Influence of the maternal high-intensity-interval-training on the cardiac Sirt6 and lipid profile of the adult male offspring in rats

Reihaneh Mohammadkhani; Neda Khaledi; Hamid Rajabi; Iraj Salehi; Alireza Komaki

doi:10.1371/journal.pone.0237148

. 2020 Aug 3;15(8):e0237148. doi: 10.1371/journal.pone.0237148

Influence of the maternal high-intensity-interval-training on the cardiac Sirt6 and lipid profile of the adult male offspring in rats

Reihaneh Mohammadkhani ¹, Neda Khaledi ^1,^*, Hamid Rajabi ¹, Iraj Salehi ^2,^#, Alireza Komaki ^2,^#

Editor: Christopher Torrens³

PMCID: PMC7398538 PMID: 32745152

Abstract

The susceptibility to cardiovascular disease in offspring could be reduced prior to birth through maternal intervention, before and during pregnancy. We evaluated whether the initiation periods of maternal exercise in preconception and pregnancy periods induce beneficial effects in the adult male offspring. Thirty-two female rats were divided into control and exercise groups. The exercise groups involve exercise before pregnancy or the preconception periods, exercise during pregnancy, and exercise before and during pregnancy. The mothers in the exercise groups were run on the treadmill in different periods. Then the birth weight and weekly weight gain of male offspring were measured, and the blood and left ventricle tissue of samples were collected for analysis of the Sirtuin 6 (Sirt6) and insulin growth factor-2 (IGF-2) gene expression, serum levels of low-density lipoprotein (LDL), high-density lipoprotein (HDL), cholesterol (Cho), and triglycerides (TG). There was no significant difference in the birth weight of offspring groups (P = 0.246) while maternal HIIT only during pregnancy leads to reduce weekly weight gain of offspring. Our data showed that Sirt6 and IGF-2 gene expression was increased (P = 0.017) and decreased (P = 0.047) by maternal exercise prior to and during pregnancy, respectively. Also, the serum level of LDL (p = 0.002) and Cho (P = 0.007) were significantly decreased and maternal exercise leads to improves the running speed of the adult male offspring (p = 0.0176). This study suggests that maternal HIIT prior to and during pregnancy have positive intergenerational consequence in the health and physical readiness of offspring.

Introduction

Based on cardiovascular research, the risk factors which are independent of genetics could be altered by maternal behavior during pregnancy [1]. Exercise is a widely accepted positive intervention during pregnancy, which could reduce the risk of cardiovascular disease (CVD) development before birth [2].

Although previous studies have shown the positive effect of maternal exercise on vascular function and heart rate in adult offspring [3, 4], little is known about the benefits of maternal exercise during pregnancy to reduce the risk of heart disease in offspring. Brito et al. confirmed that low intensity exercise during pregnancy increases the expression of cardiac Sirtuin 6 (Sirt6) and maternal exercise has a cardioprotective effect in the hearts of progeny [5]. There is a close link between Sirtuin activity and exercise by increasing nicotinamide adenine dinucleotide (NAD⁺) levels [6, 7]. There is evidence that Sirt6 protects the heart from developing diseases [8, 9] through negative regulation of the insulin growth factor (IGF) signaling in the myocardial cells [10]. The IGF system has an important role in cardiac growth during pregnancy and it is established that there is a link between the low birth weight and an increase IGF-2 gene expression in the heart that leads to an increased risk of cardiovascular disease in later life [11]. Indeed, the Sirt6 could be negatively regulated low-density lipoprotein (LDL) levels [12]. The proper level of serum lipid profile LDL, high-density lipoprotein (HDL), cholesterol (Cho), and triglycerides (TG) are considered as common biomarkers for cardiovascular health. Therefore, it seems that Sirt6 and blood lipid profile could be used as prediction components for cardiovascular health that could be altered by maternal exercise.

A recent study has shown that high-intensity exercise during pregnancy not only has no negative effects on maternal cardiac function, but it could also be tolerated by pregnant rats, which activated the protective mechanism in fetal offspring by increasing the total antioxidant capacity [13]. The high-intensity interval training (HIIT) is defined as a short and repeated period of intense exercise close to 85% to 95% of maximal heart rate with less intense recovery periods [13–15]. Recent studies support the notion that cardiovascular adaptations of exercise linked to improved cardiac health consequences are intensity-dependent and there is a significant difference in the cardiovascular benefits of strenuous intensity versus moderate intensity [15, 16]. Because regular physical activity in the preconception periods should be an important component of a healthy pregnancy [17], improving preconception health of mothers can result in improved pregnancy related-outcomes and offspring health.

However, we hypothesized whether the initiation time of maternal HIIT is a significant factor for health consequences in the offspring. To test the beneficial effect of maternal HIIT on the health of offspring, we evaluated the serum level of lipid profiles, the cardiac Sirt6 and IGF-2 gene expression as cardioprotection factors. Also, we measured birth weight and weekly weight gain and performed an exercise test on the male offspring.

Material and method

Animal

Thirty-two female Wistar rats which never experienced a pregnancy, aged 8 weeks, were purchased from the animal house of Hamadan University of Medical Sciences; The animals were kept in cages with 4 rats in each cage. The rats were first acclimatized to the treadmill exercise for one week before the experiment. The animals were housed in an air-conditioned room at 22 ± 2°C with a 12-h light/dark cycle. Standard animal chow and water were freely available. Animal care and experimental protocols were approved by the Veterinary Ethics Committee of Hamadan University of Medical Sciences (AEC: IR.UMSHA.REC.1397.528) and The National Institutes of Health Guide for Care and Use of Laboratory Animals (NIH Publication no. 85–23, revised1985) was complied with.

Study design

A summary of the present study is depicted in Fig 1. High-intensity-interval training was performed in two parts, the first part included six weeks HIIT prior to mating and the second part included 3 weeks HIIT after mating. Animals were randomly divided into two maternal groups; maternal sedentary (control group, n = 8) and maternal exercise groups (n = 24). Maternal exercise groups were also divided into 3 subgroups (BP, BDP and DP groups) based on the initiation period of the maternal exercise; BP group included mothers who exercised only before pregnancy, BDP group included mothers who exercised both before and during pregnancy, and DP group included mothers who exercised only during pregnancy.

Prior to initiating the exercise protocol, all animals were familiarized with treadmill exercise (model: 2016 Tajhiz Gostar Omid Iranian, Iran) for four days (10 mins with 10 m/min at 0^° inclination) to reduce stress. The exercise test was accessed individually for maternal groups before and after six weeks of HIIT and also for pup groups at 10 weeks old. Also, animals were known to run on the treadmill through physical handling. After the last session of six weeks exercise two females with one male were housed for two days mating and then they were separated and exercise during pregnancy was initiated. After three weeks, pregnant rats were kept individually per cage to notice the birth process, number, and birth weight of pups. some pups were given to other mothers, who were not related to present study to create uniform condition per groups and male rats were separated from their mothers at the end of the breastfeeding duration (3 weeks old) and were allocated in pup groups (Pc, Pbp, Pbdp, and Pdp); Pc group included pups of maternal control group, Pbp group included pups of mothers who exercised before pregnancy, Pbdp group included pups of mothers who exercised before and during pregnancy, and Pdp group included pups of mothers who exercised during pregnancy. The number of pups per group were based on the number of pregnant mothers. The body weight expressed in grams was recorded immediately after birth and evaluated weekly until the 8^th week in the pup groups. After measuring the final body weight of pups (10 weeks old), the rats were anesthetized with Xylazine (3 mg/kg) and Ketamine (30 mg/kg), the heart was weighed, and the left ventricle was separated after whole blood extraction. Left ventricle harvest was washed with Phosphate-buffered saline (PBS); snap-frozen in liquid nitrogen and also Blood samples were centrifuged at 2500 g for 15 min; the serum and tissue aliquots were stored at -80° for more analysis.

Mating

Two females who mate with one male were separated after two days and then the vaginal plug was checked for pregnancy. As the presence or absence of a vaginal plug does not guarantee pregnancy, all rats were kept for three weeks for more certainty and afterward, nonpregnant rats were delivered to the animal house of Hamadan University of Medical Sciences for further use.

Exercise test

The maximum running capacity was performed individually for maternal (before and after six weeks HIIT period) and pup groups (at 10 weeks old) on the treadmill. First, the warm-up initiated with 5 mins at speed of 5m/min and then each rat started exercise test by 3 mins at speed of 8m/min with 0° inclines. The speed of running gradually increased by 3m/min every 3 mins, until signs of exhaustion. Those who refused to run or have uncoordinated steps are determining factors in the detection of exhaustion [18]. Maximum speed and total distance were recorded and used to determine the appropriate speed in HIIT sessions in maternal groups.

High-intensity-interval training

Training protocol started immediately after 5 mins warm-up and High-intensity-interval-training consist to running on treadmill with the speed of 18m/min at 10° inclination for 3 mins (85–95% of VO2max), switching with active recovery, and the speed of 13m/min (65% of VO2max) at 0° inclination for 5 days/week which is in accordance with the overload principle [19]; the duration and number of bouts were increased every week. The control group was kept in the training room during the training session to resemble the exercise groups. The HIIT protocol is depicted in Table 1.

Table 1. Characteristic of HIIT protocol prior to and during pregnancy.

HIIT protocol prior to pregnancy
First week	Speed: 18 (m/min), 10 bouts
Second week	Speed: 18 (m/min), 11 bouts
Third week	Speed: 20 (m/min), 12 bouts
Fourth week	Speed: 22 (m/min), 13 bouts
Fifth week	Speed: 24 (m/min), 14 bouts
Sixth week	Speed: 26 (m/min), 15 bouts
HIIT protocol during pregnancy
First week	Speed: 18 (m/min), 10 bouts
Second week	Speed: 18 (m/min), 11 bouts
Third week	Speed: 20 (m/min), 12 bouts

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Sirt6 and IGF-2 gene expression by RT-PCR method

The total RNA was extracted from the left ventricle heart using RNA isolation of Kiazol protocol (Kiazist Life Sciences, Iran) and the quantity and quality of extracted RNA were evaluated by Nano-Drop and gel electrophorus. Following, the HyperScript^TM synthesis kit (GeneAll, Korea) was utilized for synthesizing the reaction of the first-strand cDNA from total RNA according to the manufacturer's order. Then, the resulting cDNA was applied to Real-time quantitative polymerase chain reaction (PCR) using RealQ Plus 2X Master Mix Green (AMPLIQON, Denmark) in a three-step PCR program and a total 20 reaction volumes were performed on Real-Time PCR system (Roche Life Sciences, Germany). Relative expression of examined genes was quantified and normalized by b-actin as the internal control gene. Also, conditions used of PCR and upstream and downstream primers were respectively described in Tables 2 and 3.

Table 2. The PCR program.

Cycles	Duration of cycle	Temperature
1°	15 minutes	95°
40	15 seconds	95°
	30 seconds	60°
	30 seconds	72°

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Table 3. Conditions and sequence of primers.

Gene	Sequence		PCR PRODUCT SIZE	AMPLICON T_m
Sirt6	Forward	GAC CTA ACG CTC GCT GAT GA	163	60°C
Sirt6	Reverse	CCT GGC GGT CAT GTT TTG TG	163	60°C
IGF-2	Forward	GAG GGG AGC TTG TTG ACA C	155	60°C
IGF-2	Reverse	GGC ACA GTA TGT CTC CAG G	155	60°C
b-actin	Forward	ATC AGC AAG CAG GAG TAC GAT	94	60°C
b-actin	Reverse	AAA GGG TGT AAA ACG CAG CTC	94	60°C

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Lipid serum by the colorimetric method

Fasting blood samples of offspring were collected from inferior vena cava at 10 weeks old and it was centrifuged at 600 g for 10 min. serum high-density lipoprotein (HDL), low-density lipoprotein (LDL), cholesterol (Cho) and triglycerides (TG) were measured using Pars Azmon kit (Tehran, Iran) and Alpha-classic autoanalyzer.

Statistical analysis

The distribution of data was normal when examined using the Kolmogorov-Simonov test. All data were presented as mean ± SEM. Data were analyzed using GraphPad Prism® 6.0 (GraphPad, La Jolla, CA, USA). One-way and Two-way ANOVA with Dunnett’s posthoc test was used for statistical analysis. A probability of 0.05 was considered as the criterion for significance.

Results

The outcome of maternal characteristics

The maternal characteristics are shown in Table 4. There was no significant difference in maternal weight at the start of work. Our result showed that maternal weight gain was not affected by HIIT prior to or during pregnancy and we did not find a significant difference among the pregnant rats in body weight (P = 0.29). Also, we assessed the exercise test before and after six weeks of HIIT to ensure the effectiveness of HIIT on mothers that are characterized by enhanced distance and speed on the treadmill. As expected, distance (P≤0.0001, F(3,28) = 143.3) and speed (P≤0.0001, F(3,28) = 148.3) of exercised mothers were significantly greater than non-exercised mothers.

Table 4. Maternal characteristics.

	c	BP	BDP	DP
Maternal’ weight (gr)
before 6 weeks	194.54 ± 3.2	193.17 ± 2.3	192.25 ± 3.3	194.12 ± 3.31
after 6 weeks	212.87 ± 4	208.81 ± 4.77	207.12 ± 4.67	211.1 ± 4.43
The distance of maternal Exercise test (m)
before 6 weeks HIIT	131.50	141.4	139.4	-
after 6 weeks HIIT	166.3	877.9^****	861.4^****	-
The speed of maternal Exercise test (m/min)
before 6 weeks HIIT	15.88	17.50	16.63	-
after 6 weeks HIIT	17.75	40.63^****	40.25^****	-
Number of pregnant rats/total rats	5/8	6/8	4/8	6/8
Number of pups	7 ± 0.92	5 ± 1.71	9 ± 0.98	8 ± 0.61
Birthweight of pups (gr)	6.435 ± 0.17	6.020 ± 0.25	6.325 ± 0.20	6.045 ± 0.13
Sex distribution
Female	4.60 ± 0.50	2.83 ± 1.07	5.25 ± 0.47	4.5 ± 0.76
Male	3.80 ± 0.58	3.16 ± 0.70	4.50 ± 0.64	5.33 ± 0.49

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Values represented as the mean ± standard error. Abbreviations: C group, sedentary maternal (control); BP group, maternal that exercised before pregnancy; BDP group, maternal that exercised before and during pregnancy; DP group, maternal that exercised during pregnancy.

**** p<0.0001 = significant statistically difference as compared to the Control group. One-way ANOVA followed by Dunnett's multiple comparisons test.

Besides, the number of pups and the sex distribution of pups are shown in Table 4 and the outcomes of the number of pups (P = 0.16) and sex distribution (P = 0.10) were not statistically significant.

The outcome of offspring

Maternal HIIT had effect on the weekly weight gain of the offspring whose mothers exercised only during pregnancy

The offspring were divided into four groups according to the initiation time of maternal exercise. As shown in Table 4 and Fig 2, the initiation time of maternal HIIT does not affect offspring birth weight (P = 0.246); however it could change the weight of offspring at 6^th, 7^th and 8^th weeks, as two-way ANOVA revealed a significant interaction between the initiation time of maternal HIIT and weekly weight gain of offspring (p<0.001, F(21,119) = 8).

Therefore, this data indicated that maternal HIIT only during pregnancy leads to a significant decrease in the weekly weight gain of pups.

Maternal HIIT prior to and during pregnancy improve running speed and distance of the male offspring

We compared the offspring exercise test among the pup groups to determine the influence of maternal exercise on the running performance of the male offspring in terms of running speed and tolerance to fatigue. As shown in Fig 3, there was a significant difference among offspring groups. This result indicated that the pups of mothers who exercised prior to and during pregnancy had more running distance (P = 0.0026, F(3,17) = 7.161) and running speed (P = 0.0176, F(3,17) = 4.446) on the treadmill than other pups.

Fig 3 — (A) A maximum speed of adult male offspring, (B) A maximum distance of adult male offspring. Abbreviations: Pc group (n = 5), pups of sedentary mothers; Pbp group (n = 6), pups of mothers who exercise only before pregnancy; Pbdp group (n = 4), pups of mothers who exercise prior to and during pregnancy; Pdp group (n = 6), pups of mothers who exercise only during pregnancy. Data presented as means ± S.E.M. ** p<0.01 vs. Pup Control group. One-way ANOVA followed by Dunnett's multiple comparisons test.

The initiated period of maternal HIIT has an impact on the heart mass of offspring

There has not been any difference between the heart mass (P = 0.25) and the heart mass/ body weight ratio (P = 0.31) in pup groups (Table 5).

Table 5. Pups weight characteristics.

	Pc	Pbp	Pbdp	Pdp
	(n = 5)	(n = 6)	(n = 4)	(n = 6)
Pup’s Heart mass(gr)	1.136 ± 0.040	1.087 ± 0.041	1.190 ± 0.034	1.083 ± 0.023
Pup’s Heart mass/Body weight(gr) percentage	0.389 ± 0.006	0.387 ± 0.008	0.393 ± 0.007	0.371 ± 0.003

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Data presented as means ± S.E.M. Abbreviations: Pc group, pups of sedentary mothers; Pbp group, pups of mothers who exercise only before pregnancy; Pbdp group, pups of mothers who exercise prior to and during pregnancy; Pdp group, pups of mothers who exercise only during pregnancy.

Maternal HIIT prior to and during pregnancy have effects on the Sirt6 and IGF-2 gene expression in the heart and serum lipid profile of the offspring

In order to realize the importance of initiation time of maternal HIIT to confer adult offspring health, we evaluated the cardiac Sirt6 and IGF-2 gene expression and serum lipid profile in pup groups. As shown in Fig 4, there was seen an enhancing effect of maternal HIIT on cardiovascular factors due to a significant increase in Sirt6 gene expression) P = 0.017), which in turn, decreased IGF-2 (P = 0.0473) gene expression. The gene expression results of the pup groups suggested that maternal exercise prior to and during pregnancy has beneficial effects on the offspring. Thus, the present study proposes a promising approach in improving the factors involved in the cardiac health of adult offspring as a result of the maternal HIIT.

Fig 4 — (A) cardiac Sirt6 expression in adult male pup groups. (B) cardiac IGF-2 expression in adult male pup groups. Abbreviations: Pc group (n = 5), pups of sedentary mothers; Pbp group (n = 6), pups of mothers who exercise only before pregnancy; Pbdp group (n = 4), pups of mothers who exercise prior to and during pregnancy; Pdp group (n = 6), pups of mothers who exercise only during pregnancy. Data presented as means ± S.E.M. * p<0.05 vs. Pup Control group. One-way ANOVA followed by Dunnett's multiple comparisons test.

Evaluation of the lipid profile of male pups showed that maternal exercise prior to and during pregnancy and only during pregnancy has a positive impact on the lipids profile of adult offspring by decreasing serum levels of LDL and Cho.

As shown in Fig 5, the comparison of HDL and TG among the offspring groups show no significant differences while the comparison of LDL (P = 0.0029) and Cho (P = 0.0074) revealed significant differences between Pc, Pbdp and Pdp groups.

Discussion

Maternal exercise during pregnancy as a positive maternal behavior has been extensively investigated [20–23]. The present study aimed to investigate the effect of physical activity of mothers as a healthy lifestyle on the health of offspring. Our finding indicated that firstly, strenuous intensity could be done in pregnancy without detrimental effect on the birth weight of offspring, and secondly, the ideal time for initiating high-intensity-interval training is prior to and during pregnancy that leads to the increase of Sirt6 gene expression and the decrease of IGF-2 gene expression in the heart, and the decrease LDL and Cho levels in the serum of offspring.

To date, only one other study evaluated HIIT during pregnancy which is consistent with our observation [13]. Our study indicates that pregnant rats are capable to perform HIIT on the treadmill throughout pregnancy. To our knowledge, the present study is the first study that compares the effects of preconception and prenatal periods of maternal HIIT or directly both of them on the weight and cardiovascular health of offspring. Scientific evidence supports a relationship between birth weight and postnatal health outcomes [24] and mentions the birth weight as an important factor in adult disease [25, 26]. Consistent with the previous literature [27], our study found that maternal exercise did not affect the birth weight of offspring. The weighted result of animals was notable in that the birth weight of pups was not affected by the initiation period of maternal HIIT, but maternal HIIT has a significant effect on the weekly weight gain of pups at 6^th-8^th weeks. Also, the result of heart weight and heart weight/ body weight ratio of offspring indicated that maternal HIIT only during pregnancy could lead to a significant decrease as compared to the pup of mothers who exercised prior to and during pregnancy. Our observations reveal that high-intensity exercise only during pregnancy leads to the reduction of the body weight of pups at 6^th -8^th weeks, while this weight loss was not observed in pups of mothers who were compatible with this intensity before pregnancy. This decrease may be due to a stress effect of exposing the mother to exercise, commencing only in pregnancy - which induces a stress response (unlike the Pbdp group, whose mothers were exercised prior to pregnancy as well).

Thus, the current result highlighted the importance of the preconception period for maternal physiological adaptation to high-intensity-interval training before pregnancy. It is noteworthy that the observed decrease in the adult weight of pups does not mean a negative impact and needs to be evaluated with anthropometric indicators, cardiac function and histologic examination in future works.

The epidemiological observations highlighted that the developmental changes of the pups related to health were associated with the prenatal environment. Given that HIIT could be tolerated by pregnant rats without an effect on the birth weight of pups, the main question is whether strenuous exercise could modify the intrauterine environment to provide rich conditions concerning the cardiovascular factors which affect the developmental status. Voluntary maternal exercise on the first day of pregnancy could modulate mitochondrial gene expression of the fetal heart [28], although it is not clear if elevated gene expression would persist in adulthood. Another study suggested a molecular positive change in the hippocampus of rats by controlled maternal exercise during pregnancy [29]. Raipura et al. have shown that voluntary exercise during pregnancy seems to decrease the metabolic risk in offspring [27]. The previous studies on humans and animals have shown a positive effect of maternal exercise on offspring; however, the optimal timing of initiated exercise to confer cardiovascular benefits to the next generation has not been addressed. The findings of Brito et al. demonstrated the protective effects of moderate maternal exercise in the hearts of progeny by modulating the oxidative stress and Sirtuin6 protein levels [5]. One of the well-known pathways of exercise for cardioprotective effects is the activation of the sirtuin family [9, 30]. There is evidence that down-regulation IGF-AKT signaling directly by Sirt6 is capable of blocking heart failure and myocardial disease [10]. Also, studies on the fetal left ventricle have shown that an elevation of IGF-2 gene expression levels and its receptor can increase the risk of cardiovascular disease in adulthood [31]. Darby et al. have suggested that maternal undernutrition has negative consequences for cardiac health due to an up-regulation of IGF-2 signaling in the fetal heart [32]. Therefore, the present study assesses the possibility of activation of the cardiac gene expression related to cardioprotection through the maternal exercise in adult offspring. Consistent with our hypothesis, it is indicated that maternal HIIT both prior to and during pregnancy or only during pregnancy compared with maternal exercise before pregnancy could increase Sirt6 gene expression and decrease IGF-2 gene expression in the hearts of the offspring. Thus, it was concluded that maternal HIIT during pregnancy helps giving birth to offspring with enhanced conditions in regard to the factors involved in the heart in later life.

Additionally, given that the cardiovascular adaptations to strenuous exercise related to health are due, in part, to the regulation of the level of serum lipids [33–35], the offspring's serum lipid profile as the index of cardiovascular health was evaluated. Maternal voluntary exercise prior to and during pregnancy was found to improve glucose homeostasis in adult offspring [36]. A similar study has shown that, in C57BL/6 mice, maternal exercise before and during gestation has effects on offspring metabolic health without changes in body composition [36]. A study of rabbits demonstrated that cholesterol-lowering interventions during pregnancy have profound effects on the vascular health of adult offspring [37]. The obtained data show that maternal HIIT prior to and during pregnancy could decrease LDL and Cho levels of offspring, but do not have a significant effect on serum HDL and TG levels. It is noted that maternal exercise prior to and during pregnancy is the same, as only during pregnancy could alter the lipid profile of offspring (LDL and Cho). Thus, the present study indicates that maternal HIIT as a suitable intervention to reduce the lipid profile has long-term beneficial effects in adult male offspring.

Moreover, as expected, the offspring of mothers who exercised prior to and during pregnancy can attain higher speed and greater distance in comparison to other pups. Since the maximal oxygen uptake (VO₂max) could be predicted by the data of the submaximal exercise test [38, 39], the results of the exercise test of offspring could indirectly indicate the effect of maternal exercise before and during pregnancy on the increase of offspring’s physical readiness through the increased running speed and distance. Offspring's exercise test data which was in accordance with the previous study [23] showed that the mother’s activity prior to and during pregnancy increased running speed and time to fatigue of offspring.

To summarize, the present study reinforces the beneficial effects of maternal HIIT prior to and during pregnancy as optimal periods of promoting health of offspring by changes in serum lipid profile, cardiac gene expression and running performance. Therefore, based on the results of present study maternal exercise could be suggested as an intervention to improve the health and physical readiness of male offspring.

Supporting information

S1 Fig. Amplification plots and melting cure of the selected genes.

A) melting cure of sirt6, B) melting cure of igf2, C) melting cure of b-actin, D) Amplification plots of sirt6, E) Amplification plots of igf2 and F) Amplification plots of b-actin.

(TIF)

Click here for additional data file.^{(2.5MB, tif)}

S1 File. Minimal data set of the pup’s exercise test.

Abbreviations: Pc group (n = 5), pups of sedentary mothers; Pbp group (n = 6), pups of mothers who exercise only before pregnancy; Pbdp group (n = 4), pups of mothers who exercise prior to and during pregnancy; Pdp group (n = 6), pups of mothers who exercise only during pregnancy.

(XLSX)

Click here for additional data file.^{(9.9KB, xlsx)}

S2 File. Minimal data set of the pup’s heart weight.

(XLSX)

Click here for additional data file.^{(10.2KB, xlsx)}

S3 File. Minimal data set of the pup’s weekly weight gain.

(XLSX)

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

S4 File. Minimal data set of the pup’s gene expression.

(XLSX)

Click here for additional data file.^{(13.1KB, xlsx)}

S5 File. Minimal data set of the pup’s lipid profile.

(XLSX)

Click here for additional data file.^{(12KB, xlsx)}

Acknowledgments

The authors would like to thank the Neurophysiology Research Center because of helping this project and Dr. Seyed Asaad Karimi for the reading of the manuscript.

Data Availability

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

Funding Statement

This work was supported by a grant of Hamadan University of Medical Sciences, Iran. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0237148.r001

Decision Letter 0

Christopher Torrens

4 Feb 2020

PONE-D-19-33225

Influence of maternal high-intensity-interval training prior to and during pregnancy on the cardiovascular health of the male offspring

PLOS ONE

Dear Dr. khaledi,

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There are some concerns about the methods or reporting of the methods. Equally the conclusions are far to much to be justified on the data provided

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Additional Editor Comments (if provided):

Mating: The methods state that 32 Wistar rats were purchased, giving 8 per maternal exercise group. Later in the mating section it states that non-pregnant rats were discarded. So, either more than 32 rats were obtained or no rats were discarded for being non-pregnant making the statement redundant. Please clarify.

Line 107: "...some pups were eliminated...". Please give the method of this (e.g. cervical dislocation). Also if litters were standardised, what were they standardised to?

Line 130: "those that refused to run...". How many refused to run, per group per protocol?

line 153: "...normalised by b-actin." It is preferable to use multiple housekeeping reference genes to account for treatment-induced changes in reference gene expression. Since there is only one here, do you know is b-actin expression is stable across exposures of maternal exercise?

Line 199: Two-way ANOVA is not mentioned in the methods but is mentioned here

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

Reviewers' comments:

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Reviewer #1: No

Reviewer #2: Partly

**********

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Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #2: Yes

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Reviewer #1: Mohammadkhani et al. present an interesting manuscript investigating the effect of maternal HIIT on cardiovascular health of male offspring.

The authors conclude that maternal exercise reduces the risk of heart disease and may have an intergenerational effect – there is no data to support these claims.

Studies showing that maternal exercise increases Sirt6 and Igf2 in the heart have already been published.

The authors present data showing that heart mass is different in offspring from dams that exercised only during pregnancy – what does this mean?

The authors state that, “The results of the gene expression of groups of pups suggested that maternal exercise only prior to pregnancy or prior to and during pregnancy has a cardioprotective effect on the next generation. Thus, the present study highlights the pregnancy period as a vital period with a long-lasting effect on the cardiac health of offspring.” This is a huge overstatement. There is no way to conclude this based on expression of 2 genes. Further, it is not stated at what age gene expression is measured in the offspring.

It is also not clear what age the mice were when LDL/HDL/etc. was measured.

Reviewer #2: The goal of the study by Reihaneh et al was to determine the effects of maternal high-intensity-interval training (HIIT) on cardiovascular health of male offspring. The authors found that maternal HITT before and during pregnancy increased exercise capacity and decreased serum levels of LDL and cholesterol in male offspring at 10 weeks of age. Maternal training also improved two cardioprotective factors in offspring hearts, increasing mRNA expression of Sirt6 and decreasing mRNA expression of Igf2. The topic and data are interesting and have potentially important clinical relevance. However, the very limited data are insufficient to prove that maternal exercise results in cardiovascular protection in offspring. To prove this the authors would need to perform many additional experiments including direct measurements of cardiac function. The manuscript would be much stronger if there were also data on Sirt family gene expression, cardiac and skeletal muscle histology, and more detailed serum analysis. In addition, the lack of n for each figure legend and table is concerning.

1. In addition to function studies, does maternal HIIT affect the histology of hearts? Longitudinal sections of whole mouse hearts and microscopic views of the inter-ventricular septa and left ventricles may suggest the improvement of cardiovascular function in offspring heart.

2. Sirt1, Sirt2, Sirt4, Sirt5, and Sirt6 are highly expressed in heart and known to be involved in cardiovascular disease protection. Does maternal HIIT change these gene expression? Also, the expression of Sirt6 targets (Nrf2 and NFkappaB) and the change of histone acetylation (ex. H3K9ac, H3K18ac) will support the importance of Sirt6 in the effects of maternal HIIT on offspring heart.

3. What is the n for each experiment? It is very odd that this is not included in each figure legends. Table 4 on Maternal Characteristics is not informative in terms of understanding the n for studies of offspring groups. Is the n for all of the offspring experiments the number of pregnant rats? The n for studies of offspring is based on the number of mothers studied, not the number of offspring. Thus, if for example in the BP group there are 6 mothers that each have a litter of 3 males, the n=6. If this information is not provided, the data cannot be evaluated.

4. On page 5 it is stated that you eliminated some pups to create uniform conditions. Explain this uniform condition. How many pups were kept per litter? Did you study all pups or pooled?

5. Why were the offspring only studied until 8-10 weeks of age? This is considered young adulthood, and at this age there is less chance of developing cardiovascular disease.

6. Is serum level of IGF2 changed in offspring?

7. Does maternal HIIT affect the expression of Insulin receptor, IGF1 receptor, and IGF2 receptor in offspring heart? Are total and phosphorylated levels of Akt changed by maternal HIIT in offspring heart?

8. Can the authors explain the increase of exercise capacity in offspring by a skeletal muscle phenotype? For example, endurance strength, muscle weight, and fiber composition (cross-sectional area and fiber distribution in muscle).

9. There is no rationale for only studying male offspring.

10. Page 8. Fasting blood samples were collected; how long were the animals fasted?

11. Wistar rats, not Wister (page 4 line 81).

12. Page 12. The gene expression data in the text do not seem correct (line 239).

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Aug 3;15(8):e0237148. doi: 10.1371/journal.pone.0237148.r002

Author response to Decision Letter 0

17 Apr 2020

Dear Christopher Torrens

Academic Editor

PLOS ONE

The authors would like to Thank you for giving me the opportunity to submit a revised draft of our manuscript titled [Influence of the maternal high-intensity-interval-training on the cardiac Sirt6 and lipid profile of the adult male offspring in rats, No. PONE-D-19-33225] to [PLOS ONE]. We appreciate the time and effort that you and the reviewers have dedicated to providing your feedback on our manuscript. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewers. We have highlighted the changes within the manuscript. Here is a point-by-point response to the comments and concerns. We hope that this new version of the manuscript is satisfactory for the presentation and we are thankful once again to the reviewers for their useful comments.

Sincerely yours

Neda Khaledi (Ph. D)

Corresponding Author

Academic editor' comments:

We truly appreciate the Editor’s suggestion, and we recognize the benefits improving the quality of the manuscript.

Point 1: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Response: The manuscript was modified based on PLOS ONE's style.

Point 2: To comply with PLOS ONE submissions requirements, please provide methods of sacrifice (for both mothers and offspring, including the ones mentioned at line 108) in the Methods section of your manuscript.

Response: We have added the method of sacrifice in Materials and Methods section. Following the animal care guidelines of the university, some were delivered to the animal house of Hamadan University of Medical Sciences; however, on the other hand some other which could still provide further data were used in other investigations. while some other that could still provide further data was used in other investigations.

Point 3: Please consider modifying your title to ensure that it is specific, descriptive and concise, by specifying that the study was performed in rats.

Response: In accordance with your suggestion, the title has been changed and it is now “Influence of the initiated period of maternal high-intensity-interval-training on the profile lipid and cardiac sirt6 of the adult male offspring in rats”.

Point 4: We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.

Response: By getting the help of a native English-speaking associate we improved our manuscript.

Point 5: Mating: The methods state that 32 Wistar rats were purchased, giving 8 per maternal exercise group. Later in the mating section it states that non-pregnant rats were discarded. So, either more than 32 rats were obtained or no rats were discarded for being non-pregnant making the statement redundant. Please clarify.

Response Apologies if the part was not clear enough in the original manuscript. 32 female Wistar rats were purchased. Animals were randomly divided into two maternal groups; maternal sedentary (control group, n=8) and maternal exercise groups (n=24). Maternal exercise groups were also divided into 3 subgroups (BP, BDP and DP groups) based on the initiation period of the maternal exercise. Mothers that were not pregnant, at the end of three weeks pregnancy period, were eliminated from the study. As a result, five pregnant rats in the control group, six pregnant rats in the BP group, four pregnant rats in the BDP group and six pregnant rats in the DP group remained in the study with their five offspring (to create uniform condition). At first, we randomly select two male pups from each pregnant rat per group but in accordance with the suggestion of reviewer 2, n for offspring group were modified based on the pregnant rat.

Point 6: Line 107: "...some pups were eliminated...". Please give the method of this (e.g. cervical dislocation). Also if litters were standardised, what were they standardised to?

Response: following the animal care guidelines of the university, some were delivered to the animal house of Hamadan University of Medical Sciences; however, on the other hand some other which could still provide further data were used in other investigations. while some other that could still provide further data was used in other investigations. Also, the litter size was standardized based on the lowest litter size (the BP group: 2 female and 3 male) on the first day to create uniform conditions for postnatal growth and development of the pups.

Point 7: Line 130: "those that refused to run...". How many refused to run, per group per protocol?

Response: These conditions were observed during the exercise test (not during the main protocol of exercise. An exercise test is the one that is performed by progressively increasing workloads up to limiting fatigue caused by exhaustion. Finally, all rats refused to run in the exercise test and the maximum speed obtained from this test were used as an indicator of speed in the main protocol exercise.

Point 8: line 153: "...normalised by b-actin." It is preferable to use multiple housekeeping reference genes to account for treatment-induced changes in reference gene expression. Since there is only one here, do you know is b-actin expression is stable across exposures of maternal exercise?

Response: The b-actin gene has been used as a reference for maternal exercise in several studies resembling our work [1-4].

Point 9: Line 199: Two-way ANOVA is not mentioned in the methods but is mentioned here.

Response: It was corrected.

Reviewers' comments:

Reviewer #1: Manuscript Number: PONE-D-19-33225

We are grateful to the reviewer 1 for their insightful comments on this article. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewer.

Reviewer #1: Mohammadkhani et al. present an interesting manuscript investigating the effect of maternal HIIT on the cardiovascular health of male offspring.

Response: Thank you for the positive comment.

Point 1: The authors conclude that maternal exercise reduces the risk of heart disease and may have an intergenerational effect – there is no data to support these claims.

Response: You have raised an important point here. It is well known that calorie restriction and physical exercise can effectively modulate the activity of Sirtuins towards well-being and cardiovascular health [5, 6]. We used the results of Sundaresan et al. which indicated an antihypertrophic effect of sirt6 in cardiomyocytes and showed a low level of sirt6 be a risk factor for heart failure concluded that maternal exercise with increase sirt6 and decrease igf2 genes reduces the risk of heart disease [7]. Also, others studies showed that SIRT6 has a major role in regulating the expression of IGF signaling–related genes and the dysregulation of this pathway might contribute to the pathophysiology of several diseases, including heart failure [4, 8, 9]. However, in accordance with your suggestion, the conclusion has been revised.

Point 2: Studies showing that maternal exercise increases Sirt6 and Igf2 in the heart have already been published.

Response: The effects of maternal exercise in offspring heart are not well understood. Chung et al. demonstrated that maternal voluntary exercise initiated at day 1 of gestation could transfer the positive mitochondrial phenotype to fetal hearts [1]. Also, Songstand et al. showed that the genes related to oxidative stress were altered by maternal HIIT training in the fetal heart [10]. Britro et al concluded intergenerational protective effects of maternal treadmill exercise by increasing the sirt6 protein levels in the hearts of progeny [11]. Most research evaluated the effects of voluntary wheel running or treadmill training with moderate intensity on the heart of fetal. However, the effect of maternal high-intensity interval training on the lipid profile and cardioprotective genes (sirt6 & igf2) of adult male offspring have not been investigated.

Point 3: The authors present data showing that heart mass is different in offspring from dams that exercised only during pregnancy – what does this mean?

Response: Given that heart mass results from the complex interaction between genetic, environmental, and lifestyle factors, the studies indicated high-intensity interval training could be an effective exercise program for improving cardiac function [12] and could change the heart mass [13, 14]. So, we sought to know whether maternal high-intensity interval training leads to changes in the heart mass of offspring. Thus, it was found that the initiated period of maternal HIIT is an important factor in the heart mass of offspring. Further studies are required to elucidate the echocardiographic parameters offspring underlying the maternal high-intensity interval training correlations for heart mass.

Point 4: The authors state that, "The results of the gene expression of groups of pups suggested that maternal exercise only prior to pregnancy or prior to and during pregnancy has a cardioprotective effect on the next generation. Thus, the present study highlights the pregnancy period as a vital period with a long-lasting effect on the cardiac health of offspring." This is a huge overstatement. There is no way to conclude this based on the expression of 2 genes. Further, it is not stated at what age gene expression is measured in the offspring.

Response: We agree with your suggestion and have revised our conclusion as follows: “The gene expression results of the pup groups suggested that maternal exercise prior to and during pregnancy has beneficial effects on the next generation. Thus, the present study proposes a promising approach in improving the factors involved in cardiac health of the adult offspring as a result of the maternal HIIT”. Also, all pups were sacrificed at 10 weeks old and the left ventricle was extracted.

Point 5: It is also not clear what age the mice were when LDL/HDL/etc. was measured

Response: Apologies if the part was not clear enough in the original manuscript. All pups were sacrificed at 10 weeks old and the whole of blood was extracted.

Reviewer #2: The goal of the study by Reihaneh et al was to determine the effects of maternal high-intensity-interval training (HIIT) on cardiovascular health of male offspring. The authors found that maternal HITT before and during pregnancy increased exercise capacity and decreased serum levels of LDL and cholesterol in male offspring at 10 weeks of age. Maternal training also improved two cardioprotective factors in offspring hearts, increasing mRNA expression of Sirt6 and decreasing mRNA expression of Igf2. The topic and data are interesting and have potentially important clinical relevance. However, very limited data are insufficient to prove that maternal exercise results in cardiovascular protection in offspring. To prove this the authors would need to perform many additional experiments including direct measurements of cardiac function. The manuscript would be much stronger if there were also data on Sirt family gene expression, cardiac and skeletal muscle histology, and more detailed serum analysis. In addition, the lack of n for each figure legend and table is concerning.

Response: Thank you for the positive comment. We agree with you that additional experiments including direct measurements of cardiac function are very useful and strengthen our work, but unfortunately due to the lack of facilities and the lack of time we are unable to perform further test at this time. Also, more detailed serum analysis and n for each group were added as advised.

Point 1: In addition to function studies, does maternal HIIT affect the histology of hearts? Longitudinal sections of whole mouse hearts and microscopic views of the inter-ventricular septa and left ventricles may suggest the improvement of cardiovascular function in offspring heart.

Response: Thank you for this suggestion. It would have been interesting to explore this aspect. However, this is beyond the scope of this study, and we will certainly incorporate this in future projects.

Point 2: Sirt1, Sirt2, Sirt4, Sirt5, and Sirt6 are highly expressed in heart and known to be involved in cardiovascular disease protection. Does maternal HIIT change these gene expression? Also, the expression of Sirt6 targets (Nrf2 and NFkappaB) and the change of histone acetylation (ex. H3K9ac, H3K18ac) will support the importance of Sirt6 in the effects of maternal HIIT on offspring heart.

Response: You have raised a valuable point and we agree with your suggestion that the expression of Sirt6 targets will support the importance of our work. We will certainly use your suggestion in future work. However, in cardiovascular diseases, Sirtuins have gained interest in their protective effects. We believe that Sirt6 would be more appropriate than other families because sirt6 appears to have an important role in the heart [15] and cardiovascular disease including cardiac hypertrophy, heart failure and myocardial hypoxic damage [7, 16, 17]. Moreover, physical exercise can effectively modulate the activity of Sirtuins, particularly Sirt6 [15, 18, 19]. The previous experimental study demonstrated that maternal moderate-intensity training during pregnancy could increase the cardiac protein levels of Sirt6 in neonatal of the rat. Thus, we evaluated the effect of maternal strenuous intensity training in the different initiated period on the adult offspring heart.

Point 3: What is the n for each experiment? It is very odd that this is not included in each figure legends. Table 4 on Maternal Characteristics is not informative in terms of understanding the n for studies of offspring groups. Is the n for all of the offspring experiments the number of pregnant rats? The n for studies of offspring is based on the number of mothers studied, not the number of offspring. Thus, if for example in the BP group there are 6 mothers that each have a litter of 3 males, the n=6. If this information is not provided, the data cannot be evaluated.

Response: You have raised a valuable fact here. In accordance with your point, we changed the number of offspring for each group based on the number of pregnant mothers and analyzed again. The n for each experiment has been added to legend of each table and figure.

Point 4: On page 5 it is stated that you eliminated some pups to create uniform conditions. Explain this uniform condition. How many pups were kept per litter? Did you study all pups or pooled?

Response: The litter size was standardized based on the lowest litter size (BP group: 2 female and 3 male) on the first day to create uniform conditions for postnatal growth and development of the pups. All male pups were involved in the study.

Point 5: Why were the offspring only studied until 8-10 weeks of age? This is considered young adulthood, and at this age, there is less chance of developing cardiovascular disease.

Response: We appreciate the reviewer's comment and are agree with that. Given this, the majority of studies evaluated the effect of maternal exercise in neonatal and demonstrated the levels of Sirt6 expression were increased in the neonatal cardiomyocytes from exercised mothers during pregnancy [11]. We aimed to evaluate at a mature age that many developmental processes are ongoing.

Point 6: Is the serum level of IGF2 changed in offspring?

Response: In accordance with our purpose IGF2 was measured only in heart tissue.

Point 7: Does maternal HIIT affect the expression of Insulin receptor, IGF1 receptor, and IGF2 receptor in offspring heart? Are total and phosphorylated levels of Akt changed by maternal HIIT in offspring heart?

Response: Thank you for your suggestion. It would have been interesting to explore this aspect. However, in our case, it was not possible due to lack of time. We will certainly measure this in future projects.

Point 8: Can the authors explain the increase of exercise capacity in offspring by a skeletal muscle phenotype? For example, endurance strength, muscle weight, and fiber composition (cross-sectional area and fiber distribution in muscle).

Response: Given that the maximal exercise test is a useful method for physical capacity, it was demonstrated a strong relationship between treadmill running speed and vo2max in a rat model [20]. Also, the maximum exercise test , a simple methodology and low cost, highlighted as an indicator of exercise capacity to investigate the benefits of exercise training in male rats [21]. So, our study aimed to determine the effect of the initiated period of maternal exercise before and during pregnancy on maximum exercise test of male offspring so the skeletal muscle phenotype was not measured.

Point 9: There is no rationale for only studying male offspring.

Response: The majority of studies investigating the effects of maternal exercise on offspring have primarily studied the male offspring [22, 23] and Gaini et al demonstrated that improving the mother's physical fitness before and during pregnancy had positive changes in the lipid profile of male offspring. Also, in this study female offspring were used for separate experiments, and only data from male offspring were reported here.

Point 10: Page 8. Fasting blood samples were collected; how long were the animals fasted?

Response: Fasting blood samples were collected after 12 hours of fasting.

Point 11: Wistar rats, not Wister (page 4 line 81).

Response: This mistake was corrected. Thank you.

Point 12: Page 12. The gene expression data in the text do not seem correct (line 239).

Response: Thank you for pointing this out. It was corrected.

1. Chung, E., et al., Maternal exercise upregulates mitochondrial gene expression and increases enzyme activity of fetal mouse hearts. Physiological Reports, 2017. 5(5): p. e13184.

2. Dayi, A., et al., Maternal aerobic exercise during pregnancy can increase spatial learning by affecting leptin expression on offspring's early and late period in life depending on gender. The Scientific World Journal, 2012. 2012.

3. Park, J.-w., et al., Maternal exercise during pregnancy affects mitochondrial enzymatic activity and biogenesis in offspring brain. International Journal of Neuroscience, 2013. 123(4): p. 253-264.

4. Winnik, S., et al., Protective effects of sirtuins in cardiovascular diseases: from bench to bedside. European heart journal, 2015. 36(48): p. 3404-3412.

5. Vitiello, M., et al., Multiple pathways of SIRT6 at the crossroads in the control of longevity, cancer, and cardiovascular diseases. Ageing research reviews, 2017. 35: p. 301-311.

6. Xu, S., P. Bai, and Z.G. Jin, Sirtuins in cardiovascular health and diseases. Trends in endocrinology and metabolism: TEM, 2016. 27(10): p. 677.

7. Sundaresan, N.R., et al., The sirtuin SIRT6 blocks IGF-Akt signaling and development of cardiac hypertrophy by targeting c-Jun. NATURE MEDICINE, 2012. 18(11): p. 1643.

8. Chu, C.-H., et al., Activation of insulin-like growth factor II receptor induces mitochondrial-dependent apoptosis through Gαq and downstream calcineurin signaling in myocardial cells. Endocrinology, 2008. 150(6): p. 2723-2731.

9. Li, Z., et al., SIRT6 suppresses NFATc4 expression and activation in cardiomyocyte hypertrophy. Frontiers in pharmacology, 2019. 9: p. 1519.

10. Songstad, N.T., et al., Effects of High Intensity Interval Training on Pregnant Rats, and the Placenta, Heart and Liver of Their Fetuses. PloS one, 2015. 10(11): p. e0143095.

11. Brito, V.B., et al., Exercise during pregnancy decreases doxorubicin-induced cardiotoxic effects on neonatal hearts. Toxicology, 2016. 368: p. 46-57.

12. Wisløff, U., Ø. Ellingsen, and O.J. Kemi, High-intensity interval training to maximize cardiac benefits of exercise training? Exercise and sport sciences reviews, 2009. 37(3): p. 139-146.

13. Hafstad, A., et al., High intensity interval training alters substrate utilization and reduces oxygen consumption in the heart. heart, 2011. 111: p. 1235-1241.

14. Matsuo, T., et al., Low-volume, high-intensity, aerobic interval exercise for sedentary adults: $$\\dot {V} $$ O2max, cardiac mass, and heart rate recovery. European journal of applied physiology, 2014. 114(9): p. 1963-1972.

15. Hassanieh, S. and R. Mostoslavsky, Multitasking Roles of the Mammalian Deacetylase SIRT6, in Introductory Review on Sirtuins in Biology, Aging, and Disease. 2018, Elsevier. p. 117-130.

16. Cai, Y., et al., Nmnat2 protects cardiomyocytes from hypertrophy via activation of SIRT6. FEBS letters, 2012. 586(6): p. 866-874.

17. Maksin-Matveev, A., et al., Sirtuin 6 protects the heart from hypoxic damage. Experimental cell research, 2015. 330(1): p. 81-90.

18. Huang, C.-C., et al., Effect of exercise training on skeletal muscle SIRT1 and PGC-1α expression levels in rats of different age. International journal of medical sciences, 2016. 13(4): p. 260.

19. Koltai, E., et al., Exercise alters SIRT1, SIRT6, NAD and NAMPT levels in skeletal muscle of aged rats. Mechanisms of ageing and development, 2010. 131(1): p. 21-28.

20. Høydal, M.A., et al., Running speed and maximal oxygen uptake in rats and mice: practical implications for exercise training. European Journal of Cardiovascular Prevention & Rehabilitation, 2007. 14(6): p. 753-760.

21. Rodrigues, B., et al., Maximal exercise test is a useful method for physical capacity and oxygen consumption determination in streptozotocin-diabetic rats. Cardiovascular diabetology, 2007. 6(1): p. 38.

22. Stanford, K.I., et al., Exercise before and during pregnancy prevents the deleterious effects of maternal high-fat feeding on metabolic health of male offspring. Diabetes, 2015. 64(2): p. 427-433.

23. Sheldon, R.D., et al., Gestational exercise protects adult male offspring from high-fat diet-induced hepatic steatosis. Journal of hepatology, 2016. 64(1): p. 171-178.

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PLoS One. doi: 10.1371/journal.pone.0237148.r003

Decision Letter 1

Christopher Torrens

25 Jun 2020

PONE-D-19-33225R1

Influence of the maternal high-intensity-interval-training on the cardiac Sirt6 and lipid profile of the adult male offspring in rats

PLOS ONE

Dear Dr. khaledi,

Experimentally this is fine, but the conclusions do not necessarily follow from the results. The result show an impact on maternal HIIT on some parameters in the offspring but are not enough on their own to talk about general cardiovascular help. The options would either by to add more data to support such broader conclusions or to change the focus of the conclusion so as they better fit the data presented.

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We look forward to receiving your revised manuscript.

Kind regards,

Christopher Torrens

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The paper has been improved but there are still some outstanding issues.

Table 4

Is the birthweight the mean per litter, which in turn is meaned for the group?

Looking at this table again, I don't undertstand the sex distribution values given. It seems to correspond to the number of offspring but that value is presented as a mean +/- SEM. For example the number of pups in the BP group is given as 5 +/- 1.71, which suggests quite a large variation in pups number, if so what does it mean that the split was 3:2 M:F?

Line 219: Subsection on offspring physical activity. The title here says "Maternal HIIT...could improve physical activity...". This is not really what you measured though and this becomes ambiguous. Your data show they could run longer and faster but this is not the same as being more active, which you acknowledge because the title of the section says "could improve".

Table 5 needs standardised the decimal points. The raw mass is given to three decimal places but the error is only to two, while the percentage is give to two with the error given to three places. The two measure have difference accuracies and don't need to be standardised as such, but the error for each measurement should be in the same format as the measurement it is taken.

Line 242 "Beneficial effects" This is not the you found result. The result is a change in sirt6 and IGF-2 mRNA expression in response to maternal HIIT protocols. Any interpretation of what this means, be it good or bad belongs in the discussion not the results.

[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: All comments have been addressed

Reviewer #2: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

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: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have addressed my comments, I have 2 very minor concerns.

It should be made clear that “Maternal HIIT prior to and during pregnancy have beneficial effects on the heart of the next generation” are referring to the offspring – as written, it is hard to determine if the authors mean the offspring (F1) or the next generation (F2).

Line 181: Should be “vena cava” instead of Veno Cova

Reviewer #2: The authors did not perform most of the experiments I proposed in my revision. They conclude that maternal exercise may have effects on cardiovascular health, yet they fail to do studies of cardiac function.

**********

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PLoS One. 2020 Aug 3;15(8):e0237148. doi: 10.1371/journal.pone.0237148.r004

Author response to Decision Letter 1

3 Jul 2020

Dear Christopher Torrens

Academic Editor

PLOS ONE

The authors would like to Thank you for giving me the opportunity to submit a revised draft of our manuscript titled [Influence of the maternal high-intensity-interval-training on the cardiac Sirt6 and lipid profile of the adult male offspring in rats, No. PONE-D-19-33225] to [PLOS ONE]. We appreciate the time and effort that you and the reviewers have dedicated to providing your feedback on our manuscript. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewers. We have highlighted the changes within the manuscript. We hope that this new version of the manuscript is satisfactory for the presentation and we are thankful once again to the reviewers for their useful comments.

Sincerely yours

Neda Khaledi (Ph. D)

Corresponding Author

Academic editor' comments:

We truly appreciate the Editor’s suggestion, and we recognize the benefits improving the quality of the manuscript.

Point 1: Is the birthweight the mean per litter, which in turn is meaned for the group?

Response: Yes, this birthweight was reported based on the mean of all litter per group for example in the BP group is given as 6.020 ± 0.25, which this value is obtained from the meaning of pups’s birth weight of 6 mothers.

We agree with your suggestion about sex distribution and we have added SEM for it.

Point 2: Line 219: Subsection on offspring physical activity. The title here says "Maternal HIIT...could improve physical activity...". This is not really what you measured though and this becomes ambiguous. Your data show they could run longer and faster but this is not the same as being more active, which you acknowledge because the title of the section says "could improve". Response: In accordance with your suggestion, we have revised this section.

Point 3: Table 5 needs standardised the decimal points. The raw mass is given to three decimal places but the error is only to two, while the percentage is give to two with the error given to three places. The two measure have difference accuracies and don't need to be standardised as such, but the error for each measurement should be in the same format as the measurement it is taken.

Response: You have raised an important point. It was corrected.

Point 4: Line 242 "Beneficial effects" This is not the you found result. The result is a change in sirt6 and IGF-2 mRNA expression in response to maternal HIIT protocols. Any interpretation of what this means, be it good or bad belongs in the discussion not the results.

Response: Thank you for a valuable point. It was corrected.

Reviewers' comments:

Reviewer #1: Manuscript Number: PONE-D-19-33225

We are grateful to the reviewer 1 and 2 for their insightful comments on this article.

Point 1: It should be made clear that “Maternal HIIT prior to and during pregnancy have beneficial effects on the heart of the next generation” are referring to the offspring – as written, it is hard to determine if the authors mean the offspring (F1) or the next generation (F2).

Response: Thank you for pointing this out. It was changed to offspring.

Point 2: Line 181: Should be “vena cava” instead of Veno Cova

Response: This mistake was corrected. Thank you.

Reviewers' comments:

Reviewer #2: Manuscript Number: PONE-D-19-33225

Point 1: The authors did not perform most of the experiments I proposed in my revision. They conclude that maternal exercise may have effects on cardiovascular health, yet they fail to do studies of cardiac function.

Response: Although faced with limitation just as you said, we believe that additional experiments including direct measurements of cardiac function are very useful and will strengthen our work, but unfortunately due to the lack of funding time we are unable to investigate further at this time. accurate assessment of cardiac function, which is one of the limitations of our research, need to be known in further work and this limitation was added in the manuscript (line 299). Indeed, in accordance with your suggestion we have removed the term of cardiovascular health and have revised the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0237148.r005

Decision Letter 2

Christopher Torrens

22 Jul 2020

Influence of the maternal high-intensity-interval-training on the cardiac Sirt6 and lipid profile of the adult male offspring in rats

PONE-D-19-33225R2

Dear Dr. khaledi,

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.

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Kind regards,

Christopher Torrens

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0237148.r006

Acceptance letter

Christopher Torrens

24 Jul 2020

PONE-D-19-33225R2

Influence of the Maternal High-Intensity-Interval-Training on the Cardiac Sirt6 and Lipid Profile of the Adult Male Offspring in Rats

Dear Dr. Khaledi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Christopher Torrens

Academic Editor

PLOS ONE

Associated Data

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

Supplementary Materials

S1 Fig. Amplification plots and melting cure of the selected genes.

A) melting cure of sirt6, B) melting cure of igf2, C) melting cure of b-actin, D) Amplification plots of sirt6, E) Amplification plots of igf2 and F) Amplification plots of b-actin.

(TIF)

Click here for additional data file.^{(2.5MB, tif)}

S1 File. Minimal data set of the pup’s exercise test.

(XLSX)

Click here for additional data file.^{(9.9KB, xlsx)}

S2 File. Minimal data set of the pup’s heart weight.

(XLSX)

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S3 File. Minimal data set of the pup’s weekly weight gain.

(XLSX)

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

S4 File. Minimal data set of the pup’s gene expression.

(XLSX)

Click here for additional data file.^{(13.1KB, xlsx)}

S5 File. Minimal data set of the pup’s lipid profile.

(XLSX)

Click here for additional data file.^{(12KB, xlsx)}

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Submitted filename: Response to Reviewers.docx

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Submitted filename: Response to Reviewers.docx

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Data Availability Statement

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

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PERMALINK

Influence of the maternal high-intensity-interval-training on the cardiac Sirt6 and lipid profile of the adult male offspring in rats

Reihaneh Mohammadkhani

Neda Khaledi

Hamid Rajabi

Iraj Salehi

Alireza Komaki

Roles

Abstract

Introduction

Material and method

Animal

Study design

Fig 1. Schematic experimental design of the study.

Mating

Exercise test

High-intensity-interval training

Table 1. Characteristic of HIIT protocol prior to and during pregnancy.

Sirt6 and IGF-2 gene expression by RT-PCR method

Table 2. The PCR program.

Table 3. Conditions and sequence of primers.

Lipid serum by the colorimetric method

Statistical analysis

Results

The outcome of maternal characteristics

Table 4. Maternal characteristics.

The outcome of offspring

Maternal HIIT had effect on the weekly weight gain of the offspring whose mothers exercised only during pregnancy

Fig 2. Comparison of the weekly weight gain among the pup groups.

Maternal HIIT prior to and during pregnancy improve running speed and distance of the male offspring

Fig 3. Comparison of exercise test among the pup groups.

The initiated period of maternal HIIT has an impact on the heart mass of offspring

Table 5. Pups weight characteristics.

Maternal HIIT prior to and during pregnancy have effects on the Sirt6 and IGF-2 gene expression in the heart and serum lipid profile of the offspring

Fig 4. Comparison of cardiac gene expression among the pup groups.

Fig 5. The comparison of lipid profile among the pup groups.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Christopher Torrens

Roles

Author response to Decision Letter 0

Decision Letter 1

Christopher Torrens

Roles

Author response to Decision Letter 1

Decision Letter 2

Christopher Torrens

Roles

Acceptance letter

Christopher Torrens

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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