand, improvements in endothelium-dependent vasodilation would occur earlier and in response to exercise performed with lower intensities. All this rationale was added to the Discussion section (Pages 14-15, Lines 307-336).

4. The English language needs improvement throughout the manuscript.

Answer: We double-checked the manuscript for spelling and grammar flaws.

Reviewer #2: Effects of moderate and high intensity isocaloric aerobic training upon microvascular reactivity and myocardial oxidative stress in rats.

The question posed by the authors of this study is interesting. Intensity of exercise and adaptations on endothelial function or myocardium antioxidant protection is an area of scientific interest. Also, studies on intensity and volume of exercise on animal models are scarce. The study is generally well written with good presentation of the data needed. I will proceed to a few suggestions to the authors.

Introduction

1) Line 57: “seems to be”

Answer: The correction was made.

Methods

2) Line 130: A title is needed

Answer: The correction was made

Results/ tables/ graphs

3) It is not clear what the overall diet of rats is. There is data to suggest that microvascular reactivity and oxidative stress markers are influenced by diet. It would be good to mention at least the overall energy intake value (kcal/day) and the macronutrient content of the diet. Was it isocaloric and specific among the two groups of intervention? All groups gained the same kg at the end of the study; however, was quality the same? What was the diet of control group?

Answer: We thank the reviewer for the opportunity to clarify this question. Indeed, there are several evidences demonstrating that diet composition can affect oxidative stress (please refer to Int J Vitam Nutr Res. 2001; 71:339-46; Nutr J 2011; 10: 122; Redox Biol 2016; 8: 216-25). High-fat diets, for example, are able to increase the efflux of non-esterified fatty acids, the production of ROS and the levels of lipid peroxidation, resulting in oxidative imbalance and endothelial cell impairment. In the present experiment, only a manufactured standard rat chow (Nuvilab CR-1, NuvitalTM) was offered to animals assigned in SC, HI and MI. Considering that this standard chow provided 3.78 kcal/g (56% carbohydrates, 22% proteins and 4% lipids), it is possible to claim that all groups were kept in isocaloric diet conditions. In order to elucidate this issue, we have included in the Results section data of body mass evolution (new Figure 1) and overall energy intake (kcal/day) (Page 9, Lines 206-208).

4) It is not clear whether measurements shown in graphs are taken on 4 wks. This should be written on the legends.

Answer: We added this information in the legends, as follows (Page 23, Lines 591-605):

“Fig. 1 – Body mass during the experimental period (4 weeks). Data are expressed as mean ± SEM (n = 24). No significant difference was found between the groups (P = 0.59).

Fig. 2 − Microvascular reactivity in vivo of arterioles in the cremaster muscle at the end of the experimental period (4th week). Data are expressed as median (1st − 3rd quartile) (n = 24). (a) Endothelium-dependent vasodilation; (b) endothelium-independent vasodilation. * Significant difference vs. SC (P < 0.05).

Fig. 3 – Biomarkers of oxidative stress analyzed on left ventricle samples at the end of the experimental period (4th week). Data are expressed as mean ± SEM (n = 24). (a) CAT Activity; (b) SOD Activity; (c) GPX Activity; (d) TBARS - MDA content; (e) Protein Carbonyls. *: significant difference vs. SC (P < 0.05); **: significant difference vs. SC (P < 0.01).

Fig. 4 – Western blot analysis of aortic eNOS at the end of the experimental period (4th week). Data are expressed as mean ± SEM (n = 24). No significant difference was found between the groups (P = 0.44)”.

5) Line 228: “significantly lower”

Answer: The correction was made.

6) Line 230: “significantly increased”

Answer: The correction was made.

Discussion

7) Limitations are well pointed. Possibly it would be of interest to see some sentences regarding the speculated implications of the results on human subjects.

Answer: This suggestion is pertinent. Even though transferring the meaning of data obtained in rodents to humans is always to a certain degree speculative, we can at least suggest potential implications. Mice and rats can share up to 98% of DNA with humans – as a result, in appropriate experimental conditions these animals may exhibit similar cardiovascular problems that afflict human beings, as well as benefit of specific interventions (e.g. pharmacological treatment, exercise, or diet). In contrast to humans, research with rodents allows investigating in detail the isolate effects of those interventions upon blood vessels and cardiac tissues (please refer to Biomed Res Int. 2015; 2015: 528757). On the other hand, there are many physiological differences between rats and humans that must be considered when extrapolating the results of experiments with rodents to humans (i.e., fiber types, speed of muscle contraction, muscle metabolic activity, energy cost of exercise, heart and respiratory rate, oxygen consumption, and lipid metabolism) (please refer to Physiol Rep. 2015; 3(2): e12293). In order to address this particular issue, we have included some sentences in the limitation’s paragraph, as follows: “Firstly, it is difficult to fully extrapolate the findings of experiments developed with animals to humans. Although biologically similar in many aspects, rats may fail to entirely mimic cardiovascular adaptations observed in humans, since they have different dynamics for the heart rate, oxygen consumption, metabolism, energy cost during exercise, or lipid metabolism (Biomed Res Int 2015; 2015: 528757; Physiol Rep 2015; 3: e12293)” (Page 15, Lines 337-341).

REFERENCES

Boa, B.; Costa, R.; Souza, M. et al. Aerobic exercise improves microvascular dysfunction in fructose fed hamsters. Microvasc Res. 2014; 93: 34-41.

Bloomer, R.; Effect of exercise on oxidative stress biomarkers. Adv Clinic Chem. 2008; 46:1-50.

Davies, K. Oxidative Stress, Antioxidant Defenses, and Damage Removal, Repair, and Replacement Systems. Life. 2000; 50: 279–89.

Dalle-Done, I.; Rossi, R.; Giustarini, D. et al. Protein carbonyl groups as biomarkers of oxidative stress. Clin Chim Acta. 2003; 329: 23–38.

Francescomarino, S.; Sciartilli, A.; Valerio, V. et al. The Effect of Physical Exercise on Endothelial Function. Sports Med. 2009; 39: 797-812.

Georgios Goutianos, G.; Tzioura, A.; Kyparos, A. et al. The rat adequately reflects human responses to exercise in blood biochemical profile: a comparative study. Physiol Rep. 2015; 3(2): e12293.

Gul, M.; Demircan, B.; Taysi, S. et al. Effects of endurance training and acute exhaustive exercise on antioxidant defense mechanisms in rat heart. Comp Biochem Physiol. 2006, 143: 239-45.

Haram, P.; Kemi, O.; Lee, S. et al. Aerobic interval training vs. continuous moderate exercise in the metabolic syndrome of rats artificially selected for low aerobic capacity. Cardio Res. 2009; 81, 723–32.

Harris, P.; Joshua, I.; Miller, F. Decreased vascular sensitivity to norepinephrine following exercise training. J Appl Physiol Respir Environ Exerc Physiol. 1981; 51: 282-7.

Jasperse, J.; Laughlin, H. Endothelial function and exercise training: Evidence from studies using animal models. Med Sci Sports Exerc. 2006; 38: 445-54.

Kakimoto, P.; Kowaltowski, A. Effects of high fat diets on rodent liver bioenergetics and oxidative imbalance. Redox Biol 2016; 8: 216–25.

Kemi, O.; Haram, P. Loennechen, J. et al. Moderate vs. high exercise intensity: Differential effects on aerobic fitness, cardiomyocyte contractility, and endothelial function. Cardio Res. 2005; 67: 161-72.

Leong, X.; Ng, C.; Jaarin, K. Animal Models in Cardiovascular Research: Hypertension and Atherosclerosis. Biomed Res Int. 2015; 2015: 528757.

Liu, J.; Yeo, H.; O¨Vervik-Douki, E.; et al. Chronically and acutely exercised rats: biomarkers of oxidative stress and endogenous antioxidants. J Appl Physiol. 2000; 89: 21–8.

Lu, Y.; Chiang, C. Effect of Dietary Cholesterol and Fat Levels on Lipid Peroxidation and the Activities of Antioxidant Enzymes in Rats. Int J Vitam Nutr Res. 2001; 71: 339-46.

Lu, K.; Wang, L. Wang, C. et al. Effects of high-intensity interval versus continuous moderate‑intensity aerobic exercise on apoptosis, oxidative stress and metabolism of the infarcted myocardium in a rat model. Mol Med Rep. 2015; 12: 2374-82.

Orth, T.; Allen, J.; Wood, J. et al. Exercise training prevents the inflammatory response to hypoxia in cremaster venules. J Appl Physiol. 2005; 98: 2113-8.

Peairs, A.; Rankin, J.; Lee, Y. Effects of acute ingestion of different fats on oxidative stress and inflammation in overweight and obese adults. Nutr J. 2011; 10: 122.

Powers, S.; Criswell, D.; Lawler, J. et al. Rigorous exercise training increases superoxide dismutase activity in ventricular myocardium. Am J Physiol. 1993; 265(6 Pt 2): H2094-8.

Radak, Z.; Zhao, Z.; Koltai, E. et al. Oxygen Consumption and Usage During Physical Exercise: The Balance Between Oxidative Stress and ROS-Dependent Adaptive Signaling. Antioxid. Redox Signal. 2013; 18: 1208-46.

Rius, C.; Sanz, M. Intravital Microscopy in the Cremaster Muscle Microcirculation for Endothelial Dysfunction Studies. Methods Mol Biol. 2015; 1339: 357-66.

Tjønna, A.; Lee, S.; Rognmo, Ø. et al. Aerobic interval training versus continuous moderate exercise as a treatment for the metabolic syndrome: a pilot study. Circulation. 2008; 118: 346-54.

Sawyer, B.; Tucker, W.; Bhammar, D. et al. Effects of high-intensity interval training and moderate-intensity continuous training on endothelial function and cardiometabolic risk markers in obese adults. J Appl Physiol. 2016; 121: 279-88.

Submitted filename: Response to Reviewers_Lorena.docx

Click here for additional data file.

28 Oct 2019

PONE-D-19-14652R1

Effects of moderate and high intensity isocaloric aerobic training upon microvascular reactivity and myocardial oxidative stress in rats

PLOS ONE

Dear Prof Farinatti,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please address all comments raised by the reviewers as well as the following comments of the editor:

1. Please label appropriately  the western blot images provided. I have examined the revised paper as well as the figure in question and the raw data. The bands of fig. 4 are very blurry as they are in the original blot. As it seems, authors actually measured the protein levels of three different groups of rats following the intervention. There is no ladder or control in the original figure. The original figure has 9 columns that I am not sure what they represent since there are no labels. Each column must represent a different animal from one of the three experimental groups. Authors need to provide the original membranes from all the animals (24 in total) and label each column to let us know which column represents a specific animal of a specific group. Therefore, I invite you to provide the original membranes from all the animals (ideally, a control and ladder should also be provided).

2. Please explain why the study design did not include a  measurement of the dependent variables at baseline, to allow the evaluation  of delta differences between pre- and post-training values in the three groups.

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19 Nov 2019

Rio de Janeiro, November 14th, 2019

- Code: PONE-D-19-14652R1

- Title: “Effects of moderate and high intensity isocaloric aerobic training upon microvascular reactivity and myocardial oxidative stress in rats"

- Corresponding Author: Paulo Farinatti

- e-mail: ptvf1964@gmail.com

Dear Editor,

Please find below our responses to the reviewers’ comments concerning our manuscript (PONE-D-19-14652R1), entitled “Effects of moderate and high intensity isocaloric aerobic training upon microvascular reactivity and myocardial oxidative stress in rats”. Thank you for the opportunity of resubmitting our manuscript.

We have addressed all the issues raised by the reviewers. The manuscript has been rewritten according to the reviewers’ suggestions, and an itemized, point-by-point response to each of the reviewers’ comments has been provided. Changes on the manuscript are marked in the “Revised Manuscript with Track Changes”, as per the received instructions.

Yours Sincerely,

Paulo Farinatti, PhD

University of Rio de Janeiro State

Salgado de Oliveira University

1) Please label appropriately the western blot images provided I have examined the revised paper as well as the figure in question and the raw data. The bands of fig. 4 are very blurry as they are in the original blot. As it seems, authors actually measured the protein levels of three different groups of rats following the intervention. There is no ladder or control in the original figure. The original figure has 9 columns that I am not sure what they represent since there are no labels. Each column must represent a different animal from one of the three experimental groups. Authors need to provide the original membranes from all the animals (24 in total) and label each column to let us know which column represents a specific animal of a specific group. Therefore, I invite you to provide the original membranes from all the animals (ideally, a control and ladder should also be provided).

Answer: We have sent the figures with the molecular weight and labels for each group. Below the pictures we placed the overlay of blot images with the colorimetric image showing the weight markers. We hope that this information will be enough to satisfy the Editor’s demand.

2) Please explain why the study design did not include a measurement of the dependent variables at baseline, to allow the evaluation of delta differences between pre- and post-training values in the three groups

Answer: Measurements of some outcomes at baseline were not feasible, since animals should be sacrificed. Alternatively, we could have added three more groups of rats to be sacrificed in control conditions (surgical procedures to expose cremaster muscle, and extraction of aorta and left ventricle). However, the value of this “control assessment” would be limited, since pre vs. post-training comparisons would not be inter-individual. The refinement of the experiment by doing so would not be questionable and the unnecessary sacrifice of animals would violate the ethical requisite of 3R's (Replacement, Reduction and Refinement) (Please refer to Bate S, Karp N. PLoS One 2014; 9: e114872). Given these ethical reasons, repeated measurements to evaluate deltas were performed only for variables which assessment was not invasive, and when surgery procedures were not necessary (e.g. VO2 max, body weight). In the case of variables invasively assessed, instead of performing repeated measurements we adopted an approach widely used in prior research with animals, by including only one control group (sedentary animals) that directly contrasted with both experimental groups (exercised animals) (Please refer to Johnson P, Besselsen D. ILAR Journal, 2002; 43: 202-6). In fact, this procedure is usually applied in studies investigating cardiovascular outcomes, particularly microcirculation (Please refer to Machado M et al. Exp Physiol, 2017 102: 1716–28; Boa B et al. PLoS ONE 2014, 9: e102554). Finally, we could claim that outcomes in our study have not been affected by potential confounding variables (as aging, diet, or exercise protocol). Thus, a single control group seemed to be enough to provide data corresponding to “baseline condition” (i.e. without any intervention).

Submitted filename: Response letter 14-11-19.docx

Click here for additional data file.

24 Jan 2020

Effects of moderate and high intensity isocaloric aerobic training upon microvascular reactivity and myocardial oxidative stress in rats

PONE-D-19-14652R2

Dear Dr. Farinatti,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The authors have successfully addressed my comments in the revised version of the manuscript. Therefore, I recommend the publication of the manuscript.

Reviewer #2: Comments have been addressed and authors made changes in the text. Some details missing are now added (eg time points of the measurements are now added in figure legends). Also explanation in answers is backed with literature.

**********

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Reviewer #1: No

Reviewer #2: No

31 Jan 2020

PONE-D-19-14652R2

Effects of moderate and high intensity isocaloric aerobic training upon microvascular reactivity and myocardial oxidative stress in rats

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