Resistance training prevents right ventricle hypertrophy in rats exposed to secondhand cigarette smoke

Ana Caroline Rippi Moreno; Gisele Alborghetti Nai; Caroline Pancera Laurindo; Karen Cristina Rego Gregorio; Tiago Olean-Oliveira; Marcos Fernando Souza Teixeira; Patricia Monteiro Seraphim

doi:10.1371/journal.pone.0236988

. 2020 Aug 7;15(8):e0236988. doi: 10.1371/journal.pone.0236988

Resistance training prevents right ventricle hypertrophy in rats exposed to secondhand cigarette smoke

Ana Caroline Rippi Moreno ¹, Gisele Alborghetti Nai ², Caroline Pancera Laurindo ¹, Karen Cristina Rego Gregorio ¹, Tiago Olean-Oliveira ¹, Marcos Fernando Souza Teixeira ³, Patricia Monteiro Seraphim ^1,^*

Editor: Stanton A Glantz⁴

PMCID: PMC7413484 PMID: 32764771

Abstract

Exposure to secondhand cigarette smoke is associated with the development of diverse diseases. Resistance training has been considered one of the most useful tools for patients with pulmonary disease, improving their quality of life. This study aimed to evaluate the effect of resistance training (RT) on the prevention of thickening of the right ventricle wall of rats exposed to secondhand cigarette smoke. Thirty-two Wistar rats were divided into four groups: Control (C), Smoker (S), Exercised (E) and Exercised Smoker (ES). The smoker groups were exposed to the smoke of four cigarettes for 30 min, twice daily, five days a week, for 16 weeks. The exercised groups climbed on a vertical ladder with progressive load, once a day, five days a week, for 16 weeks. The heart, trachea, lung, liver and gastrocnemius muscle were removed for histopathological analysis. Pulmonary emphysema (S and ES vs C and E, P < 0.0001) and pulmonary artery thickness enlargement (S vs C and E, P = 0.003, ES vs C, P = 0.003) were detected in the smoking groups. There was an increase in the right ventricle thickness in the S group compared with all other groups (P < 0.0001). An increase in resident macrophages in the liver was detected in both smoking groups compared with the C group (P = 0.002). Additionally, a relevant reduction of the diameter of the muscle fibers was detected only in ES compared with the C, S and E groups (P = 0.0002), impairing, at least in part, the muscle mass in exercised smoking rats. Therefore, it was concluded that resistance training prevented the increase of thickness of the right ventricle in rats exposed to secondhand cigarette smoke, but it may be not so beneficial for the skeletal muscle of smoking rats.

1. Introduction

Smoking is a preventable disease responsible for a large number of deaths worldwide and approximately 6 million deaths per year [1]. The use of cigarettes exposes users to approximately 4720 noxious substances [2], impairing the action and function of several tissues [3, 4]. According to the World Health Organization (WHO), smoking is considered a chronic and epidemic disease and is predicted to cause more than 10 million deaths by 2030 [5].

Secondhand smoke is the combination of two smokes: the mainstream exhaled by smokers plus the burning end of a cigarette (sidestream). Its smoke contains many noxious substances, with hundreds toxic, and some can trigger câncer [6]. Exposure to secondhand cigarette smoke is related to the development of more than 50 diseases, including pulmonary emphysema and chronic bronchitis, which compose chronic obstructive pulmonary disease (COPD) [7]. Smokers are 14 times more likely to develop COPD, and 90% of the deaths caused by COPD occur in smokers or former smokers [8]. COPDs are the fourth leading cause of death in the world, and are expected to reach the third position in 2020 [7]. Socioeconomic costs for the treatment of these and other diseases related to smoking are high, and it is crucial to find tools capable of preventing and/or reducing the ill effects of this habit.

Patients with COPD often have pulmonary hyperinflation [8]. The hyperinflation consists of persistent elevation of pulmonary arterial pressure, which may be caused by increased pressure in the venous and arterial segments of the pulmonary circulation, resulting in increased pulmonary artery thickness [9]. An increase in pulmonary artery thickness is not a specific disease but a pathophysiological condition. However, it can lead to right ventricle overload, hypertrophy and dilatation of the right ventricle free wall developing pulmonary arterial hypertension [10].

Recently, exercise in the form of resistance training (RT) was classified as one of the best and most useful options for the treatment of patients with pulmonary diseases [11, 12]. It is also an effective tool for preventing insulin resistance, controlling arterial and pulmonary hypertension, in addition to improving quality of life [13]. The predominant metabolism in RT is anaerobic, involving adenosine triphosphate and phosphocreatine (ATP-PC) and the glycolytic pathway [14]. This type of metabolism ensures proper muscular endurance associated with the production of maximum strength and power, with pauses for recovery during the performance, among the series [15].

Although several benefits of RT have already been described in the literature, there is no consensus about its real contribution to the prevention of tissue alterations provoked by exposure to secondhand cigarette smoke. The present study aimed to evaluate the effect of resistance training on anatomopathological changes in the right and left ventricles, trachea, lung, gastrocnemius muscle and liver of rats exposed to secondhand smoke.

2. Material and methods

2.1 Animals

Thirty-two male Wistar rats, aged 45 days, were separated into cages with four animals per cage and kept under a controlled temperature (23 ± 2°C), in a light/dark cycle (12 h/12 h) room [16], with free access to water. The cages were filled with environmental enrichment, and all procedures were carefully done to reduce stress level, and ensure the welfare of the animals [17]. Every day the animals were monitored by observing the food intake behavior, and no alteration was noticed during all procedures with the animals.

Forty grams of commercial chow (Supralab—Alisul, Maringa, PR, Brazil) were offered to each rat, every Monday, Wednesday and Friday. The rest of the chow was weighed and subtracted from the initial offer (40 g) for monitoring. After the adaptation period, resistance training and exposure to secondhand cigarette smoke were performed for 16 weeks.

The weight gain was determined by subtracting the initial weight from the final weight on the euthanasia day.

All procedures complied with the ethical principles of animal research and were approved by the Ethical Committee for Animal Research of the School of Sciences and Technology, Sao Paulo State University, Presidente Prudente (# 02/2017).

2.2 Experimental design

The animals were divided into four groups: Control (C / n = 8)—no intervention; Exercised (E / n = 8)—performed RT; Smoker (S / n = 8)—exposed to secondhand cigarette smoke; Exercised Smoker (ES / n = 8)—exposed to secondhand smoke exposure and performed RT.

2.3 Smoking exposure

The smoking rats (S and ES) were exposed to secondhand cigarette smoke for 16 weeks [18, 19]. In the first adaptation week, all animals in the S and ES groups were exposed to secondhand cigarette smoke from combustion of 2 cigarettes for 10 min per day. A specific gas detector (ToxiPro^® from Biosystems) placed inside the chamber measured 250 ppm (parts per million) of CO (carbon monoxide) during this adaptation period to the secondhand exposure, as described in the literature [20]. After adaptation week, the smoking rats (S and ES) were exposed to further 16 weeks [18, 21]. During the experimental protocol, the smoking rats were exposed to secondhand smoke from the combustion of 4 cigarettes for 30 min long, twice a day, 5 days a week, with 350 ppm of CO / exposure. The referred dose of CO was similar to the previous studies, which avoid the risk of mortality [18–21].

The inhalation system was a custom-built system composed by a closed glass box (100 x 44 x 44 cm), divided in 2 different compartments: one compartiment (A) allocated the burning cigarettes, and the second compartment (B) allocated one cage with 4 rats to be exposed to the cigarette smoke. A ventilator was coupled to the lateral wall of the compartment (A) to push the smoke of the smoldered cigarette to the compartment (B). An exhauster was coupled to the lateral wall of the compartment (B) drawing the air through the chamber (Fig 1). Four cigarettes were lit and the complete combustion occurred during 10 minutes with no puff in the compartment (A). Additionally, the rats spent 20 minutes remaining in the compartment (B) inhaling the air saturated with the smoke of the smoldered cigarette in the compartment (A). Although the cigarettes were not pre-balanced with consistent moisture before beginning of the exposure, the sequence of the cages exposed in each day was alternated, to ensure similar characteristics of the air inhaled by all smoking rats until the end of the intervention period. Commercial cigarettes (Malboro Red^®, Philip Morris International, Brazil) containing 10mg of tar, 0.8 mg of nicotine, and 10 mg of carbon monoxide during the combustion were used, as already found in the literature [21, 22].

2.4 Resistance training

The exercised groups (E and ES) performed the climbing protocol previously reported in the literature by Horberger and Farrar [23], which mimics progressive resistance exercises in humans. Two phases were included: adaptation and experimental.

The adaptation phasis comprised the first 4 days of the intervention, in which the animals adapted to the climbing exercise by being stimulated by a manual stimulus in their rostral portion to climb to a cage present at the top of a ladder, where they were able to rest for 60 seconds. The protocol was repeated 4 x a day.

2.4.1 Maximum Supported Load (MSL) test

On the fifth day, the rats performed the maximum supported load test to determine which initial load each animal could lift [24, 25]. A glove containing washers inside was used as load, and it was attached to the tail of the animal by adhesive tape. The initial load was fixed at 75% of body weight for each animal. After each complete climb, 30 grams of weight were added. The test was interrupted if the animal performed more than eight repetitions and if, after three consecutive stimuli, the animal could not perform the climb. The maximum load recorded was the weight corresponding to the last complete climb [23–25] (see S1 Table).

2.4.2 Experimental phase

The training consisted of four series of climbs on a ladder, with intervals of 60 seconds, five times a week (on consecutive days), once per day. The training started with 50% of 01 MSL for each animal with the load increasing to 75%, 90% and 100% of 01 MSL every 4 weeks, resulting in an experimental period with 16 weeks of intervention, characteristic of long-term exercise [23–25].

2.5 Euthanasia

Anesthesia with intraperitoneal ketamine hydrochloride (60 mg / kg body weight) and xylazine hydrochloride (10 mg / kg body weight) was injected after 12 h of fasting, 24 h after the last RT and smoking sessions for removal of organs (liver, trachea, lung, heart and gastrocnemius skeletal muscle). Euthanasia occurred by exsanguination (Fig 2). Naso-anal length was measured with a ruler and body mass was weighed in the scale for calculation of Lee Index (weight^1/3/naso-anal length).

Fig 2 — 1 MSL = maximum supported load; RT = resistance training; min = minutes.

2.6 Histopathological analysis

Liver, trachea, lung, heart and gastrocnemius skeletal muscle were weighed on a precision scale; the relative weight was calculated [26] (tissue weight/final weight x10^-2), and then the tissue was stored in 10% buffered formalin. The lungs, heart and trachea were collected together. After withdrawal of the heart, 10% buffered formalin was injected slowly through the trachea for fixation of the pulmonary parenchyma. After 24 h, fragments of the right and left lung of each animal were removed. The heart was coronally sectioned at the level of the atria and ventricles, and the liver, trachea, and gastrocnemius muscle were sectioned in transverse sections.

The sections were submitted to normal histological processing, with inclusion in paraffin (Dynamics Analytical Reagents, São Paulo, Brazil). Three serial cuts of 5 μm with a spacing of 15 μm were performed on all slides. Some slides were stained by hematoxylin-eosin (HE) (Dolles, São Paulo, Brazil), others by Masson’s (Merck KgaA, Darmstadt Germany) and Alcian Blue—PAS Staining (Merck, Germany), as described in detail below.

The histopathological analysis was blinded and performed by a single experienced observer using an optical microscope (NIKON Labophot, Japan). The original stained image was captured by a Leica photomicroscope (Leica Microssistems, Switzerland) using ImageJ^® software from the National Institute of Health (NIH, USA). The parameters evaluated were as follows:

Two stainings were performed for the trachea: the first was HE staining to identify inflammatory infiltrate (0 = absent, 1 = mild, 2 = moderate, 3 = severe) and the inflammatory cell type (polymorphonuclear and / or mononuclear). The second was Alcian Blue—PAS staining, with ten high-power fields (HPFs) and a magnification of 40x; 10 photos corresponding to approximately 1 mm² were taken to identify the number of goblet cells [27, 28].
Two stainings were performed for the lung: the first was HE staining to identify interstitial inflammatory infiltrate (0 = absent, 1 = mild, 2 = moderate, 3 = severe); the inflammatory cell type (polymorphonuclear and / or mononuclear) and location (intra-alveolar, interstitial and peribronchial); tissue congestion (0 = absent, 1 = mild, 2 = moderate, 3 = severe); interstitial fibrosis (0 = absent, 1 = focal, 2 = diffuse); and emphysema [0 = absent, 1 = focal (compromising only part of the pulmonary parenchyma), 2 = diffuse (compromising all lung parenchyma)]. The second was Alcian Blue-PAS staining for analysis of pulmonary artery thickness, with two photos per animal and two measurements per photo (magnification of 200x) [28, 29]. Pulmonary emphysema was diagnosed when the alveolar spaces were enlarged and the alveolar septa retracted.
Two stainings were used in the heart: the first was HE staining to measure the thickness of the left and right ventricular free walls and the interventricular septum [28, 29]. One photo of each area (magnification of 100x) was taken with two measurements per picture. The second staining was by Masson's trichrome staining for quantification of collagen fibers, as described in detail below [30].
HE staining was used in the gastrocnemius skeletal muscle. Sections were taken from the central area of the muscle. The analysis of the diameter of the muscle fibers was performed in 50 intact fibers in the HPF photos [31].
HE staining was used in the liver to identify tissue congestion (0 = absent, 1 = mild, 2 = moderate, 3 = severe); inflammatory interstitial infiltrate (0 = absent, 1 = mild, 2 = moderate, 3 = severe) and the type of inflammatory cell present (polymorphonuclear and / or mononuclear); necrosis (0 = absent; 1 = present); cholestasis (0 = absent, 1 = present); presence and type of steatosis (0 = absent, 1 = present: microvesicular and / or macrovesicular). The Kupffer cells (resident macrophages) were counted in 10 HPF, corresponding to approximately 1 mm² in each sample.

2.7 Collagen density (Fractal dimension)

To perform the analysis of the fractal dimension of the right and left ventricles, the slides stained with Masson's trichrome were photographed (one photo for each area for each animal) and passed through the binarization process to read and analyze the fractal dimension with the box-counting method, using free ImageJ (NIH) software (http://rsbweb.nih.gov/ij/).

ImageJ software performs box counting in two dimensions, allowing the quantification of the distribution of pixels in the space. The fractal analysis of the histological slides is the relation between the resolution and the evaluated scale: DF = (Log Nr / log r-1), with Nr being the amount of equal elements needed to fill the original object and r being the scale applied to the object. Therefore, the fractal dimension calculated with ImageJ software will always be between 0 and 2, relative to the density of cardiac collagen [30].

2.8 Statistics

The results are expressed as the mean ± standard error of the mean (SEM). Two-way ANOVA was used for the quantitative results, with Tukey's as a posttest, and Kruskal-Wallis was used for analysis of categorical variables. P values less than 5% were considered statistically significant. GraphPad Prism software version 6.0 was used.

3. Results

The smoking groups had lower body mass gains compared to the C group (P = 0.005) but a similar Lee Index. However, only the ES group showed a significant reduction in feed intake compared to the C and E groups (P = 0.0005), as well as a reduction in final body weight compared to the C group (P = 0.001) (Table 1).

Table 1. Characteristics of the animals.

	C	E	S	ES
Body mass gain (g)	230.50±10.80	217.25±13.22	184.75±6.35*	169.12±9.69^#
Final body weight (g)	482.62±12.71	463.87±15.30	435.12±6.80	414.01±16.04*
Feed consumption (g)	29.52±0.58	29.33±0.60	28.48±0.42	26.59±0.40^#
Lee Index	320.19±3.34	317.49±3.13	317.48±2.74	312.63±2.04
Skeletal muscle gastrocnemius (g)	2.8±0.1	2.7±0.1	2.7±0.1	2.6±0.1

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Lee Index (weight ^1/3/naso-anal length in cm). Body mass gain (final body weight—initial body weight): *P = 0.005 vs C; ^#P = 0.005 vs C and E. Final body weight: *P = 0.001 vs C. Feed consumption (day / animal): ^#P = 0.0005 vs C and E. Values expressed as the mean ± SEM (n = 8 / group).

The trachea showed no inflammatory infiltrate or change in goblet cell counts (see S1 Fig). However, although the pulmonary tissue showed no inflammation, tissue congestion or interstitial fibrosis, pulmonary emphysema was observed in the smoking groups (Fig 3) (S and ES, P < 0.0001). Almost all smoking animals, sedentary and exercised, presented diffuse emphysema, except one ES rat.

Fig 3 — Upper left image—Typical Pulmonary parenchyma without alteration in C group. Upper right image—Typical Pulmonary parenchyma without alteration in E group. Bottom left image—Typical Pulmonary emphysema in S group. Note the dilated alveolar spaces. Bottom right image—Typical Pulmonary emphysema in ES group. Note the dilated alveolar spaces.

In the evaluation of pulmonary artery thickness, there was a significant increment in the smoking groups, with an increase in the S group compared to the control groups (C and E) and in the ES group compared to the C group (P = 0.003) (Fig 4).

Fig 4 — Upper left image—C group, Upper right image—E group, Bottom left image—S group and Bottom right image—ES group. Graph: Pulmonary artery thickness. *P = 0.003 vs C and E groups, **P = 0.003 vs C. Values are expressed as the mean ± SEM (n = 8 / group).

The S group presented increased right ventricle thickness compared to all other groups (Fig 5) (P < 0.0001). However, no significant alteration was observed in the evaluation of left ventricle thickness or septum. In the fractal analysis of both ventricles, no significant alteration was detected among the groups (see S2 Fig).

The diameter of the fibers of the gastrocnemius muscle was significantly reduced in the ES group compared to all other groups (P = 0.0002) (Fig 6).

In the liver, no inflammatory infiltrate, necrosis, cholestasis, or steatosis were observed in the groups (Fig 7). However, there was a significant increase of approximately 30% in the smoking groups (S and ES, P = 0.002) compared to the C group in the Kupffer cell counting, which indicates the number of resident macrophages of this tissue.

4 Discussion

In the present study, we observed that exposure to secondhand cigarette smoke decreased body mass, provoked pulmonary emphysema, and increased pulmonary artery thickness and the amount of resident macrophages in the liver. Resistance training in the smoking rats reduced the final body weight, which was associated with lower feed intake, reduced muscle fiber diameter of the gastrocnemius muscle, and maintenance of right ventricle thickness, which likely prevented pulmonary arterial hypertension development.

Cigarettes are the main licit drug consumed worldwide. It is estimated that there are 1,2 billion smokers worldwide and that one in five people has this harmful habit [5]. Smoking, in addition to causing dependence, is related to the appearance of several chronic diseases in the lung, heart, liver and skeletal muscle, in addition to malnutrition and emotional illness [3, 4].

A systematic review examined the effects of cigarette smoke on the body weight of smokers, concluding that low- and medium-intensity smokers (up to 1 pack per day) have a significant reduction of body weight due to nicotine causing an increase in metabolic rate and a reduction in caloric absorption and appetite [32]. In the present study, rats exposed to secondhand smoke had reduced body mass gain. In the ES group, a significant reduction in weight gain was observed compared to both control and exercised groups, accompanied by a significant reduction in final body weight and feed consumption, as described in the literature [32].

Among the COPD spectrum is pulmonary emphysema, which is characterized by the destruction of the alveolar walls and an increase in the air spaces distal to the terminal bronchioles [33]. Patients with COPD often present nutritional changes due to smoking, resulting in a weight reduction, as well as negative prognoses: survival is reduced by 13 years on average [34]. In the present study, the presence of pulmonary emphysema was detected in both groups of smokers (S and ES) groups that had reduced weight gain. In addition, the pulmonary emphysema detected was in agreement with the study by Kozma et al. [7], who concluded that exposure to secondary cigarette smoke is capable of developing a model of oxidative lung injury and inflammation, accelerating functional and morphological alterations, and limiting gas exchange [35].

It is known the worsening of pulmonary emphysema can impairs the functional capacity of skeletal muscle, such as reduction of type I motor units; atrophy of motor units; reduced capillarity and altered levels of metabolic enzymes [7]. Thus, COPD is can considered a systemic disease that is capable of resulting in musculoskeletal changes, such as weakness, muscular dysfunction and weight reduction, due to chronic oxidative stress, which also contributes to reduced caloric intake [36]. The gastrocnemius muscle was evaluated in the present study because it has mixed skeletal muscle fibers, with characteristics of type I and II motor units. This muscle is considered the prime driver of extension at the hip and knee joints and of plantarflexion at the ankle, all of which are essential movements for stair climbing [37]. A reduction in the diameter of the gastrocnemius muscle fiber was detected in the ES group. Additionally, ES rats showed reduced final body weight, reduced feed consumption, lower weight gain, pulmonary emphysema and increased macrophages in the liver. We can suggest that the reduction in the diameter of the muscle fiber can be associated with the diagnosis of pulmonary emphysema, which as previously mentioned [36], can develop negative skeletal muscle changes. In this case, although we have not studied any type of reactive oxygen species (ROS), we know that the practice of physical activity and smoking habit can develop a state of chronic oxidative stress [36], which would impair the development of muscle fibers. Thus, we suggest that the intensity of the training of smokers should be evaluated, and that RT should be performed with considerable caution in smokers.

In addition to peripheral muscle changes, exposure to smoking can also cause changes in respiratory muscles, which are responsible for pulmonary hyperinflation, a characteristic of COPD patients [38]. Pulmonary hyperinflation can change the shape of the chest wall and reduces the number of muscle fibers in the diaphragm, causing the muscle to work under increased mechanical load due to airflow limitation [39]. Thus, changes in the structure of the diaphragm in smokers include an increase in type I motor units and a decrease in type II motor units, possibly increasing the oxidative capacity of the muscle fibers of this muscle [40]. This is an aerobic adaptation of the diaphragm, which is insufficient to restore a normal contraction force, which may result in a physiopathological condition of hyperinflation [40].

Pulmonary hypertension (PH) is a hemodynamic disorder defined by an abnormal increase in pulmonary arterial pressure (PAPm), classified as PAPm ≥ 25 mmHg [41]. The three main features of PH are as follows: 1—dysfunctional pulmonary hemodynamics (i.e., exacerbated vasoconstriction and reduced vasodilation); 2—structural changes in the pulmonary vascularization (i.e., wall remodeling and hypertrophy), leading to elevated pulmonary arterial pressure; and 3- long-term right ventricular (RV) pressure overload and subsequent RV failure [42]. In the present study, we observed a significant increase in the pulmonary artery thickness in both groups of smokers. The smoking but not exercised rats developed an increase in the thickness of the pulmonary artery and of the right ventricle. On the other hand, although RT had not prevented the increase in the thickness of the pulmonary artery, it seemed to prevent the thickening of the right ventricle. It is likely that although RT in smoking rats had not prevented the second stage of PH (remodeling of the pulmonary artery wall), trained smoking rats have not shown an increase in RV, causing a delay in the prognosis of PH. This suggests that the main causal factor in this model is the lack of resistance training, which can contribute for the triggering of pulmonary hypertension in the future. We did not perform right cardiac catheterization (RCC) and transthoracic echocardiography (ET), which are relevant tools for the detection and screening of PH [41]. Further studies can be developed with the intention of verifying whether resistance training prevents the development of PH using CCD and ET techniques.

Increasingly in the literature, it is shown that liver and heart are two organs that can influence each other. There are heart diseases striking the liver, liver diseases striking the heart, and diseases striking the heart and the liver simultaneously [43]. In the present study, when we analyzed the increase of hepatic resident macrophages in the smoking groups (S and ES), we also noticed that although this group did not present steatosis and/or inflammatory infiltrate in the liver, there was innate immune system action, which is always present, ready to defend against microorganisms and eliminate damaged cells [44]. In this case, hepatocytes can have been injured by exposure to secondhand smoke, once smoking can affect organs even that it has no direct contact with cigarette smoke in fact, as liver. Smoking can provoke three effects on the liver: oncogenic effects, indirect or direct toxic effects and immunological effects [45] by agents with cytotoxic potential released during the combustion. These cytotoxic agents trigger oxidative stress, lipid peroxidation activating stellate cells and development of fibrosis in the liver [46]. Although we did not find fibrosis in our model, we find significant increased Kupffer cells after cigarette smoke exposure, suggesting anormal condition in the liver of smoking rats. Once heart was impaired in sedentary smoking rats only, it is likely that this study has been performed in the beginning of the emergence of the complications of the hepatocardiac disease [47]. Further studies characterizing the type of macrophages in the liver and analyzing the ROS reactive oxygen species in smokers could contribute to a better understanding of the mechanisms involved in resistance training effects in this model.

In summary, we suggest that despite the benefits of RT, caution is required considering the intensity of protocol of strength training in smokers, since RT reduced the muscle fibers in the gastrocnemius muscle, which did not occur with nonsmokers. However, we pointed out although resistance training did not prevent the development of pulmonary emphysema, an increase of macrophages in the liver and pulmonary artery hypertrophy, as observed in secondhand smoking rats, helped to prevent increased right ventricle thickness.

Supporting information

S1 Fig. Number of goblet cells in the trachea (per mm2).

Values of the sum of 10 fields expressed as the mean ± SEM (n = 8 / group).

(PPTX)

Click here for additional data file.^{(48.7KB, pptx)}

S2 Fig. Histopathological evaluation of the heart.

In A, thickness of the left ventricle (in μm). In B, thickness of the septum (in μm). In C, fractal dimension of the left ventricle (Log Nr / log r-1). In D, fractal dimension of the right ventricle (Log Nr / log r-1). No significant alteration was observed. Values expressed as the mean ± SEM (n = 8 / group).

(PPTX)

Click here for additional data file.^{(111.8KB, pptx)}

S1 Table. MSL weight—Details exercise capacity and MSL weight for E and ES groups in the beginning of the resistance training protocol.

(DOCX)

Click here for additional data file.^{(12.5KB, docx)}

S1 File

(DOCX)

Click here for additional data file.^{(122.5KB, docx)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. 2020 Aug 7;15(8):e0236988. doi: 10.1371/journal.pone.0236988.r001

Author response to previous submission

9 Dec 2019

Attachment

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PLoS One. doi: 10.1371/journal.pone.0236988.r002

Decision Letter 0

Stanton A Glantz

6 Feb 2020

PONE-D-19-31709

Resistance training prevents right ventricle hypertrophy in rats exposed to secondhand cigarette smoke

PLOS ONE

Dear Dr Seraphim,

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.

I agree with Reviewer 1 that you should name the brand of cigarettes. There would only be a conflict of interests if you had some financial or other relationship with the manufacturer, that would need to be disclosed.

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We look forward to receiving your revised manuscript.

Kind regards,

Stanton A. Glantz

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript by Moreno et al. reports that RV hypertrophy resulting from secondhand smoke exposure of rats can be prevented by resistance training (RT). The finding that RT completely prevented the otherwise substantial increase in wall thickness is interesting and supported by the data, and suggests that not only smokers, but people exposed frequently to high levels of secondhand smoke, would gain a health benefit from an RT exercise program in addition to the expected general health benefits of exercise. It’s less clear how to interpret the change in skeletal muscle fiber diameter. The histology is convincing and the main conclusions of the paper are solid. I do have many concerns, however, including lack of sufficient information about the smoke exposure, as follows.

Major concerns:

The authors state in their response to previous evaluation that they have opted to not identify the commercial cigarette brand that they have used, to avoid conflict of interest. This is not acceptable; they need to identify the cigarette because not all commercial cigarettes are the same; it’s important information. There should be no concern about conflict of interest here; identification of cigarettes used in such studies is routinely done and the only potential conflict of interest that I can see is if one of the authors works for or is funded by the tobacco company in question or the tobacco industry in general; is this is the case, then it needs to be disclosed but the cigarette still needs to be identified.

There are some essential details about the smoke exposure that are missing and should be added. Please provide some measure of smoke concentrations in the exposure chamber, as either total particulate matter or respirable suspended particles (<2.5 µm) (and potentially CO if that was measured). Is the smoking system a commercial system (if so, which one) or a custom built system? I don’t entirely follow the part about the chamber used for placement of a lit cigarette; despite describing airflow to bring smoke to the rats, there’s no mention of smoke being sucked through the cigarette to generate the smoke (what puff protocol was used; e.g., puff volume, puff duration, puff frequency). If no smoke was sucked through the cigarettes, and they were simple smoldering in place such as sitting in an ashtray, then that is a different model from what is typically used for secondhand smoke and should be explained. Are the multiple cigarettes all lit simultaneously or successively? (I’m guessing it was successively one at a time, since a cigarette probably can’t last for a whole 30 minutes, but please clarify.) Were they pre-equilibrated to consistent humidity levels before use, as is common practice?

I understand that table 1 is being provided to address concerns about the weight bearing protocol, but it looks more like Results than Methods to me.

Discussion: Regarding the sentence starting on line 324: “It is known that exposure to secondhand smoke intensifies the harmful effects of the smoke due to the absence of a filter; secondhand smoke has nicotine and CO2 concentrations three times higher than smoke experienced 336 by a smoker, and it has 50 times more carcinogenic substances (6,7).” I am not aware that this is the case, although it is popularly assumed. To the best of my knowledge, the filter does not filter out very much of the material that gets deep into the airways, and the mainstream smoke inhaled by a smoker is far more concentrated than secondhand smoke. (By CO2, did they mean CO?) They cite reference 6 and 7; 6 is a meta-analysis and 7 is a study about cluster headaches and neither supports the statement. I would remove this highly questionable sentence. This in no way minimizes the importance of secondhand smoke as a major source of adverse health effects.

Minor comments

While the nomenclature of SC, EC, SS, and ES makes sense, it’s REALLY hard to follow because S stands for both Sedentary and Smoker. Yes, it matters if it’s the first or second letter of the pair, but it just makes it much less intuitive to follow along for the reader. Can the authors come up with a similarly informative but more reader-friendly way of denoting the permutations?

Abstract line 37: it will be much easier to follow if a comma is added after EC. Otherwise, it’s not immediately clear whether EC belongs to the first comparison or the second. Even then, it’s a confusing line to read.

The sentence starting on abstract line 41 would be more useful if there was an indication of what the finding means (i.e., tell the reader if reduction of the muscle fiber diameter good or bad).

I rarely comment on keywords, but it seems to me that including “tobacco” as a keyword would be useful.

Line 57: It’s not accurate to say that the “consumption” of a cigarette is divided into particulate and gas phases; consumption is the verb. Better to say cigarette smoke consists of particulate and gas phases. Actually, it may be a language issue, but the entire paragraph is problematic, and references 6 and 7 seem like odd choices to use here. Are the authors attempting to describe the mainstream smoke that is inhaled through the filter and the sidestream smoke that is generated (not “exhaled”) by the burning tip? This is all irrelevant to gas or particle phases; the phrase on line 58 that the gas phase consists of two types of smoke does not make sense. I suggest that they replace this paragraph (and get rid of those two references) with a simple statement that the smoke inhaled by smokers is mainstream smoke, the smoke generated by the burning tip is sidestream smoke, the secondhand smoke in the air around smokers consists of a mixture of mostly sidestream smoke with some exhaled mainstream smoke, and that secondhand smoke contains high concentrations of noxious components.

Line 71: In “…it is fundamental to find tools…”, fundamental should probably be replaced by something like crucial or important.

Line 74: “pulmonary pressure”: do they mean pulmonary arterial pressure or increased pressure in the lungs?

Line 80: I suggest clarifying the first time they say “resistance training” to make it more obvious to readers who aren’t familiar with the topic, especially given that “insulin resistance” shows up a few words later. Perhaps “Recently, exercise in the form of resistance training (RT) was classified…”

In Table 1 in the Methods; please add units (presumably g).

Line 237, I think “tricomium” is supposed to be “trichrome”

Line 257: what is a “Lee index”? Please clarify in the text. It’s in the figure legend but the reader has not yet encountered that when the term is first used.

Line 325: Cigarette smoking is not a drug; cigarettes are the drug.

Line 414: “strength” is misspelled.

In Figure 1, the word “cigarettes” is misspelled twice, and it is missing a space in “1x day(4 days)”

The legends for Figures 2-6 include magnification denotations of the form “100x” etc., which are meaningless unless the final print size of the figure is known and not useful when viewing on a monitor. Scale bars are better, and the figures actually have scale bars already but they are much too small to be seen, so it will be a simple matter to make standard visible scale bars based on them. If a magnification number is desired, please state as “10x objective” etc.

In figures 2-5, where quartets of histology panels are shown, it will be much simpler for the reader if panels are labeled as ES, SS, etc. rather than a, b, etc.; it saves the reader from having to look at the legend to understand the figure.

I suggest making the asterisks in the bar graphs larger; they are tiny and look like lint on the computer monitor.

Reviewer #2: The manuscript by Seraphim et al. aims at determining the effect of resistance training on various parameters in rats exposed to second hand cigarette smoke. Their main conclusion is that the training prevents right ventricle hypertrophy. They provided data that are somehow related to development of right ventricle hypertrophy such as pulmonary artery thickness and lungs histopathology, as well as some supportive information such as body mass gain, food consumption, and masses of the different tissues that they included in their study. Not being an expert on the field, this reviewer cannot say how novel and interesting the findings are for the scientific community. However, there are definitely a number of issues that have to be addressed before the manuscript can be considered for publication.

General observations:

1. This reviewer was invited as an animal experiment ethics specialist. In this respect, the study is fine; there seems to be no ethical concern.

2. Although a language correction has been performed on the manuscript, clarity of the text is still below publishable quality. To illustrate it, this reviewer performed editing of a 1.5 pages section of Materials and Methods. a/ line 120-122: “The smoking rats (SS and ES) were exposed to secondhand cigarette smoke (19) for 16 weeks, which was predetermined to characterize long-term exposure (20).” The second part of the sentence may mean that 16 weeks of exposure is considered as a long term one. b/ line 125: Change “characterizing” to “representing”. c/ line 138: Cigarettes never contain carbon monoxide, which is a gas. The indicated 10 mg should be the average dose a smoker receive during a smoke. d/ line 153-154: “This test was performed only in this moment.” ???? Better delete.

Specific comments:

1. The overall design of the study indicates that more results have been anticipated than have actually been resulted. That creates some problems. The organ masses were properly presented as not significantly altered in line 260. However, according to PLOS ONE’s publishing policies “data not shown” is not permissible. Either the data should be presented, or the observation should be omitted. In this case, I suggest presenting the data as a supplementary table.

2. line 271: On what basis was pulmonary emphysema diagnosed? Put it in the Methods section.

3. Fig 4: Exercise increased right ventricle thickness in non-smoker rats while decreased it to BELOW THE CONTROL LEVEL in smoker ones. These data are very hard to understand.

4. The number of Kupffer cells in the liver is presented in Fig 6. Either move this figure to the supplementary section or explain in the Discussion section the relevance of the data presented in it to right ventricle hypertrophy.

5. The Discussion section is poor. Please, interpret the data presented in the Results section regarding right ventricle hypertrophy and related studies on the field. Also, please avoid drawing conclusions on unsupported data.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Aug 7;15(8):e0236988. doi: 10.1371/journal.pone.0236988.r003

Author response to Decision Letter 0

2 Apr 2020

Dear Stanton A. Glantz,

Firstly, we would like to thank for the appreciattion and comments in oour paper titled “Resistance training prevents right ventricle hypertrophy in rats exposed to secondhand cigarette smoke”. The review was done, and the authors accepted the suggestions as well as implemented the information needed.

The answers for the reviwers´s questions are above, as following:

Reviewer #1:

1- “The authors state in their response to previous evaluation that they have opted to not identify the commercial cigarette brand that they have used, to avoid conflict of interest. This is not acceptable; they need to identify the cigarette because not all commercial cigarettes are the same; it’s important information. There should be no concern about conflict of interest here; identification of cigarettes used in such studies is routinely done and the only potential conflict of interest that I can see is if one of the authors works for or is funded by the tobacco company in question or the tobacco industry in general; is this is the case, then it needs to be disclosed but the cigarette still needs to be identified.”

Answer: The commercial cigarette brand was added to the text in Material and Methods Section of the manuscript.

2- “There are some essential details about the smoke exposure that are missing and should be added. Please provide some measure of smoke concentrations in the exposure chamber, as either total particulate matter or respirable suspended particles (<2.5 µm) (and potentially CO if that was measured). Is the smoking system a commercial system (if so, which one) or a custom built system? I don’t entirely follow the part about the chamber used for placement of a lit cigarette; despite describing airflow to bring smoke to the rats, there’s no mention of smoke being sucked through the cigarette to generate the smoke (what puff protocol was used; e.g., puff volume, puff duration, puff frequency). If no smoke was sucked through the cigarettes, and they were simple smoldering in place such as sitting in an ashtray, then that is a different model from what is typically used for secondhand smoke and should be explained. Are the multiple cigarettes all lit simultaneously or successively? (I’m guessing it was successively one at a time, since a cigarette probably can’t last for a whole 30 minutes, but please clarify) Were they pre-equilibrated to consistent humidity levels before use, as is common practice?”

Answer: The inhalation system was a custom built system composed by a closed glass box (100 x 44 x 44 cm), divided in 2 different compartments: one compartiment for allocation of the cage with 04 rats to be exposed to the cigarette smoke, and the other for the allocation of the cigarettes to be burned. A compressor of 10 L/min-air was coupled to the cigarette compartment to push the smoke to the other compartment of the box. The compartment where the animal was allocated presented a hole for the exhaust of the smoke. Four cigarettes were lit and the complete combustion occurred during 10 minutes, however the rats spent 30 minutes inside this system. So, during the 10 first minutes inside the chamber, the cigarettes were burned, but in the rest of the time the rats were exposed only to the polluted air of the environment. The dose used in the smoke exposure is equivalent to 10 to 20 cigarettes for a chronic human smoker.

4) “I understand that table 1 is being provided to address concerns about the weight bearing protocol, but it looks more like Results than Methods to me.”

Answer: Table 1 was included as Supplementary material – see Suppl 1.

Answer: The sentence and the references were deleted of the manuscript.

Minor comments

1) While the nomenclature of SC, EC, SS, and ES makes sense, it’s REALLY hard to follow because S stands for both Sedentary and Smoker. Yes, it matters if it’s the first or second letter of the pair, but it just makes it much less intuitive to follow along for the reader. Can the authors come up with a similarly informative but more reader-friendly way of denoting the permutations?

Answer: The nomenclature was changed for the groups in the manuscript. Control (C) - without interventions; Exercised (E) - performed RT; Smoker (S) exposed to secondhand cigarette smoke; Exercised Smoker (ES) - exposed to secondhand smoke exposure and performed RT.

2) Abstract line 37: it will be much easier to follow if a comma is added after EC. Otherwise, it’s not immediately clear whether EC belongs to the first comparison or the second. Even then, it’s a confusing line to read.

Answer: The sentence was changed.

3) The sentence starting on abstract line 41 would be more useful if there was an indication of what the finding means (i.e., tell the reader if reduction of the muscle fiber diameter good or bad).

Answer: The sentence was changed.

4) I rarely comment on keywords, but it seems to me that including “tobacco” as a keyword would be useful.

Answer: The word was included as keyword.

5) Line 57: It’s not accurate to say that the “consumption” of a cigarette is divided into particulate and gas phases; consumption is the verb. Better to say cigarette smoke consists of particulate and gas phases. Actually, it may be a language issue, but the entire paragraph is problematic, and references 6 and 7 seem like odd choices to use here. Are the authors attempting to describe the mainstream smoke that is inhaled through the filter and the sidestream smoke that is generated (not “exhaled”) by the burning tip? This is all irrelevant to gas or particle phases; the phrase on line 58 that the gas phase consists of two types of smoke does not make sense. I suggest that they replace this paragraph (and get rid of those two references) with a simple statement that the smoke inhaled by smokers is mainstream smoke, the smoke generated by the burning tip is sidestream smoke, the secondhand smoke in the air around smokers consists of a mixture of mostly sidestream smoke with some exhaled mainstream smoke, and that secondhand smoke contains high concentrations of noxious components.

Answer: The sentence was substitute by “Secondhand smoke is the combination of two smokes: the mainstream exhaled by smokers plus the burning end of a cigarette (sidestream). It smoke contains more noxious substances, with hundreds toxic, and some can trigger câncer”

6) Line 71: In “…it is fundamental to find tools…”, fundamental should probably be replaced by something like crucial or important.

Answer: The word “fundamental” was substituted for “crucial”, as suggested.

7) Line 74: “pulmonary pressure”: do they mean pulmonary arterial pressure or increased pressure in the lungs?

Answer: The correct is pulmonary arterial pressure.

8) Line 80: I suggest clarifying the first time they say “resistance training” to make it more obvious to readers who aren’t familiar with the topic, especially given that “insulin resistance” shows up a few words later. Perhaps “Recently, exercise in the form of resistance training (RT) was classified…”

Answer: The sentence was changed, as suggested.

9) In Table 1 in the Methods; please add units (presumably g).

Answer: The Table 1 was deleted of the manuscript.

10) Line 237, I think “tricomium” is supposed to be “trichrome”.

Answer: The word was changed.

11) Line 257: what is a “Lee index”? Please clarify in the text. It’s in the figure legend but the reader has not yet encountered that when the term is first used.

Answer: The term was explained in details in Methods section (section 2.5).

12) Line 325: Cigarette smoking is not a drug; cigarettes are the drug.

Answer: The sentence was changed.

13) Line 414: “strength” is misspelled.

Answer: The word was corrected.

14) In Figure 1, the word “cigarettes” is misspelled twice, and it is missing a space in “1x day(4 days)”

Answer: The figure was corrected.

15) The legends for Figures 2-6 include magnification denotations of the form “100x” etc., which are meaningless unless the final print size of the figure is known and not useful when viewing on a monitor. Scale bars are better, and the figures actually have scale bars already but they are much too small to be seen, so it will be a simple matter to make standard visible scale bars based on them. If a magnification number is desired, please state as “10x objective” etc.

Answer: The legends of the figures were changed.

16) In figures 2-5, where quartets of histology panels are shown, it will be much simpler for the reader if panels are labeled as ES, SS, etc. rather than a, b, etc.; it saves the reader from having to look at the legend to understand the figure.

Answer: The labeled was changed for the abbreviation of the groups.

17) I suggest making the asterisks in the bar graphs larger; they are tiny and look like lint on the computer monitor.

Answer: The asterisks are larger.

General observations:

1) This reviewer was invited as an animal experiment ethics specialist. In this respect, the study is fine; there seems to be no ethical concern.

2) Although a language correction has been performed on the manuscript, clarity of the text is still below publishable quality. To illustrate it, this reviewer performed editing of a 1.5 pages section of Materials and Methods. a/ line 120-122: “The smoking rats (SS and ES) were exposed to secondhand cigarette smoke (19) for 16 weeks, which was predetermined to characterize long-term exposure (20).” The second part of the sentence may mean that 16 weeks of exposure is considered as a long-term one.

Answer: Sixty weeks of exposure means long-term exposure, considering rats can llive during 24 months.

According the literature (https://doi.org/10.1590/S0102-67202012000100011) each rat month in adulthood is similar to around 2.5 human years.

So we have:

1 month in adulthood rat corresponding to 2.5 human years and

4 months (16 weeks) in adulthood rat corresponding to around 10 human years,

So, 16 week smoke exposure should be considered long-term exposure.

Anyway, the sentence was removed from the text, stopping at 16 weeks in the paragraph, as following:

“The smoking rats (S and ES) were exposed to secondhand cigarette smoke for 16 weeks”.

b/ line 125: Change “characterizing” to “representing”.

Answer: The sentence was rewritten.

c/ line 138: Cigarettes never contain carbon monoxide, which is a gas. The indicated 10 mg should be the average dose a smoker receive during a smoke.

Answer: The sentence was changed for: “Commercial cigarettes were used in the study, containing 10mg of tar, 0.8 mg of nicotine, and 10 mg of carbon monoxide during the combustion”.

d/ line 153-154: “This test was performed only in this moment.” ???? Better delete.

Answer: The sentenced was deleted.

Specific comments:

1) The overall design of the study indicates that more results have been anticipated than have actually been resulted. That creates some problems. The organ masses were properly presented as not significantly altered in line 260. However, according to PLOS ONE’s publishing policies “data not shown” is not permissible. Either the data should be presented, or the observation should be omitted. In this case, I suggest presenting the data as a supplementary table.

Answer: The Table with the organ masses is presented as supplementary material.

2) line 271: On what basis was pulmonary emphysema diagnosed? Put it in the Methods section.

Answer: The sentence was added in Methods section: “Pulmonary emphysema was diagnosed when the alveolar spaces were enlarged and the alveolar septa retracted”.

3) Fig 4: Exercise increased right ventricle thickness in non-smoker rats while decreased it to BELOW THE CONTROL LEVEL in smoker ones. These data are very hard to understand.

Answer: In fact, smoking caused increase of the right ventricle thickness (see S group). Resistive training did not change the thickness of left and right ventricles nor in non-smoking (E) nor in smoking (ES) rats.

Answer: The paragraph was changed correlating liver and heart diseases and the figure 6 was maintained in the manuscript.

Answer: Thanks for the suggestion, changes were included in the Discussion Section.

Attachment

Submitted filename: Response to reviewers.doc

Click here for additional data file.^{(53KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0236988.r004

Decision Letter 1

Stanton A Glantz

29 Apr 2020

PONE-D-19-31709R1

Resistance training prevents right ventricle hypertrophy in rats exposed to secondhand cigarette smoke

PLOS ONE

Dear Dr Seraphim,

While both reviewers have said that the manuscript is improved, there are still a lot of problems, including sloppy presentation of the results and various inconsistencies in the manuscript.

I am giving you one more chance to clean up this manuscript. I will then send it back to the reviewers. If they are happy, it will be accepted. Otherwise, it will be rejected.

I strongly suggest that you have some people who are not authors on this paper carefully review everything before you resubmit the paper. It is not the reviewers' job to write the paper for you.

We would appreciate receiving your revised manuscript by Jun 13 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Stanton A. Glantz

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: No

**********

6. Review Comments to the Author

Reviewer #1: First of all, an important note: there are two versions of the revised manuscript, one clean and the other with changes tracked, but the two version are not identical. I realized in the middle of reviewing that at least one change made in the tracked changes version is not changed in the untracked version (see SS in line 304 of the clean version that has been changed to S (with the line through the deleted extra S) in corresponding line 314 of the tracked changes version. I don’t know if more slipped through, but it means that the tracked changes version is the one that should be used, or else some changes might become unchanged.

The authors have made various changes to respond to previous critiques. I think the new nomenclature for the four groups is much clearer than the previous one; a good solution. Note that the changes in group designations all show up in the changes tracked version but as mentioned above, at least one of them is not in the untracked version.

One remaining issue that we still need to fix is the lack of clarity about the smoke exposure system, despite the authors having added more details. The problem is that with a custom-built system, details of how the system works are important so that the results can be put in context of other smoke exposure literature. From the new description added, it’s still not clear to me how the smoke is being generated, and they refer to one of their previous papers but it is in Portuguese. Another new reference is to a review article about various effects of smoke on the lung; there’s no procedural detail there that is relevant to this paper. Most studies that produce secondhand smoke follow some variation of a pretty consistent workflow: air is sucked through a cigarette by some mechanism and according to a specific puff protocol (e.g. ISSO, Health Canada Intense), and the smoke from the burning tip is collected. Cigarettes are frequently pre-equilibrated to consistent humidity before use. These conditions provide not just consistency between experiments, but also the ability to place the results in context of the rest of the body of smoke research.

For example, there’s no mention in the description of air going through the cigarettes; just that the cigarettes are in a compartment and air goes from that compartment to the rats. Does that mean the cigarettes are sitting on the floor of the compartment simply smoldering in place, with no periodic puffing? If so, it would not invalidate the system, but it also would not be consistent with how such studies are typically accomplished and that should be specifically mentioned. The chemistry is different if the cigarette is smoldering in an ashtray for 10 minutes vs. if someone is actually smoking it and puffing occasionally. Please show a photograph or a diagram, and make the air path clear. It’s also not clear to me what they mean when they say that the dose of smoke exposure is equivalent to 10 to 20 cigarettes for a chronic human smoker, because their model is secondhand exposure; that can’t be directly compared with the number of cigarettes being smoked by people.

The other changes have all been made satisfactorily. I noted a misspelling in the newly added material, “simoustaneouly” in the second-to-last paragraph of the discussion (line 460 in the tracked changed version).

Reviewer #2: Although the English of the manuscript has improved (at least the first 15 pages), it is still far from tolerable (at least by this reviewer). The manuscript either has not been revised grammatically, or the proof-reader has made a very poor job. This reviewer could not evaluate the revised manuscript because of the quality of English. Proof-reading is outside the reviewers' responsibilities, although the other reviewer very kindly has also suggested a number of such mistakes in the first round. In an original submission, the mistakes could be tolerated, however, not in a revised manuscript. This reviewer has got as far as the top of page 16 before quitting, and does not want to waste his/her time on submissions that do not reflect due care from the authors' side.

page 14, line81-83: Clarify the sentence. Most probably the first comma is unnecessary. Regardless, as presented, the third part of the sentence does not confer comprehensible meaning.

page 14, line 98: Change “monitoring” to “monitored”. Change “intake food” to “intake of food”.

page 15, line 120: Change “04” to “4”, then do the same throughout the text.

page 15, line 121: Delete “only”.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. 2020 Aug 7;15(8):e0236988. doi: 10.1371/journal.pone.0236988.r005

Author response to Decision Letter 1

12 Jun 2020

We would like to start by acknowledging the thoroughness of the revision and the importance of the reviewer’s comments that greatly improved our manuscript.

The answers for the reviewers’ questions are below, as following:

Reviewer #1:

Comment 1) First of all, an important note: there are two versions of the revised manuscript, one clean and the other with changes tracked, but the two version are not identical. I realized in the middle of reviewing that at least one change made in the tracked changes version is not changed in the untracked version (see SS in line 304 of the clean version that has been changed to S (with the line through the deleted extra S) in corresponding line 314 of the tracked changes version. I don’t know if more slipped through, but it means that the tracked changes version is the one that should be used, or else some changes might become unchanged.

Response to comment 1 -: I would like to apologize because the final file of the Manuscript with untracked changes was really different of the version with tracked changes. I have made a mistake during the submission process, changing the files. I apologize about this mistake. But I am sure that this error did not compromise the relevance of the manuscript and the comprehension of the text in the manuscript.

Comment 2) The authors have made various changes to respond to previous critiques. I think the new nomenclature for the four groups is much clearer than the previous one; a good solution. Note that the changes in group designations all show up in the changes tracked version but as mentioned above, at least one of them is not in the untracked version.

Answer to comment 2 – We agreed with the reviewer. The designation of the groups was much better. This time, we took care about using the same designation for all groups in the untracked version. We have checked all nomenclature before sending the new version.

Comment 3) A) One remaining issue that we still need to fix is the lack of clarity about the smoke exposure system, despite the authors having added more details. The problem is that with a custom-built system, details of how the system works are important so that the results can be put in context of other smoke exposure literature. From the new description added, it’s still not clear to me how the smoke is being generated, and they refer to one of their previous papers but it is in Portuguese. Another new reference is to a review article about various effects of smoke on the lung; there’s no procedural detail there that is relevant to this paper. Most studies that produce secondhand smoke follow some variation of a pretty consistent workflow: air is sucked through a cigarette by some mechanism and according to a specific puff protocol (e.g. ISSO, Health Canada Intense), and the smoke from the burning tip is collected. Cigarettes are frequently pre-equilibrated to consistent humidity before use. These conditions provide not just consistency between experiments, but also the ability to place the results in context of the rest of the body of smoke research.

3 B) For example, there’s no mention in the description of air going through the cigarettes; just that the cigarettes are in a compartment and air goes from that compartment to the rats. Does that mean the cigarettes are sitting on the floor of the compartment simply smoldering in place, with no periodic puffing? If so, it would not invalidate the system, but it also would not be consistent with how such studies are typically accomplished and that should be specifically mentioned. The chemistry is different if the cigarette is smoldering in an ashtray for 10 minutes vs. if someone is actually smoking it and puffing occasionally. Please show a photograph or a diagram, and make the air path clear.

3 C) It’s also not clear to me what they mean when they say that the dose of smoke exposure is equivalent to 10 to 20 cigarettes for a chronic human smoker, because their model is secondhand exposure; that can’t be directly compared with the number of cigarettes being smoked by people.

Answer to comment 3 - A) The reference 20 in Portuguese was changed for another reference published in English by the same group: Garcia BC, Bonfim MR, Camargo RCT, Souza DRS, Abreu LC, Filho, JCSC. Effects of passive smoking associated with physical exercise in the skeletal muscle of rats during pregnancy and lactation. Int J Morphol. 2015;33:497-506. This study is from a research group that used the same chamber of exposure. We also added the reference # 18 in the line 138 (tracked version) or line 130 (untracked version), to justify the use of the model of chamber of exposure to cigarette smoke. But, in both articles, there is no adittional detail how the air flow was generated and the direction of this flow. As it is a custom-built system, we agreed with the reviewer, and more details are crucial to understand how the system works. In this new version we are adding the Fig. 1 showing the detail of the air flow inside the chamber and in the text we are describing the system more detaily. Because of this, we had to change the number of all figures in the manuscript as well.

Answer to comment 3 - B) During the exposure, the cigarettes were lighted and were left burning with no periodic puffing. Cigarettes were not pre-equilibrated to consistent humidity before the use. But 4 animals per time were let in the exposure chamber. And in the next day, we changed the sequence of the cages that would begin the exposure. A more detailed description was added in the Methods (lines 131-144 in the untracked version).

Answer to comment 3 - C) We agreed with the reviewer when he said that the dose of smoke exposure can’t be directly compared with the number of cigarettes being smoked by people, because of the model is secondhand exposure. The sentence was withdrawn (see lines 131-133 in the tracked manuscript).

Comment 4) The other changes have all been made satisfactorily. I noted a misspelling in the newly added material, “simoustaneouly” in the second-to-last paragraph of the discussion (line 460 in the tracked changed version).

Answer to comment 4 – The word was corrected to simultaneously (line 432).

Reviewer #2: Comment 1) Although the English of the manuscript has improved (at least the first 15 pages), it is still far from tolerable (at least by this reviewer). The manuscript either has not been revised grammatically, or the proof-reader has made a very poor job. This reviewer could not evaluate the revised manuscript because of the quality of English. Proof-reading is outside the reviewers' responsibilities, although the other reviewer very kindly has also suggested a number of such mistakes in the first round. In an original submission, the mistakes could be tolerated, however, not in a revised manuscript. This reviewer has got as far as the top of page 16 before quitting, and does not want to waste his/her time on submissions that do not reflect due care from the authors' side.

Answer: -: We would like to apologize about the grammar mistakes. We have sent the certificate of the English review made in the first version of the manuscript in attachment. After this, we added some new sentences in the manuscript to answer the reviewers, and to adequate the manuscript. However, we did not submit under an English review again, but we believe in that our inclusion in the text did not compromise the relevance of the study. We really believe in our results in this manuscript. We really believe in that the manuscript is adequate now.

Comment 2) - page 14, line81-83: Clarify the sentence. Most probably the first comma is unnecessary. Regardless, as presented, the third part of the sentence does not confer comprehensible meaning. We really believe in the manuscript is adequate now.

Answer: The third part of the setence explains the difference between anaerobic and aerobic training. If the pauses of recovery among the series were not done, the exercise would become aerobic. High-intensity and short-term duration with pauses among series characterize anaerobic exercise. The sentence was modified for better comprehension (see lines 82-84 in the both untracked and tracked versions).

Comment 3) -page 14, line 98: Change “monitoring” to “monitored”. Change “intake food” to “intake of food”.

Answer: all changes were done.

Comment 4) -page 15, line 120: Change “04” to “4”, then do the same throughout the text.

Answer: all changes were done

Comment 5) -page 15, line 121: Delete “only”.

Answer: all changes were done.

Attachment

Submitted filename: Response to reviewers.doc

Click here for additional data file.^{(51KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0236988.r006

Decision Letter 2

Stanton A Glantz

20 Jul 2020

Resistance training prevents right ventricle hypertrophy in rats exposed to secondhand cigarette smoke

PONE-D-19-31709R2

Dear Dr. Seraphim,

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

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Stanton A. Glantz

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: All my comments have been adequately addressed, including those ones of the previous revision round.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Number of goblet cells in the trachea (per mm2).

Values of the sum of 10 fields expressed as the mean ± SEM (n = 8 / group).

(PPTX)

Click here for additional data file.^{(48.7KB, pptx)}

S2 Fig. Histopathological evaluation of the heart.

(PPTX)

Click here for additional data file.^{(111.8KB, pptx)}

S1 Table. MSL weight—Details exercise capacity and MSL weight for E and ES groups in the beginning of the resistance training protocol.

(DOCX)

Click here for additional data file.^{(12.5KB, docx)}

S1 File

(DOCX)

Click here for additional data file.^{(122.5KB, docx)}

Attachment

Submitted filename: Response.docx

Click here for additional data file.^{(13.9KB, docx)}

Attachment

Submitted filename: Response to reviewers.doc

Click here for additional data file.^{(53KB, doc)}

Attachment

Submitted filename: Response to reviewers.doc

Click here for additional data file.^{(51KB, doc)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Resistance training prevents right ventricle hypertrophy in rats exposed to secondhand cigarette smoke

Ana Caroline Rippi Moreno

Gisele Alborghetti Nai

Caroline Pancera Laurindo

Karen Cristina Rego Gregorio

Tiago Olean-Oliveira

Marcos Fernando Souza Teixeira

Patricia Monteiro Seraphim

Roles

Abstract

1. Introduction

2. Material and methods

2.1 Animals

2.2 Experimental design

2.3 Smoking exposure

Fig 1. Custom-built system of chamber enable to expose animals to the cigarette smoke.

2.4 Resistance training

2.4.1 Maximum Supported Load (MSL) test

2.4.2 Experimental phase

2.5 Euthanasia

Fig 2. Timeline of the interventions performed over 18 weeks.

2.6 Histopathological analysis

2.7 Collagen density (Fractal dimension)

2.8 Statistics

3. Results

Table 1. Characteristics of the animals.

Fig 3. Histopathological assessment of the lung with HE staining.

Fig 4. Typical histopathological evaluation of the pulmonary artery thickness with Alcian blue—PAS.

Fig 5. Histopathological evaluation of the right ventricle thickness with HE staining.

Fig 6. Typical histopathological evaluation of the gastrocnemius muscle with HE staining.

Fig 7. Number of Kupffer cells in the liver (per mm2).

4 Discussion

Supporting information

Data Availability

Funding Statement

References

Author response to previous submission

Decision Letter 0

Stanton A Glantz

Roles

Author response to Decision Letter 0

Decision Letter 1

Stanton A Glantz

Roles

Author response to Decision Letter 1

Decision Letter 2

Stanton A Glantz

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Fig 7. Number of Kupffer cells in the liver (per mm²).