Hypercaloric diet models do not develop heart failure, but the excess sucrose promotes contractility dysfunction.

Several diseases are associated with excess of adipose tissue, and obesity is considered an independent risk factor for the development of cardiac remodeling and heart failure. Dietary aspects have been studied to elucidate the mechanisms involved in these processes. Thus, the purpose was the development and characterization of an obesity experimental model from hypercaloric diets, which resulted in cardiac remodeling and predisposition to heart failure. Thirty- day-old male Wistar rats (n = 52) were randomized into four groups: control (C), high sucrose (HS), high-fat (HF) and high-fat and sucrose (HFHS) for 20 weeks. General characteristics, comorbidities, weights of the heart, left (LV) and right ventricles, atrium, and relationships with the tibia length were evaluated. The LV myocyte cross sectional area and fraction of interstitial collagen were assayed. Cardiac function was determined by hemodynamic analysis and the contractility by cardiomyocyte contractile function. Heart failure was analyzed by pulmonary congestion, right ventricular hypertrophy, and hemodynamic parameters. HF and HFHS models led to obesity by increase in adiposity index (C = 8.3 ± 0.2% vs. HF = 10.9 ± 0.5%, HFHS = 10.2 ± 0.3%). There was no change in the morphological parameters and heart failure signals. HF and HFHS caused a reduction in times to 50% relaxation without cardiomyocyte contractile damage. The HS model presented cardiomyocyte contractile dysfunction visualized by lower shortening (C: 8.34 ± 0.32% vs. HS: 6.91 ± 0.28), as well as the Ca2+ transient amplitude was also increased when compared to HFHS. In conclusion, the experimental diets based on high amounts of sugar, lard or a combination of both did not promote cardiac remodeling with predisposition to heart failure under conditions of obesity or excess sucrose. Nevertheless, excess sucrose causes cardiomyocyte contractility dysfunction associated with alterations in the myocyte sensitivity to intracellular Ca2+.

Introduction

Obesity is a disease associated with several disorders and the intensity and duration of this condition are associated with cardiac remodeling with presence or absence of heart failure [1-4]. The excess of adipose tissue requires increased metabolic demands, which leads to chronic volume overload and, consequently, cardiac remodeling. In addition, changes in cardiovascular hemodynamics, can modify the cardiac morphology and function. Thus, the association of obesity with comorbidities, some neurohormonal and metabolic alterations may result in heart failure [5, 6]. In this context, dietary aspects have been studied to explain the possible mechanisms related to the emergence of this condition, which can modulate cardiovascular risk factors.

High-fat intake is not only related to lipid metabolism, but the type of ingested fat can also influence insulin resistance and promote alterations in blood pressure [7]. Diets with high intakes of simple carbohydrates may result in increased heart exposure to insulin, which activates cardiac protein synthesis and may promote left ventricular hypertrophy [8, 9].

Studies have shown that obesity induced by hypercaloric diets promotes cardiac remodeling with changes in morphology and/or cardiac function [10, 11]. Evidence from experimental studies indicates that high-fat feeding promotes cardiac contractile function damage [12, 13]. In our laboratory, myocardial dysfunction was demonstrated in basal conditions and physiological cardiac remodeling in obese rats [14-16]. In contrast, results on the effects of diets with high simple carbohydrate content (sucrose and/or fructose) are inconsistent. Sharma et al. [9] observed that hypertensive animals fed by fructose diet for eight weeks resulted in an increase in LV wall thickness and mortality, while Salie et al. [17] demonstrated cardioprotective effect after ischemia reperfusion in Wistar rats, using diet with sucrose supplementation for 16 weeks. Moreover, high-fat diets associated with simple carbohydrates have demonstrated cardiac damage, ventricular hypertrophy, interstitial fibrosis, increased rigidity and impairment in cardiac relaxation [17, 18].

Despite much research and evidence of cardiovascular damage caused by high-fat and high-sucrose diets, it is still unclear whether these diets, isolated or combined, promote cardiac remodeling with predisposition to heart failure. Thus, this study aimed to investigate the cardiac remodeling process in an experimental model induced by different types of hypercaloric diets (high-fat (lard), high-sugar and the combination of both) and their effects in the cardiac function. The hypothesis was that hypercaloric diets would promote cardiac remodeling, cardiovascular damage and predispose to heart failure, being most evident in the HFHS model.

Material and methods

Animal care and experimental design

Thirty-day-old male Wistar rats (≅110 g) obtained from the Animal Quarters of the Federal University of Espírito Santo (Brazil) were individually caged and subjected to different dietary regimens. All animal experiments were approved by the Ethics Review Committee of Federal University of Espírito Santo (CEUA-UFES 08/2016) and conducted in accordance with current Brazilian laws.

Rats were randomly assigned in control diet (C; n = 12), high-sugar diet (HS; n = 14), high-fat diet (HF; n = 13) and high-fat and high-sugar diet (HFHS; n = 13). All animals had free access to water and chow (40 g/day). To analyze whether dietary-induced obesity was associated with alterations in nutritional behavior, food consumption (FC) was measured daily. Calorie intake (CI) was calculated weekly by the average weekly FC × dietary energetic density. Feed efficiency (FE), the ability to transform consumed calories into body weight, was determined by following the formula: mean body weight gain (g)/total calorie intake (kcal). The HS group had water supplemented with sugar (300 g/l) in alternate weeks. For the calculation of the caloric intake of the HS group, the caloric energy from the water supplemented with sugar was also quantified (1.2 kcal/mL consumed).

The experimental diets provided sufficient amounts of protein, vitamins and minerals according to the Nutrient Requirements for Laboratory Animals. The diets used in the current study were formulated by Nutriave Alimentos® (Vitória, Espírito Santo, Brazil) [19]. The feed ingredients were blended, homogenized and extruded (Extru-Tech Extruder, Model E-750, Sabetha, KS, USA) in the form of pellets. Then, the pellets were dried on a horizontal conveyor dryer (20 minutes, temperature: ±70°C). The composition (g/kg) and nutrients for each experimental diet (%) are described in Table 1. The duration of the experimental protocol was 20 consecutive weeks.

Table 1

Composition and nutritional values of diets.

Components (g/kg)	Diets
	C	HS	HF	HFHS
Corn	200	200	180	80
Rice	200	200	200	200
Bone meal	120	120	120	120
Sugar	-	100	-	100
Soy oil	75	75	-	-
Lard	-	-	200	200
Gluten	200	200	200	200
Salt	3.5	3.5	3.5	3.5
Mineral Mix**	35	35	30	30
Vitamin Mix**	16.5	16.5	16.5	16.5
Inert Material***	150	50	50	50
Total (g)	1000	1000	1000	1000
Nutrient Composition (%)
Protein	24.8	21.8	17.8	19.2
Carbohydrate	49.6	52.3	44.6	43.4
Lipids	25.6	25.9	37.6	37.4
Energy Density (Kcal/g)	3.55	3.65	4.59	4.49

Diets. C: normal rodent chow; HS: High-sugar; HF: high-fat; HFHS: high fat and high sugar. In order to calculate the caloric intake of HS, the caloric value of the sugar diet (3.65 kcal/g) plus the caloric value of water intake with sugar (1.2 kcal/ml) was computed ** Vitamin and Mineral Mix: vit. A, vit. C., vit. D3, vit. E, vit. K3, vit. Complex B, pantothenic acid, folic acid, biotin, choline; selenium, iron, copper, manganese, iodine, zinc, cobalt, calcium, and phosphorus.

*** Bentonite: inert material, with no nutritional value and calories.

Characterization of obesity

The adiposity index, used to assess obesity, was calculated by adiposity index [body fat (BF)/final body wt] ×100. BF was measured from the sum of the individual fat pad weights as follows: BF = epididymal fat + retroperitoneal fat + visceral fat.

Metabolic and hormonal measurements

After 20 weeks, the animals were subjected to 12–15 h of fasting, and blood samples were collected in dry tubes. The serum was separated by centrifugation at 10,000 rpm for 10 min. (Heraeus Megafuge 16R Centrifuge, Thermo Scientific, Massachusetts, USA) and stored at −80 °C for subsequent analysis (Coldlab Ultra Freezer CL374-86V, Piracicaba, São Paulo, Brazil). Serum glucose concentration was measured using specific kit (Bioclin Bioquímica®, Belo Horizonte, Minas Gerais, Brazil and Synermed do Brasil Ltda., São Paulo, Brazil) and analyzed by automated biochemical equipment BS-200 (Mindray do Brasil-Comércio and Distribuição de Equipamentos Médicos Ltda., São Paulo, Brazil). Insulin was determined using an enzyme-linked immunosorbent assay (ELISA) using specific kit (Linco Research Inc., St. Louis, MO, USA). The reading was carried out using a microplate reader (Asys Expert Plus Microplate Reader, Cambourne, Cambridge, UK).

Cardiac remodeling assessment

The cardiac remodeling process was assessed by determination of heart weight (HW), left ventricle, HW and LV/tibia length ratios. At the end of the experimental protocol, the animals received an intraperitoneal injection (IP) of sodium heparin (1000 U/Kg/IP; Heparamax-s, Blau Pharmaceutic S.A., São Paulo, Brazil) for cardiomyocyte analysis. After 30 minutes, rats were anesthetized with ketamine (90 mg/kg, IP) plus xylazine (10 mg/kg, IP) and euthanized by decapitation. Subsequently, their chests were opened by mid-thoracotomy, and the heart, ventricles, fat pads, and tibia were separated, dissected, weighed or measured.

Hemodynamic measurements

After 20 weeks of experimental protocol, the rats (n = 6 per group) were anesthetized intraperitoneally (IP) with urethane (Carbamic acid ethyl ester; 1.2 g/kg/IP injection; Sigma-Aldrich, USA) and submitted to catheterization surgery. LV hemodynamic data were obtained from right carotid artery using a micromanometer (MikroTipTM, SPR 320, USA). Systolic and diastolic blood pressure (SBP and DBP), heart rate (HR); LV systolic and end-diastolic pressure (LVSP and LVEDP); maximum positive (+dP/dtmax) and negatives (-dP/dtmax) derivatives of LV pressure, LV relaxation time constant (TAU) were acquired and analyzed using a computer (Biopac System, USA).

Histological analysis

The LV transverse sectional area of animals from each group was fixed in phosphate-buffered 4% paraformaldehyde (7.4 pH) and embedded in paraffin. Sections of 6 μm were obtained and stained with hematoxylin-eosin (HE) and picrosirius red stain to determine the myocyte cross-sectional area and to collagen volume fraction, respectively.

Cardiomyocyte contractile function

Under anesthesia as described above, the hearts from rats were quickly removed by median thoracotomy, then enzymatically isolated [20]. The isolated cells were placed in an experimental chamber with a glass coverslip base mounted on the stage of an inverted microscope (IonOptix, Milton, USA) coupled with an edge detection system with a 40× objective lens (Nikon Eclipse–TS100, USA). Cells were immersed in Tyrode’s solution containing 1.8 mM CaCl2 and field stimulated at 1 Hz (20V, 5ms duration square pulses). Fractional shortening and the times to 50% contraction and relaxation were measured.

Intracellular Ca2+ measurements

Myocytes were loaded with 1.0 μM Fura2-acetoxymethyl (AM) ester (Molecular Probes, USA). Ca2+ transient amplitude was reported as F/F0, where F is the maximal fluorescence intensity average measured at the peak of [Ca2+]i transients, and F0 is the baseline fluorescence intensity measured at the diastolic phase of [Ca2+]i transients. We also analyzed the period of time until the Ca2+ transient peak and 50% Ca2+ decay were reached.

Heart failure

Heart failure was considered when three criteria were met: Pulmonary congestion: a) percentage of lung humidity above 80%; b) lung/body weight (BW) ratio greater than lung/BW approximately 2-fold in relation to C [21]; c) right lung/BW above 5 mg/g [22]. RV hypertrophy: characterized by the ratio of the weight of RV adjusted by BW above 0.8 mg/g [23]. Elevated LVEDP: LV end diastolic pressure (LVEDP) greater than 15 mmHg [24, 25]; and Systolic dysfunction: maximum positive value of the first derivative of LV systolic pressure above 7000 mmHg [22]. After removal of the lung tissue, wet weight (WW) and dry weight (DW) were determined before and after drying the samples at 60°C for 48 hours. Water content (%H2O) was estimated using the follow formula: %H2O = [(WW—DW)/WW] × 100.

Statistical analysis

Data were reported as mean ± standard error of the mean (SEM) and analyzed using one-way analysis of variance (ANOVA) followed by the Tukey post hoc, at a 5% significance level.

Results

After 20 weeks, HF rats demonstrated a final body weight 22% and 20.6% greater than C and HS, respectively (Table 2). The HF and HFHS diets promoted a substantial elevation of visceral and retroperitoneal fat pads compared to C and HS diets. Specifically, HF and HFHS rats showed an elevation of 62% and 43% in body fat, respectively, when compared to C. In addition, the adiposity index was significantly greater in these groups (31.3% and 22.9%) than in C. In relation to HS, there was an increase in body fat of 68% and 48.7% in HF and in HFHS, respectively.

Table 2

General characteristics.

Variables	Experimental Groups
	C	HS	HF	HFHS
IBW (g)	107 ± 3	110 ± 3	111 ± 3	110 ± 4
FBW (g)	533 ± 17	538 ± 13#	649 ± 34*	616 ± 22
BW gain (g)	426 ± 17	428 ± 12#	538 ± 32*	506 ± 20
Epididymal fat pad (g)	11.2 ± 0.6	10.5 ± 0.7	13.1 ± 0.9	13.5 ± 1.0
Visceral fat pad (g)	11.4 ± 0.6	10.6 ± 0.6#&	18.5 ± 1.4*	15.9 ± 1.4*
Retroperitoneal fat pad (g)	21.9 ± 1.0	21.8 ± 2.0#&	40.4 ± 4.5*	34.4 ± 2.2*
Body fat (g)	44.5 ± 1.6	42.9 ± 0.6#&	72.0 ± 6.3*	63.8 ± 4.3*
Adiposity index (%)	8.3 ± 0.2	7.9 ± 0.3#&	10.9 ± 0.5*	10.2 ± 0.3*
Food consumption (g/day)	22.3 ± 0.7	16.2 ± 0.5*#	18.9 ± 0.7*	17.2 ± 0.4*
Caloric intake (kcal/day)	79.2 ± 2.6	92.1 ± 2.1*&	86.9 ± 3.5	77.3 ± 1.8α
Feed efficiency (%)	3.84 ± 0.08	3.32 ± 0.05*	4.39 ± 0.09*#	4.64 ± 0.09*&
Glucose (mg/dL)	108 ± 2	112 ± 3	115 ± 4	115 ± 3
Insulin (ng/mL)	1.86 ± 0.13	1.77 ± 0.15	2.19 ± 0.16	2.39 ± 0.23

Data are presented as the mean ± SEM. Control diet—(C; n = 12); high-sugar diet—(HS; n = 14); high-fat diet—(HF; n = 13), and high-fat and high-sugar diet (HFHS; n = 13). IBW: initial body weight; FBW: final body weight; BW: body weight. One-way ANOVA for independent samples followed by Tukey post hoc test. p < 0.05 vs. * C

# HF vs. HS

& HFHS vs. HS

α HF vs. HFHS.

The nutritional profile of rats is summarized in Table 2. The C rats had an approximately 17.9%, 29.6% and 37.6% greater daily food consumption (g) than the HF, HFHS and HS groups, respectively, but the daily caloric intake was higher in the HS group in relation to the C and HFHS groups (HS: 92.1 ± 2.1 vs. C: 79.2 ± 2.6 and HFHS: 77.3 ± 1.8 kcal/day, p < 0.05). In addition, there was a difference in FC between rats fed the HF diet compared with the HS group since rats fed the HS diet consumed significantly less food (HG: 16.2 ± 0.5 vs. HF:18.9 ± 0.7; p < 0.05). Furthermore, HF presented a 12.4% increase in caloric intake over HFHS (p < 0.05). There was no difference in the caloric intake of C compared to HF and HFHS (p > 0.05). While the feed efficiency (%) was higher in the HF (14.3%) and HFHS groups (20.8%) than in C (Table 2), there was a lower feed efficiency in HS rats than C (HS: 3.32 ± 0.05 vs. C: 3.84 ± 0.08; p < 0.05).

In relation to cardiac remodeling, there were no differences in the parameters analyzed in groups HS, HF and HFHS when compared to C (Fig 1). However, the absolute heart and LV weights were significantly elevated in HF rats in relation to HS group (Fig 1A and 1B), representing an increase of 32% and 29%, respectively. In addition, these rats also showed elevation of HW and LVW-to-tibia length ratios when compared to HS rats (Fig 1C and 1D). Additionally, the histological analysis from LV samples revealed that CSA and interstitial collagen fraction (%) were similar among the groups (Fig 2A and 2B). There was no statistical difference between the experimental groups for the lung/FBW, right lung/FBW and RV/FBW ratios, as well as for the lung water content (%) (Fig 3A–3D).

Fig 1

Effect of different diet composition on cardiac remodeling.

Data are shown as mean ± SEM. Control diet—(C; n = 5); high-sugar diet—(HS; n = 8); high-fat diet—(HF; n = 5), and high-fat and high-sugar diet (HFHS; n = 6). HW: heart weight; LW: left weight. p < 0.05 vs. # HF vs. HS. One-way analysis of variance (ANOVA) followed by the Tukey post hoc test.

Fig 2

Histological study in myocardium.

Control diet—(C; n = 5); high-sugar diet—(HS; n = 8); high-fat diet—(HF; n = 5), and high-fat and high-sugar diet (HFHS; n = 6) after 20 weeks. (A) cross sectional area (CSA). (B): interstitial collagen of myocardium; representative picrosirius red-stained left ventricle (LV) section. Arrows: interstitial collagen. Data are shown as mean ± SEM. One-way analysis of variance (ANOVA) followed by the Tukey post hoc test. p < 0.05 vs. * C; & HFHS vs. HS; §HFHS vs. HF.

Fig 3

Effect of different diet composition on parameters of heart failure.

Data are shown as mean ± SEM. Control diet—(C; n = 12); high-sugar diet—(HS; n = 14); high-fat diet—(HF; n = 13), and high-fat and high-sugar diet (HFHS; n = 13). RV: right ventricle. (A) Lung weight/body weight ratio. (B) Right lung weight/body weight ratio. (C) RV weight/body weight. (D) Lung water content (C = 5; HS = 8; HF = 5, and HFHS = 6). One-way analysis of variance (ANOVA) followed by the Tukey post hoc test.

The different types of hypercaloric diets did not promote hemodynamic alterations (Table 3). In addition, the fractional shortening was significantly reduced by 20% and 15% in the HS group in relation to C and HFHS, respectively (Fig 4B). Although all groups showed lower values of time to 50% contraction in relation to C, there only was statistical difference between HF and C (Fig 4C). The time to 50% relaxation were reduced in HS when compared to HF and HFHS, respectively (Fig 4D), resulting in impaired of cardiomyocyte relaxation. Moreover, HF and HFHS groups had reduced times to 50% relaxation in relation to C (Fig 4D). Ca2+ transient amplitude was increased in HS compared to HFHS (Fig 4E). In contrast, no differences were observed in times to peak and 50% Ca2+ decay among other groups (Fig 4F and 4G).

Table 3

Left ventricular hemodynamics measurements.

Variables	Experimental Groups
	C	HS	HF	HFHS
SBP (mmHg)	97.8 ± 3.5	98.9 ± 4.5	92.7 ± 3.8	98.8 ± 3.5
DBP (mmHg)	58.5 ± 4.5	61.1 ± 5.2	56.5 ± 2.7	59.6 ± 5.1
HR (bpm)	288 ± 10	314 ± 17	321 ± 11	328 ± 14
LVSP (mmHg)	99.5 ± 2.9	95.8 ± 4.5	93.4 ± 3.9	100 ± 3
LVDP (mmHg)	4.02 ± 1.6	2.17 ± 0.81	2.19 ± 0.85	2.8 ± 0.79
+dP/dtmáx (mmHg/s)	6568 ± 471	7301 ± 1045	6157 ± 720	8022 ± 652
-dP/dtmáx (mmHg/s)	-6769 ± 389	-6485 ± 725	-6375 ± 661	-7749 ± 416
Tau (s)	0.014 ± 0.003	0.011 ± 0.001	0.010 ± 0.002	0.011 ± 0.001

Data are presented as the mean ± SEM (n = 6 animal per group). Control diet—(C); high-sugar diet—(HS); high-fat diet—(HF), and high-fat and high-sugar diet (HFHS). SBP: systolic blood pressure; DBP: diastolic blood pressure; HR: heart rate; LV: left ventricle; LVSP: LV systolic pressure; LVEDP: LV end-diastolic pressure; +dP/dtmáx: maximum positive derivative of LV pressure; -dP/dtmáx: maximum negative derivative of LV pressure; Tau: LV relaxation time constant. One-way ANOVA for independent samples followed by Tukey post hoc test.

Fig 4

Effect of different diet composition on contractile function and calcium transients of left ventricular cardiomyocytes.

Control diet (C; n = 5; cells = 71), high-sugar diet (HS; n = 6, cells = 115), high-fat diet (HF; n = 6, cells = 106) and high-fat and high-sugar diet (HFHS; n = 5, cells = 81). Data are shown as mean ± SEM. (A) Representative contraction traces obtained from the cardiomyocytes of rats: (B) Cell shortening expressed as % of resting cell length. (C) Time to 50% of contraction. (D) Time to 50% of relaxation. (E) Amplitude of transients. (F) Time to peak. (G) Time to from peak transient to half resting value p < 0.05 vs. * C; # HS vs. HF; & HFHS vs. HS. One-way analysis of variance (ANOVA) followed by the Tukey post hoc test.

Discussion

Obesity is associated with structural and functional changes in the heart [1]. However, the HS did not cause morphological cardiac remodeling. Chess and Stanley [26] reported that high sugar intake leads to hyperglycemia in blood circulation and cardiotoxic effects. Increased glycemia leads to elevated serum insulin levels, which in cardiac tissue induce elevated protein synthesis of cardiomyocytes and, consequently, left ventricular hypertrophy. Sharma et al. [9] proposed that the ingestion of carbohydrates, in particular sugar, associated or not with blood pressure overload, can cause LV hypertrophy, via insulin receptor stimulation and activation of Akt/mTOR pathways. These findings suggest that, in the HS model, there was no need for elevated circulating insulin in response to increased glycemic levels, since the animals remained euglycemic.

The HF model did not cause cardiac remodeling, although the animals showed higher heart weight and heart/tibia length ratio compared to C, however, this difference was not significant (p = 0.08). In addition, although the myocyte CSA of this group had a higher value in relation to C, this increase was also not significant (p = 0.07). However, the HF diet promoted cardiac remodeling in relation to HS group (Fig 1A and 1B). In relation to the HFHS, no changes in heart weight, CSA and myocardial collagen fraction were observed, which shows that this treatment did not lead to cardiac remodeling. These findings are divergent from Poudyal et al. [27], who visualized cardiac remodeling with LV hypertrophy and increased collagen fraction in Wistar rats after 32 weeks of experimental protocol. The authors reported that these changes are related, among other factors, to the aging process. Another mechanism is related to leptin that mediates the process of cardiac hypertrophy in parts by the mitogen-activated protein kinase (MaPK p38), which regulates various cellular processes.

Specifically, the HS did not promote cardiac remodeling, however, it was able to prevent the elevation on heart and LV weights in the HFHS, interfering in the effect of HF diet. According to literature, high sucrose intake [68% total energy sucrose diet, 69% total mass sucrose diet, or 20% sucrose solution] induced hyperinsulinemia without alterations in plasma glucose level in rats and mice [28-30]. Interestingly, our HS rats have not demonstrated alterations in glucose and insulin levels (Table 2). Several mechanisms have been postulated, including the prohypertrophic effects of insulin, insulin growth factor-1, and insulin resistance [31]. Initially the circulating insulin levels are increased, directly stimulating cardiomyocyte growth [32] and indirectly via binding to the insulin growth factor-1 receptor [33]. Chess et al. [26], analyzing the effects of dietary extremes (high carbohydrate and fat intake) on the remodeling process and heart failure, reported that high sugar intake causes hyperglycemia in the bloodstream and cardiotoxic effects. Increased glycemia leads to elevated serum insulin levels, which in cardiac tissue, induces increased protein synthesis of cardiomyocytes and, consequently, left ventricular hypertrophy. Sharma et al. [9] propose that carbohydrate intake, in particular sugar, associated or not with pressure overload may cause LV hypertrophy, via insulin receptor stimulation and activation of Akt/mTOR, proteins involved in protein signaling pathways. Thus, our results from HS diet suggest that the sugar intake was not able to affect the process of cardiac remodeling, probably due to the absence of hyperinsulinemia and elevation of glucose levels, as well as it was not promote cardiotoxic effect, indicating a cardioprotective effect alone or when associated with the HF diet.

Experimental models that mimic the eating habits of the population have been widely used to elucidate the mechanisms of obesity and cardiovascular disorders [34]. In this sense, there was no significant difference was observed in vivo study, indicating that the dietary interventions used did not cause cardiac functional adaptation. In addition, our results demonstrate that obesity models (HH and HFHS) preserved the cardiomyocyte contractile function with a punctual improvement in myocardial relaxation. These results differ from other studies that have shown impairment in contraction and relaxation in experimental obesity models [12, 15, 16, 35]. These authors observed a reduction in the L-type Ca2+ channels expression, lower phosphorylation of RyR2, and a decrease in SERCA2a and phospholamban phosphorylation (PLB), respectively, due to the increase of intracellular Ca2+ concentration and removal of cytosolic Ca2+. However, Fauconnier et al. [36] observed that, in the presence of palmitate, cellular shortening increased by 40%, as well as a 25% increase in the ratio between cellular shortening and the Ca2+ transient amplitude, demonstrating that this substrate was able to improve the contractile responses. Thus, to maintain contractile function, the heart requires a continuous and abundant supply of energy, which comes mainly from fatty acids and glucose.

Free fatty acid oxidation is the major source of energy for the myocardium and up to 80% of high-energy phosphates are produced, while the glucose metabolism provides the remaining quantity of energy. Glucose as glycogen are stored to be used during increased metabolic demands such as obesity and diabetes, since the glucose utilization is 20–30% more metabolic efficient than free fatty acid oxidation in producing high-energy phosphates. In this sense, an energetic dysregulation play an important role in the pathophysiology of the failing heart. According Doesn’t et al. [37] a possible cause for these metabolic derangements in HF could be related to myocardial insulin resistance, which limits the utilization of glucose and favors the increased utilization of free fatty acids for ketogenesis. These changes lead to a reduction in the production of high-energy phosphates and therefore to a metabolically inefficient heart. Therefore, the metabolic changes in HF may favor for the progression and reducing functional capacity. Nevertheless, our findings suggest that the supply of fatty acids seems to initially exert a cardioprotective effect against the aggressions imposed by the obesity condition and pressure surges in the hyperlipidic models, since the HF diet did not probably promote systemic and myocardial insulin resistance (data not evaluated). In addition, it should be noted that the occurrence of heart failure was not observed in the evaluated experimental models; literature highlights the reduced use of fatty acids in heart failure [37], maintaining normal functioning of the heart.

On the other hand, the hyperglycemic model showed a cardiomyocyte contractile dysfunction. This finding corroborates Vasanji et al. [38], who demonstrated a reduction in the fractional shortening of animals fed a sucrose-rich diet, indicating cardiac contractility damage. Balderas-Villalobos et al. [39] reported that the consumption of sucrose in Wistar rats promoted a decreased Ca2+ transient amplitude, although without modifications in the amount of Ca2+ of sarcoplasmic reticulum (SR). These researchers suggested that alterations in the activity of L-type Ca2+ channels or RyR2 receptors may contribute to reduce Ca2+ release and reuptake in SR via SERCA2a. In addition, Abel et al. [1] suggest that altered use of the myocardial substrate may be a mediator of subsequent contractile dysfunction. Within this context, when fatty acid levels are low and glucose concentrations are high, myocardial metabolism adapts to the use of glucose as an energetic substrate. This adaptive response is initially beneficial because it maintains adenosine triphosphate (ATP) levels in the face of decreased mitochondrial oxidative phosphorylation from fatty acids; however, this change in energy metabolism is not just a primary effect of cardiac remodeling, but it may, in fact, be a predictor of cardiac dysfunction [40]. Thus, the damage observed in the hyperglycemia model might be, among other factors, due to the higher glucose intake, which seems to cause some imbalance in intracellular homeostasis and, consequently, alter the mechanism of cardiac contraction and relaxation, as well as Ca2+ handling.

Heart failure (HF) is the common endpoint of most heart disease, being one of the most important clinical challenges in the health field [41]. Obesity is an independent risk factor for the development of HF and this condition causes a series of hemodynamic changes, which aim to maintain the body's homeostasis, including increased cardiac output and decreased peripheral resistance, changing the cardiac morphology that predispose to left and right ventricular dysfunction. These results suggest that the damage in the cardiac morphology and function may lead to heart failure, even in the absence of other cardiac diseases [2, 6]. Our results indicate that no differences were found between the experimental groups, demonstrating the absence of HF. The absence of HF in the experimental models used in this study might be explained by the failure in the characterization of tissue congestion, RV hypertrophy, as well as systolic and diastolic dysfunction. In this sense, there were no morphological and functional adaptations, originating from the condition of obesity or excess sucrose, capable of predisposing to HF.

Conclusion

The experimental diets based on high amounts of sugar, lard or a combination of both did not promote cardiac remodeling with predisposition to heart failure under conditions of obesity or excess sucrose. Nevertheless, excess sucrose causes cardiomyocyte contractility dysfunction associated with alterations in the myocyte sensitivity to intracellular Ca2+.

6 Nov 2019

PONE-D-19-28274

Hypercaloric diet models do not develop heart failure, but the excess sucrose promotes contractility dysfunction

PLOS ONE

Dear Mr Leopoldo,

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.

The authors need to address point-by-point responses to the reviewers' comments below before the manuscript is reconsidered for publication. 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.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Tohru Minamino, M.D., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

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

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: No

Reviewer #2: No

Reviewer #3: Partly

**********

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #3: Yes

**********

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: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

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: In this study the authors investigate the effect of HF and or HS sucrose diet on cardiac function and remodeling.

Overall the manuscript is poorly written, several sentences in particular in the introduction and discussion are either hard to understand or make no sense.

In the abstract it says 60 animals in total, 15 in each group. In the introduction it is down to 12/14/13/13 in each group, in the result part it is down to 5-8 animals. In particular when claiming absence of effect it is very important to have sufficient high n's. The entire study seems to be underpowered.

Overall, the manuscript contains very little data, maybe the data should have been combined with a article from 2018 from the same authors which seems to originate from the same study since the number of animals in each group stated in the introduction match the numbers in the current manuscript.

Reviewer #2: The manuscript by Matias et al. aims to develop a hypercaloric animal model of cardiac remodeling and predisposition to heart failure. The authors studied the effect of 3 different hypercaloric diets, one composed by high fat, other by high sucrose and the last one by the combination of high fat with high sucrose on heart and cardiomyocyte function. They found that none of the models studied promoted cardiac remodeling and predisposition to heart failure, however the sucrose diet causes cardiomyocyte contractility dysfunction. The manuscript is well written and the experiments were well design, although I believe it lacks mechanistic insights on why diets do not promote cardiac remodeling but high sucrose produces cardiomyocytes dysfunction.

1- Please describe the composition of the diets. This is an important factor as different diet composition might produce different disease phenotypes: heart failure, hypertension, among others. Note that several authors in the past has described the development of heart failure and hypertension with hypercaloric diet consumption (see e.g. Panchal and brown, 2011, Journal of Biomedicine and Biotechnology), and the composition of the diet might be of the factor that can contribute to the different results observed in the present manuscript. Also, diet composition, e.g. unsaturated fatty acids (UFA) can also contribute to the different results observed, see e.g. Carbone et al. 2017. JACC: Basic to Translational Science. Please discuss this.

2- Can the authors calculate the caloric intake of the animals? Was the caloric intake similar between groups?

3- How was the HFHsu administered? Was solid food? Or was sucrose administered in drinking water? Please describe the procedures, as this is quite important to interpret the results of the present manuscript. Usually, HFHSu models consume more sugar than fat and resemble more an HSu model than HF model.

4- Advanced glycation end-products (AGEs) can be endogenously formed as a consequence of a high dietary sugar intake and several lines of evidence suggest that AGEs are related to the development and progression of heart failure. I believe that the manuscript will truly benefit from the measurement of AGEs particularly because this might explain the differences between high fat and high sucrose diets.

5- Note, that the title and the conclusion of the manuscript depend totally on diet composition evaluated in the present study, please be more cautious on these conclusions.

Reviewer #3: This manuscript reports that the experimental hypercaloric models did not promote cardiac remodeling and predisposition to heart failure under conditions of obesity or excess sucrose. Nevertheless, excess sucrose causes cardiomyocyte contractility dysfunction associated with alterations in intracellular Ca2+. This work may add knowledge in the literature. The work in general is interesting, but there are important points and suggestion that should to be answered.

1 – The authors needs to organize the reference in the Introduction section (line 70 and 73).

2 - The authors describe in the Introduction Section that "….study aimed to investigate the cardiac remodeling process in an experimental model induced by different types of hypercaloric diets (high-fat (lard), high-sugar and the combination of both) and their effects on the development and progression of heart failure." I suggest changing the term ‘heart failure” for “...and their effects in the cardiac function”, because the experimental model do not is validated and do not develop the pathophysiology described;

3 – In the Results section, the diets association (HFHS) did induced any changes (figure 1), by the way, the HFD induces significantly changes in the cardiac parameters, So I would like to understand if the HS can interfere in the effect of HFD? This effect should be described in the Discussion section.

4 – The authors describe in the Discussion section that HS did cause morphological cardiac remodeling, but it is know the cardiotoxic effect of the high sugar. So I would like to understanding, did this diet works? Why we don not have the data of the metabolic profile? Moreover, I would like to suggest that the authors explain better, this absence of cardiotoxic effect in the HS.

5 – The author describe in the Discussion section a possible effect cardioprotetor in the initial stage of obesity, so I would like to suggest that this point be further explored and add in this section. Moreover, I suggest describing further details of the role of fatty acid reduction in heart failure.

6 – In the Discussion section, the authors explore the myocardial metabolic adapt to the use of glucose as an energetic substrate, however did not measure any parameter for this suggestion. So it is necessary describe further this point.

In addition, I would like to understand the effect of leptin in this HS experimental model due the intimal association of calcium concentration.

**********

6. 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

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

20 Dec 2019

Manuscript Number: PONE-D-19-28274

Title: Hypercaloric diet models do not develop heart failure, but the excess sucrose promotes contractility dysfunction

Review Comments to the Author

Reviewer #1:

1. In this study the authors investigate the effect of HF and or HS sucrose diet on cardiac function and remodeling. Overall the manuscript is poorly written, several sentences in particular in the introduction and discussion are either hard to understand or make no sense.

Dear Reviewer, we appreciate the commentary, but we do not specifically understand what we need to change. In addition, the manuscript was sent to the English edition (English Certificate) and corrected by a specialized company. What do we need to be clearer?

2. In the abstract it says 60 animals in total, 15 in each group. In the introduction it is down to 12/14/13/13 in each group, in the result part it is down to 5-8 animals. In particular when claiming absence of effect it is very important to have sufficient high n's. The entire study seems to be underpowered.

Dear Reviewer, we appreciate the commentary and agree that this information was confuse. Therefore, we believe a misconception has occurred and we have not expressed well about the number of animals. This information was added to the text, making it easier for readers to understand the total number of animals per group and per technique, since several techniques were used to respond to the initial study objective. Thus, each technique was performed with an appropriate number of animals to avoid absence of effect. For example, to morphological and histological analysis we used 5-8 animals from each group, because we would need the total left ventricle without going through any solution used in the cardiomyocyte technique. In addition, for the cardiomyocyte technique (5-6 animals each group), the animal's heart is fully utilized for cell dissociation and isolation. Therefore, we had to distribute the animals in different techniques. To general characteristics and to analysis of heart failure, we used all animals. It is noteworthy that current literature studies use this design strategy without asking for the effect.

The alteration was performed in the abstract. The correct sentence is “Thirty- day-old male Wistar rats (n = 52) were randomized into four groups: control (C), high sucrose (HS), high-fat (HF) and high-fat and sucrose (HFHS) for 20 weeks.” (Abstract, line 6)

3. Overall, the manuscript contains very little data, maybe the data should have been combined with a article from 2018 from the same authors which seems to originate from the same study since the number of animals in each group stated in the introduction match the numbers in the current manuscript.

Dear Reviewer, we appreciate the commentary and disagree the actual manuscript contains very little data because in the previous study the aim was to investigate the effects of different dietary interventions on nutritional, metabolic, biochemical, hormonal, and cardiovascular profiles, as well as to add to development and characterization of an experimental model of obesity [1]. In the current study, our purpose was the development and characterization of an obesity experimental model from hypercaloric diets, which resulted in cardiac remodeling and predisposition to heart failure. In this sense, our would like to study the effects of these diets on heart. Generally, we divide the projects and study into several subprojects with different purposes, mainly in order to optimize the time and design of the experimental protocols, as well as the resources due to them lack by research funding agencies in Brazil. We believe this to occur frequently in world science, as it ends up optimizing the publication and discussion of the findings.

1. Matias AM, Estevam WM, Coelho PM, Haese D, Kobi JBBS, Lima-Leopoldo AP, Leopoldo AS. Differential Effects of High Sugar, High Lard or a Combination of Both on Nutritional, Hormonal and Cardiovascular Metabolic Profiles of Rodents. Nutrients. 2018 Aug 11;10(8). pii: E1071. doi: 10.3390/nu10081071.

Reviewer #2:

The manuscript by Matias et al. aims to develop a hypercaloric animal model of cardiac remodeling and predisposition to heart failure. The authors studied the effect of 3 different hypercaloric diets, one composed by high fat, other by high sucrose and the last one by the combination of high fat with high sucrose on heart and cardiomyocyte function. They found that none of the models studied promoted cardiac remodeling and predisposition to heart failure, however the sucrose diet causes cardiomyocyte contractility dysfunction. The manuscript is well written and the experiments were well design, although I believe it lacks mechanistic insights on why diets do not promote cardiac remodeling but high sucrose produces cardiomyocytes dysfunction.

1- Please describe the composition of the diets. This is an important factor as different diet composition might produce different disease phenotypes: heart failure, hypertension, among others. Note that several authors in the past has described the development of heart failure and hypertension with hypercaloric diet consumption (see e.g. Panchal and brown, 2011, Journal of Biomedicine and Biotechnology), and the composition of the diet might be of the factor that can contribute to the different results observed in the present manuscript. Also, diet composition, e.g. unsaturated fatty acids (UFA) can also contribute to the different results observed, see e.g. Carbone et al. 2017. JACC: Basic to Translational Science. Please discuss this.

Dear Reviewer, we appreciate the comment and we agree the composition of the diet might be of the factor that can contribute to the different results observed in the present manuscript. Our group published a specific study about the composition and experimental obesity model [1], in which it is possible to identify in detail the ingredients used and the nutritional value of diets (Nutrients 2018, 10(8), 1071; https://doi.org/10.3390/nu10081071), as detailed below:

The experimental diets provided sufficient amounts of protein, vitamins, and minerals according to the Nutrient Requirements of Laboratory Animals [2]. The diets used in the current were formulated by Nutriave Alimentos® (Vitória, Espírito Santo, Brazil). The feed ingredients were blended, homogenized and extruded (Extru-Tech Extruder, Model E-750, Sabetha, KS, USA) in the form of pellets. Then, pellet was dried on a horizontal conveyor dryer (20 minutes, temperature: ± 70°C). The composition (g/kg) and nutrients of each experimental diet (%) is described in Table 1.

Table 1. Composition and nutritional values of diets

Components (g/kg) Diets

C HS HF HFHS

Corn 200 200 180 80

Rice 200 200 200 200

Bone meal 120 120 120 120

Sugar - 100 - 100

Soy oil 75 75 - -

Lard - - 200 200

Gluten 200 200 200 200

Salt 3.5 3.5 3.5 3.5

Mineral Mix** 35 35 30 30

Vitamin Mix** 16.5 16.5 16.5 16.5

Inert Material*** 150 50 50 50

Total (g) 1000 1000 1000 1000

Nutrient Composition (%)

Protein 24.8 21.8 17.8 19.2

Carbohydrate 49.6 52.3 44.6 43.4

Lipids 25.6 25.9 37.6 37.4

Energy Density (Kcal/g) 3.55 3.65 4.59 4.49

Diets. C: normal rodent chow; HS: High-sugar; HF: high-fat; HFHS: high fat and high sugar. *Rats received diet with simple carbohydrate and water supplemented with sugar (300 g/L) in alternate weeks. In order to calculate the caloric intake of HS, the caloric value of the sugar diet (3.65 kcal/g) plus the caloric value of water intake with sugar (1.2 kcal/ml).

Unfortunately, we have not the composition of unsaturated fatty acids because we did not have resources at the time for the realization of these dosages, as well as equipment for this analysis. This suggestion will be applied in future studies.

Nevertheless, as requested by Reviewer, the information about the composition of the diets was included in the text. The added sentence is “The diets used in current study were previously described by Amanda et al. [1], which provided sufficient amounts of protein, vitamins, and minerals” (Material and methods, Animal Care and Experimental design, page 4, lines 97-99)

1. Matias AM, Estevam WM, Coelho PM, Haese D, Kobi JBBS, Lima-Leopoldo AP, Leopoldo AS. Differential Effects of High Sugar, High Lard or a Combination of Both on Nutritional, Hormonal and Cardiovascular Metabolic Profiles of Rodents. Nutrients. 2018 Aug 11;10(8). pii: E1071. doi: 10.3390/nu10081071.

2. Council, N.R. Nutrient Requirements of Swine; National Academies Press: Washington, DC, USA, 1998.

2- Can the authors calculate the caloric intake of the animals? Was the caloric intake similar between groups?

As requested by Reviewer, in order to analyze if dietary-induced obesity was associated with alterations in the nutritional behavior, the food consumption (FC) was measured daily and the calorie intake (CI) was calculated weekly by the average weekly FC × dietary energetic density.

This information was previously published by our group [1]. However, the results (data not shown in the current study) showed that rats from C group had an approximate15.2%, 22.8% and 27.3% more daily food consumption (g) than HF, HFHS and HS groups, respectively, but the daily caloric intake was higher in the HS in relation to C and HFHS groups (HS: 92 ±2.0 vs. C: 79.2± 2.6and HFHS: 77.3 ± 1.7 kcal/day, p<0.05). In addition, there was different in FC between rats fed the HF diet compared with the HS group, since rats fed the HS diet consumed significantly less food (HG: 16.2 ± 0.4 vs. HF: 18.9 ± 0.7; p<0.05). Furthermore, HF presented a 12.4% increase in caloric intake than HFHS (p <0.05). There was no difference in the caloric intake of C compared to HF and HFHS (p> 0.05).

1. Matias AM, Estevam WM, Coelho PM, Haese D, Kobi JBBS, Lima-Leopoldo AP, Leopoldo AS. Differential Effects of High Sugar, High Lard or a Combination of Both on Nutritional, Hormonal and Cardiovascular Metabolic Profiles of Rodents. Nutrients. 2018 Aug 11;10(8). pii: E1071. doi: 10.3390/nu10081071.

3- How was the HFHsu administered? Was solid food? Or was sucrose administered in drinking water? Please describe the procedures, as this is quite important to interpret the results of the present manuscript. Usually, HFHSu models consume more sugar than fat and resemble more an HSu model than HF model.

Dear Reviewer, we appreciate the commentary and as requested, the information about how was the HFHsu administered was mentioned below. This information was also previously published by our group [1].

Rats received diet (HFHS) fed a saturated high-fat diet and added simple sugar in the form of pellets. In addition, these rats received water supplemented with sugar (300 g/L) in alternate weeks”. In order to calculate the caloric intake of HS, the caloric value of the sugar diet (3.65 kcal/g) plus the caloric value of water intake with sugar (1.2 kcal/ml).

1. Matias AM, Estevam WM, Coelho PM, Haese D, Kobi JBBS, Lima-Leopoldo AP, Leopoldo AS. Differential Effects of High Sugar, High Lard or a Combination of Both on Nutritional, Hormonal and Cardiovascular Metabolic Profiles of Rodents. Nutrients. 2018 Aug 11;10(8). pii: E1071. doi: 10.3390/nu10081071.

4- Advanced glycation end-products (AGEs) can be endogenously formed as a consequence of a high dietary sugar intake and several lines of evidence suggest that AGEs are related to the development and progression of heart failure. I believe that the manuscript will truly benefit from the measurement of AGEs particularly because this might explain the differences between high fat and high sucrose diets.

Dear Reviewer, we appreciate the commentary and agree that the measurement of AGEs could be very important and enriched the work, but we don’t have any sample for this analysis. Really,

as literature, this analysis is an emerging topic in the pathogenesis of metabolic diseases. AGEs can be ingested with high temperature processed foods, but also endogenously formed as a consequence of a high dietary sugar intake. We really like the suggestion and the idea will definitely be applied. Nevertheless, when this work was done, we didn´t have the prospect of carrying out the advanced glycation end-products (AGEs) in these animals because the initial aim of the current study was to investigate this study aimed to investigate the cardiac remodeling process in an experimental model induced by different types of hypercaloric diets (high-fat (lard), high-sugar and the combination of both) and their effects on the development and progression of heart failure. This suggestion will be applied in future studies and the projects in progress in our laboratory.

5- Note, that the title and the conclusion of the manuscript depend totally on diet composition evaluated in the present study, please be more cautious on these conclusions. Dear Reviewer, we appreciate the commentary and agree that the title and the conclusion of the manuscript depend totally on diet composition evaluated in the present study, but we have the measure of diet composition as demonstrated in the answer 1. However, as requested we improved the conclusion but we still find the appropriate title based on our findings.

Title: Hypercaloric diet models do not develop heart failure, but the excess sucrose promotes contractility dysfunction

Conclusion: In summary, the experimental diets based on high amounts of sugar, lard or a combination of both did not promote cardiac remodeling with predisposition to heart failure under conditions of obesity or excess sucrose. Nevertheless, excess sucrose causes cardiomyocyte contractility dysfunction associated with alterations in the myocyte sensitivity to intracellular Ca2+.

Reviewer #3:

This manuscript reports that the experimental hypercaloric models did not promote cardiac remodeling and predisposition to heart failure under conditions of obesity or excess sucrose. Nevertheless, excess sucrose causes cardiomyocyte contractility dysfunction associated with alterations in intracellular Ca2+. This work may add knowledge in the literature. The work in general is interesting, but there are important points and suggestion that should to be answered.

1 – The authors needs to organize the reference in the Introduction section (line 70 and 73).

Dear Reviewer, we appreciate the commentary and we agree that the reference in the introduction section (line 70 and 73) were disorganized. As requested, this section was reorganized, and the reformulated section is described below:

Evidence from experimental studies indicates that high-fat feeding promotes cardiac contractile function damage [12, 13]. In our laboratory, myocardial dysfunction was demonstrated in basal conditions and physiological cardiac remodeling in obese rats [14-16]. In contrast, results on the effects of diets with high simple carbohydrate content (sucrose and/or fructose) are inconsistent. Sharma et al. [9] observed that hypertensive animals fed by fructose diet for eight weeks resulted in an increase in LV wall thickness and mortality, while Salie et al. [17]. (Introduction, page 3, line 70 and 73)

2 - The authors describe in the Introduction Section that "….study aimed to investigate the cardiac remodeling process in an experimental model induced by different types of hypercaloric diets (high-fat (lard), high-sugar and the combination of both) and their effects on the development and progression of heart failure." I suggest changing the term ‘heart failure” for “...and their effects in the cardiac function”, because the experimental model do not is validated and do not develop the pathophysiology described;

As suggested by Reviewer, we changed this part of the introduction ("….study aimed to investigate the cardiac remodeling process in an experimental model induced by different types of hypercaloric diets (high-fat (lard), high-sugar and the combination of both) and their effects on the development and progression of heart failure”) and the sentence was rewritten as described below:

Thus, this study aimed to investigate the cardiac remodeling process in an experimental model induced by different types of hypercaloric diets (high-fat (lard), high-sugar and the combination of both) and their effects in the cardiac function. (Introduction, page 4, line 81-84)

3 – In the Results section, the diets association (HFHS) did induced any changes (figure 1), by the way, the HFD induces significantly changes in the cardiac parameters, So I would like to understand if the HS can interfere in the effect of HFD? This effect should be described in the Discussion section.

Dear Reviewer, we appreciate the commentary and as requested, we added in the discussion section how the HS diet can interfere in the effect of HFD, since the association between HS and HF did not promote changes in the weight of the total heart and left ventricle, as well as in the tibia ratios. Recalling that HF induced only significant changes in cardiac parameters in relation to HS. The discussion about this information is described below and it was added in the text:

Specifically, the HS did not promote cardiac remodeling, however, it was able to prevent the elevation on heart and LV weights in the HFHS, interfering in the effect of HF diet. According to literature, high sucrose intake [68% total energy sucrose diet, 69% total mass sucrose diet, or 20% sucrose solution] induced hyperinsulinemia without alterations in plasma glucose level in rats and mice [1-3]. Interestingly, our HS rats have not demonstrated alterations in glucose and insulin levels (data not shown). Several mechanisms have been postulated, including the prohypertrophic effects of insulin, insulin growth factor-1, and insulin resistance [4]. Initially the circulating insulin levels are increased, directly stimulating cardiomyocyte growth [5] and indirectly via binding to the insulin growth factor-1 receptor [6]. Chess et al. [7], analyzing the effects of dietary extremes (high carbohydrate and fat intake) on the remodeling process and heart failure, reported that high sugar intake causes hyperglycemia in the bloodstream and cardiotoxic effects. Increased glycemia leads to elevated serum insulin levels, which in cardiac tissue, induces increased protein synthesis of cardiomyocytes and, consequently, left ventricular hypertrophy. Sharma et al. [8] propose that carbohydrate intake, in particular sugar, associated or not with pressure overload may cause LV hypertrophy, via insulin receptor stimulation and activation of Akt/mTOR, proteins involved in protein signaling pathways. Thus, our results from HS diet suggest that the sugar intake was not able to affect the process of cardiac remodeling, probably due to the absence of hyperinsulinemia and elevation of glucose levels, as well as it was not promote cardiotoxic effect, indicating a cardioprotective effect alone or when associated with the HF diet. (Discussion, pages 11 and 12)

The new references were added in the text.

1. Davidoff AJ, Mason MM, Davidson MB, Carmody MW, Hintz KK, Wold LE, Podolin DA, Ren J. Sucrose-induced cardiomyocyte dysfunction is both preventable and reversible with clinically relevant treatments. Am J Physiol Endocrinol Metab. 2004 May;286(5):E718-24.

2. Gonsolin D, Couturier K, Garait B, Rondel S, Novel-Chaté V, Peltier S, Faure P, Gachon P, Boirie Y, Keriel C, Favier R, Pepe S, Demaison L, Leverve X.High dietary sucrose triggers hyperinsulinemia, increases myocardial beta-oxidation, reduces glycolytic flux and delays post-ischemic contractile recovery. Mol Cell Biochem. 2007 Jan;295(1-2):217-28. Epub 2006 Aug 31.

3. Pang X, Zhao J, Zhang W, Zhuang X, Wang J, Xu R, Xu Z, Qu W. Antihypertensive effect of total flavones extracted from seed residues of Hippophae rhamnoides L. in sucrose-fed rats. J Ethnopharmacol. 2008 May 8;117(2):325-31.

4. Phillips RA, Krakoff LR, Dunaif A, Finegood DT, Gorlin R, Shimabukuro S. Relation among left ventricular mass, insulin resistance, and blood pressure in nonobese subjects. J Clin Endocrinol Metab. 1998;83:4284–4288.

5. Hill DJ, Milner RD. Insulin as a growth factor. Pediatr Res. 1985;19:879–886.

6. Straus DS. Growth-stimulatory actions of insulin in vitro and in vivo. Endocr Rev. 1984;5:356–369.

7. Chess DJ, Stanley WC. Role of diet and fuel overabundance in the development and progression of heart failure. Cardiovasc Res. 2008;79: 269-278.

8. Sharma N, Okere IC, Duda MK, Chess DJ, O'Shea KM, Stanley WC. Potential impact of carbohydrate and fat intake on pathological left ventricular hypertrophy. Cardiovasc Res. 2007;73: 257-268.

4 – The authors describe in the Discussion section that HS did cause morphological cardiac remodeling, but it is know the cardiotoxic effect of the high sugar. So I would like to understanding, did this diet works? Why we don not have the data of the metabolic profile? Moreover, I would like to suggest that the authors explain better, this absence of cardiotoxic effect in the HS.

Dear Reviewer, we appreciate the commentary and as requested and explained above, we added in the discussion section a possible explanation why the HS diet did not promote cardiotoxic effect. In addition, the metabolic profile (glucose and area under the curve for glucose; elevated in HF and HHFS groups) has been shown in our previous study [1], but the insulin levels (data not shown) were similar among the groups (C: 1.86±0.13; HS: 1.77±0.15, HF: 2.19±0.16 and HFHS: 2.39±0.23; p = 0.052).

1. Matias AM, Estevam WM, Coelho PM, Haese D, Kobi JBBS, Lima-Leopoldo AP, Leopoldo AS. Differential Effects of High Sugar, High Lard or a Combination of Both on Nutritional, Hormonal and Cardiovascular Metabolic Profiles of Rodents. Nutrients. 2018 Aug 11;10(8). pii: E1071. doi: 10.3390/nu10081071.

The discussion about this information is described below and it was added in the text:

Specifically, the HS did not promote cardiac remodeling, however, it was able to prevent the elevation on heart and LV weights in the HFHS, interfering in the effect of HF diet. According to literature, high sucrose intake [68% total energy sucrose diet, 69% total mass sucrose diet, or 20% sucrose solution] induced hyperinsulinemia without alterations in plasma glucose level in rats and mice [2-4]. Interestingly, our HS rats have not demonstrated alterations in glucose and insulin levels (data not shown). Several mechanisms have been postulated, including the prohypertrophic effects of insulin, insulin growth factor-1, and insulin resistance [5]. Initially the circulating insulin levels are increased, directly stimulating cardiomyocyte growth [6] and indirectly via binding to the insulin growth factor-1 receptor [7]. Chess et al. [8], analyzing the effects of dietary extremes (high carbohydrate and fat intake) on the remodeling process and heart failure, reported that high sugar intake causes hyperglycemia in the bloodstream and cardiotoxic effects. Increased glycemia leads to elevated serum insulin levels, which in cardiac tissue, induces increased protein synthesis of cardiomyocytes and, consequently, left ventricular hypertrophy. Sharma et al. [9] propose that carbohydrate intake, in particular sugar, associated or not with pressure overload may cause LV hypertrophy, via insulin receptor stimulation and activation of Akt/mTOR, proteins involved in protein signaling pathways. Thus, our results from HS diet suggest that the sugar intake was not able to affect the process of cardiac remodeling, probably due to the absence of hyperinsulinemia and elevation of glucose levels, as well as it was not promote cardiotoxic effect, indicating a cardioprotective effect alone or when associated with the HF diet. (Discussion, pages 11 and 12)

The new references were added in the text.

.

2. Davidoff AJ, Mason MM, Davidson MB, Carmody MW, Hintz KK, Wold LE, Podolin DA, Ren J. Sucrose-induced cardiomyocyte dysfunction is both preventable and reversible with clinically relevant treatments. Am J Physiol Endocrinol Metab. 2004 May;286(5):E718-24.

3. Gonsolin D, Couturier K, Garait B, Rondel S, Novel-Chaté V, Peltier S, Faure P, Gachon P, Boirie Y, Keriel C, Favier R, Pepe S, Demaison L, Leverve X.High dietary sucrose triggers hyperinsulinemia, increases myocardial beta-oxidation, reduces glycolytic flux and delays post-ischemic contractile recovery. Mol Cell Biochem. 2007 Jan;295(1-2):217-28. Epub 2006 Aug 31.

4. Pang X, Zhao J, Zhang W, Zhuang X, Wang J, Xu R, Xu Z, Qu W. Antihypertensive effect of total flavones extracted from seed residues of Hippophae rhamnoides L. in sucrose-fed rats. J Ethnopharmacol. 2008 May 8;117(2):325-31.

5. Phillips RA, Krakoff LR, Dunaif A, Finegood DT, Gorlin R, Shimabukuro S. Relation among left ventricular mass, insulin resistance, and blood pressure in nonobese subjects. J Clin Endocrinol Metab. 1998;83:4284–4288.

6. Hill DJ, Milner RD. Insulin as a growth factor. Pediatr Res. 1985;19:879–886.

7. Straus DS. Growth-stimulatory actions of insulin in vitro and in vivo. Endocr Rev. 1984;5:356–369.

8. Chess DJ, Stanley WC. Role of diet and fuel overabundance in the development and progression of heart failure. Cardiovasc Res. 2008;79: 269-278.

9. Sharma N, Okere IC, Duda MK, Chess DJ, O'Shea KM, Stanley WC. Potential impact of carbohydrate and fat intake on pathological left ventricular hypertrophy. Cardiovasc Res. 2007;73: 257-268.

5 – The author describe in the Discussion section a possible effect cardioprotetor in the initial stage of obesity, so I would like to suggest that this point be further explored and add in this section. Moreover, I suggest describing further details of the role of fatty acid reduction in heart failure.

Dear Reviewer, we appreciate the commentary and as requested, we added in the discussion section a possible effect cardioprotective by reduction of utilization of fatty acids in myocardium in the initial stage of obesity, as well as we described with further details the role of fatty acid reduction in heart failure. The discussion about this information is described below and it was added in the text:

Free fatty acid oxidation is the major source of energy for the myocardium and up to 80 % of high-energy phosphates are produced, while the glucose metabolism provides the remaining quantity of energy. Glucose as glycogen are stored to be used during increased metabolic demands such as obesity and diabetes, since the glucose utilization is 20–30 % more metabolic efficient than free fatty acid oxidation in producing high-energy phosphates. In this sense, an energetic dysregulation play an important role in the pathophysiology of the failing heart. According Doesn’t et al. [1] a possible cause for these metabolic derangements in HF could be related to myocardial insulin resistance, which limits the utilization of glucose and favors the increased utilization of free fatty acids for ketogenesis. These changes lead to a reduction in the production of high-energy phosphates and therefore to a metabolically inefficient heart. Therefore, the metabolic changes in HF may favor for the progression and reducing functional capacity. Nevertheless, our findings suggest that this supply of fatty acids seems to initially exert a cardioprotective effect against the aggressions imposed by the obesity condition and pressure surges in the hyperlipidic models, since the HF diet did not probably promote systemic and myocardial insulin resistance (data not evaluated). In addition, it should be noted that the occurrence of heart failure was not observed in the evaluated experimental models; literature highlights the reduced use of fatty acids in heart failure [1], maintaining normal functioning of the heart. (Discussion, page 13, lines 8-26)

1. Doenst T, Nguyen TD, Abel ED. Cardiac metabolism in heart failure. Circ Res. 2013;113: 709-724.

6 – In the Discussion section, the authors explore the myocardial metabolic adapt to the use of glucose as an energetic substrate, however did not measure any parameter for this suggestion. So it is necessary describe further this point.

In addition, I would like to understand the effect of leptin in this HS experimental model due the intimal association of calcium concentration.

Dear Reviewer, we appreciate the commentary and, really, we did not measure any parameter for evaluation of myocardial substrate as a mediator of subsequent contractile dysfunction in HS rats. As requested, the discussion about this was reformulated and it was added in the text:

In addition, Abel et al. [1] suggest that altered use of the myocardial substrate may be a mediator of subsequent contractile dysfunction. Within this context, when fatty acid levels are low and glucose concentrations are high, myocardial metabolism adapts to the use of glucose as an energetic substrate. This adaptive response is initially beneficial because it maintains adenosine triphosphate (ATP) levels in the face of decreased mitochondrial oxidative phosphorylation from fatty acids; however, this change in energy metabolism is not just a primary effect of cardiac remodeling, but it may, in fact, be a predictor of cardiac dysfunction [2]. (Discussion, page 14, lines 8-15)

1. Abel ED, Litwin SE, Sweeney G. Cardiac remodeling in obesity. Physiol Rev. 2008;88: 389-419.

2. Alfarano C, Foussal C, Lairez O, Calise D, Attané C, Anesia R, Daviaud D, Wanecq E, Parini A, Valet P, Kunduzova O. Transition from metabolic adaptation to maladaptation of the heart in obesity: role of apelin. Int J Obes (Lond). 2015 Feb;39(2):312-20.

Leptin has been considered a key factor in the regulationof cardiovascular function, since since endogenous leptin production acts as aphysiological regulator of cardiovascular function, whereas hyperleptinemia or leptin resistance serves as pathophysiological indicator of cardiovascular diseases [3]. Leptin may reduce insulin release and enhance insulin sensitivity, consistent with elevated insulin. However, long-term hyperleptinemia commonly seen in human obesity may trigger leptin resistance, suppress insulin sensitivity and ultimately induce insulin resistance [4]. Nevertheless, our HS rats did not present hyperleptinemia that may be explained by the similar levels of adipose tissue compared to C (previously published data - 5). These results suggest that leptin did not impair the insulin release and, consequently, the myocardial glucose utilization.

3. Hintz KK, Aberle NS, Ren J. Insulin resistance induces hyperleptinemia, cardiac contractile dysfunction but not cardiac leptin resistance in ventricular myocytes. Int J Obes Relat Metab Disord. 2003 Oct;27(10):1196-203.

4. Ren J. Leptin and hyperleptinemia - from friend to foe for cardiovascular function. J Endocrinol. 2004 Apr;181(1):1-10.

5. Matias AM, Estevam WM, Coelho PM, Haese D, Kobi JBBS, Lima-Leopoldo AP, Leopoldo AS. Differential Effects of High Sugar, High Lard or a Combination of Both on Nutritional, Hormonal and Cardiovascular Metabolic Profiles of Rodents. Nutrients. 2018 Aug 11;10(8). pii: E1071. doi: 10.3390/nu10081071.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

2 Jan 2020

PONE-D-19-28274R1

Hypercaloric diet models do not develop heart failure, but the excess sucrose promotes contractility dysfunction

PLOS ONE

Dear Mr Leopoldo,

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.

Although the authors have answered some of the questions raised by the reviewers,  one reviewer still has some concerns that should be addressed. The authors need to give point-by-point responses to the reviewer's comments thoroughly before the manuscript is considered for publication.

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.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Tohru Minamino, M.D., Ph.D.

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 #2: (No Response)

**********

2. 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 #2: Partly

**********

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

Reviewer #2: Yes

**********

4. 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 #2: Yes

**********

5. 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 #2: Yes

**********

6. 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 #2: The authors have reviewed the manuscript entitled “Hypercaloric diet models do not develop heart failure, but the excess sucrose promotes contractility dysfunction” that aimed to develop a hypercaloric animal model of cardiac remodeling and predisposition to heart failure. The authors answered to some of the questions raised, however they failed to include the requested data into the manuscript and to add results that will give information on the mechanisms that may explain the differences between high fat and high sucrose diets.

Please see below my comments:

- Although some of the information requested by this reviewer was already included in a previous publication as stated by the authors “Matias AM, Estevam WM, Coelho PM, Haese D, Kobi JBBS, Lima-Leopoldo AP, Leopoldo AS. Differential Effects of High Sugar, High Lard or a Combination of Both on Nutritional, Hormonal and Cardiovascular Metabolic Profiles of Rodents. Nutrients. 2018 Aug 11;10(8). pii: E1071. doi: 10.3390/nu10081071.” e.g. composition of the diets, caloric intake, diet administration, this must be included in the present manuscript to allow the readers a full understand of the manuscript . This information is crucial to the manuscript as it main focus is to evaluate the effect of different diets on heart failure and cardiac contractility.

- Please include data of metabolic profile of the different diets, mainly insulin and glycemia values, specially in the context of the discussion of the mechanisms promoting cardiac remodeling (lines 263- 282 page 12)

- Please include descriptions for figure legends. e.g – Figure 1 – effect of different diet composition on…

**********

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 #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

21 Jan 2020

The response to Reviewers was attached in the attach files and described below.

Manuscript Number: PONE-D-19-28274R1

Title: Hypercaloric diet models do not develop heart failure, but the excess sucrose promotes contractility dysfunction

Review Comments to the Author

Reviewer #2:

The authors have reviewed the manuscript entitled “Hypercaloric diet models do not develop heart failure, but the excess sucrose promotes contractility dysfunction” that aimed to develop a hypercaloric animal model of cardiac remodeling and predisposition to heart failure. The authors answered to some of the questions raised, however they failed to include the requested data into the manuscript and to add results that will give information on the mechanisms that may explain the differences between high fat and high sucrose diets.

Please see below my comments:

1 - Although some of the information requested by this reviewer was already included in a previous publication as stated by the authors “Matias AM, Estevam WM, Coelho PM, Haese D, Kobi JBBS, Lima-Leopoldo AP, Leopoldo AS. Differential Effects of High Sugar, High Lard or a Combination of Both on Nutritional, Hormonal and Cardiovascular Metabolic Profiles of Rodents. Nutrients. 2018 Aug 11;10(8). pii: E1071. doi: 10.3390/nu10081071.” e.g. composition of the diets, caloric intake, diet administration, this must be included in the present manuscript to allow the readers a full understand of the manuscript. This information is crucial to the manuscript as it main focus is to evaluate the effect of different diets on heart failure and cardiac contractility.

Dear Reviewer, we appreciate the comment and as requested the composition of the diets, caloric intake and diet administration were added in the text. In addition, the Table 1 was added in the current manuscript.

The added sentence about the composition of the diets was “The experimental diets provided sufficient amounts of protein, vitamins and minerals according to the Nutrient Requirements for Laboratory Animals. The diets used in the current study were formulated by Nutriave Alimentos® (Vitória, Espírito Santo, Brazil). The feed ingredients were blended, homogenized and extruded (Extru-Tech Extruder, Model E-750, Sabetha, KS, USA) in the form of pellets. Then, the pellets were dried on a horizontal conveyor dryer (20 minutes, temperature: ±70° C). The composition (g/kg) and nutrients for each experimental diet (%) are described in Table 1.” (Material and Methods, Animal care and Experimental design, page 5, line 3-9)

Table 1. Composition and nutritional values of diets

Components (g/kg) Diets

C HS HF HFHS

Corn 200 200 180 80

Rice 200 200 200 200

Bone meal 120 120 120 120

Sugar - 100 - 100

Soy oil 75 75 - -

Lard - - 200 200

Gluten 200 200 200 200

Salt 3.5 3.5 3.5 3.5

Mineral Mix** 35 35 30 30

Vitamin Mix** 16.5 16.5 16.5 16.5

Inert Material*** 150 50 50 50

Total (g) 1000 1000 1000 1000

Nutrient Composition (%)

Protein 24.8 21.8 17.8 19.2

Carbohydrate 49.6 52.3 44.6 43.4

Lipids 25.6 25.9 37.6 37.4

Energy Density (Kcal/g) 3.55 3.65 4.59 4.49

Diets. C: normal rodent chow; HS: High-sugar; HF: high-fat; HFHS: high fat and high sugar. *Rats received diet with simple carbohydrate and water supplemented with sugar (300 g/L) in alternate weeks. In order to calculate the caloric intake of HS, the caloric value of the sugar diet (3.65 kcal/g) plus the caloric value of water intake with sugar (1.2 kcal/ml).

As requested by Reviewer, in order to analyze if dietary-induced obesity was associated with alterations in the nutritional behavior, the food consumption (FC) was measured daily and the calorie intake (CI) was calculated weekly by the average weekly FC × dietary energetic density. These information and description of results were added in the Table 2 and results section. (Results, page 9, line 1-9)

The added sentence about the nutritional behavior was “The nutritional profile of rats is summarized in Table 2. The C rats had an approximately 17.9%, 29.6% and 37.6% greater daily food consumption (g) than the HF, HFHS and HS groups, respectively, but the daily caloric intake was higher in the HS group in relation to the C and HFHS groups (HS: 92.1 ± 2.1 vs. C: 79.2 ± 2.6 and HFHS: 77.3 ± 1.8 kcal/day, p < 0.05). In addition, there was a difference in FC between rats fed the HF diet compared with the HS group since rats fed the HS diet consumed significantly less food (HG: 16.2 ± 0.5 vs. HF:18.9 ± 0.7; p < 0.05). Furthermore, HF presented a 12.4% increase in caloric intake over HFHS (p < 0.05). There was no difference in the caloric intake of C compared to HF and HFHS (p > 0.05).”

Table 2. General Characteristics

Variables Experimental Groups

C HS HF HFHS

IBW (g) 107 ± 3 110 ± 3 111 ± 3 110 ± 4

FBW (g) 533 ± 17 538 ± 13# 649 ± 34* 616 ± 22

BW gain (g) 426 ± 17 428 ± 12# 538 ± 32* 506 ± 20

Epididymal fat pad (g) 11.2 ± 0.6 10.5 ± 0.7 13.1 ± 0.9 13.5 ± 1.0

Visceral fat pad (g) 11.4 ± 0.6 10.6 ± 0.6#& 18.5 ± 1.4* 15.9 ± 1.4*

Retroperitoneal fat pad (g) 21.9 ± 1.0 21.8 ± 2.0#& 40.4 ± 4.5* 34.4 ± 2.2*

Body fat (g) 44.5 ± 1.6 42.9 ± 0.6#& 72.0 ± 6.3* 63.8 ± 4.3*

Adiposity index (%) 8.3 ± 0.2 7.9 ± 0.3#& 10.9 ± 0.5* 10.2 ± 0.3*

Food consumption (g/day) 22.3 ± 0.7 16.2 ± 0.5 18.9 ± 0.7 17.2 ± 0.4

Caloric intake (kcal/day) 79.2 ± 2.6 92.1 ± 2.1 86.9 ± 3.5 77.3 ± 1.8

Glucose (mg/dL) 108 ± 2 112 ± 3 115 ± 4 115 ± 3

Insulin (ng/mL) 1.86 ± 0.13 1.77 ± 0.15 2.19 ± 0.16 2.39 ± 0.23

Data are presented as the mean ± SEM. Control diet - (C; n=12); high-sugar diet - (HS; n=14); high-fat diet - (HF; n=13), and high-fat and high-sugar diet (HFHS; n=13). IBW: initial body weight; FBW: final body weight; BW: body weight. One-way ANOVA for independent samples followed by Tukey post hoc test. p < 0.05 vs. * C; # HF vs. HS; & HFHS vs. HS.

Dear Reviewer, we appreciate the commentary and as requested, the information about how was the HFHsu administered was mentioned below and added in the text.

All animals had free access to water and chow (40 g/day). To analyze whether dietary-induced obesity was associated with alterations in nutritional behavior, food consumption (FC) was measured daily. Calorie intake (CI) was calculated weekly by the average weekly FC × dietary energetic density. The HS group had water supplemented with sugar (300 g/l) in alternate weeks. For the calculation of the caloric intake of the HS group, the caloric energy from the water supplemented with sugar was also quantified (1.2 kcal/mL consumed). (Material and Methods, Animal care and Experimental design, page 4, line 2-7)

2 - Please include data of metabolic profile of the different diets, mainly insulin and glycemia values, specially in the context of the discussion of the mechanisms promoting cardiac remodeling (lines 263- 282 page 12)

Dear Reviewer, we appreciate the commentary and as requested, we included data of metabolic profile (insulin and glucose) of the different diets. This information was added in the Table 2, results and discussion sections. The sentence added was described below.

The metabolic profile parameters including glucose (p=0.26) and insulin (p=0.06) were similar among the groups (Table 2). (Results section, page 9, lines 9-10)

Table 2. General Characteristics

Variables Experimental Groups

C HS HF HFHS

IBW (g) 107 ± 3 110 ± 3 111 ± 3 110 ± 4

FBW (g) 533 ± 17 538 ± 13# 649 ± 34* 616 ± 22

BW gain (g) 426 ± 17 428 ± 12# 538 ± 32* 506 ± 20

Epididymal fat pad (g) 11.2 ± 0.6 10.5 ± 0.7 13.1 ± 0.9 13.5 ± 1.0

Visceral fat pad (g) 11.4 ± 0.6 10.6 ± 0.6#& 18.5 ± 1.4* 15.9 ± 1.4*

Retroperitoneal fat pad (g) 21.9 ± 1.0 21.8 ± 2.0#& 40.4 ± 4.5* 34.4 ± 2.2*

Body fat (g) 44.5 ± 1.6 42.9 ± 0.6#& 72.0 ± 6.3* 63.8 ± 4.3*

Adiposity index (%) 8.3 ± 0.2 7.9 ± 0.3#& 10.9 ± 0.5* 10.2 ± 0.3*

Food consumption (g/day) 22.3 ± 0.7 16.2 ± 0.5 18.9 ± 0.7 17.2 ± 0.4

Caloric intake (kcal/day) 79.2 ± 2.6 92.1 ± 2.1 86.9 ± 3.5 77.3 ± 1.8

Glucose (mg/dL) 108 ± 2 112 ± 3 115 ± 4 115 ± 3

Insulin (ng/mL) 1.86 ± 0.13 1.77 ± 0.15 2.19 ± 0.16 2.39 ± 0.23

Data are presented as the mean ± SEM. Control diet - (C; n=12); high-sugar diet - (HS; n=14); high-fat diet - (HF; n=13), and high-fat and high-sugar diet (HFHS; n=13). IBW: initial body weight; FBW: final body weight; BW: body weight. One-way ANOVA for independent samples followed by Tukey post hoc test. p < 0.05 vs. * C; # HF vs. HS; & HFHS vs. HS.

Specifically, the HS did not promote cardiac remodeling, however, it was able to prevent the elevation on heart and LV weights in the HFHS, interfering in the effect of HF diet. According to literature, high sucrose intake [68% total energy sucrose diet, 69% total mass sucrose diet, or 20% sucrose solution] induced hyperinsulinemia without alterations in plasma glucose level in rats and mice [28-30]. Interestingly, our HS rats have not demonstrated alterations in glucose and insulin levels (Table 2). Several mechanisms have been postulated, including the prohypertrophic effects of insulin, insulin growth factor-1, and insulin resistance [31]. Initially the circulating insulin levels are increased, directly stimulating cardiomyocyte growth [32] and indirectly via binding to the insulin growth factor-1 receptor [33]. Chess et al. [26], analyzing the effects of dietary extremes (high carbohydrate and fat intake) on the remodeling process and heart failure, reported that high sugar intake causes hyperglycemia in the bloodstream and cardiotoxic effects. Increased glycemia leads to elevated serum insulin levels, which in cardiac tissue, induces increased protein synthesis of cardiomyocytes and, consequently, left ventricular hypertrophy. Sharma et al. [9] propose that carbohydrate intake, in particular sugar, associated or not with pressure overload may cause LV hypertrophy, via insulin receptor stimulation and activation of Akt/mTOR, proteins involved in protein signaling pathways. Thus, our results from HS diet suggest that the sugar intake was not able to affect the process of cardiac remodeling, probably due to the absence of hyperinsulinemia and elevation of glucose levels, as well as it was not promote cardiotoxic effect, indicating a cardioprotective effect alone or when associated with the HF diet. (Discussion section, page 13, line 12)

3 - Please include descriptions for figure legends. e.g – Figure 1 – effect of different diet composition on…

Dear Reviewer, we appreciate the commentary and as requested, we included the descriptions for all figures (Figs. 1-4).

The description of figures is described below, and it was added in the text:

Figure 1. Effect of different diet composition on cardiac remodeling. Data are shown as mean ± SEM. Control diet - (C; n=5); high-sugar diet - (HS; n=8); high-fat diet - (HF; n=5), and high-fat and high-sugar diet (HFHS; n=6). HW: heart weight; LW: left weight. p < 0.05 vs. # HF vs. HS. One-way analysis of variance (ANOVA) followed by the Tukey post hoc test.

Figure 2. Histological study in myocardium from control diet - (C; n=5); high-sugar diet - (HS; n=8); high-fat diet - (HF; n=5), and high-fat and high-sugar diet (HFHS; n=6) after 20 weeks. (A) cross sectional area (CSA). (B): interstitial collagen of myocardium; representative picrosirius red-stained left ventricle (LV) section. Arrows: interstitial collagen. Data are shown as mean ± SEM. One-way analysis of variance (ANOVA) followed by the Tukey post hoc test. p < 0.05 vs. * C; & HFHS vs. HS; §HFHS vs. HF.

Figure 3. Effect of different diet composition on parameters of heart failure. Data are shown as mean ± SEM. Control diet - (C; n=12); high-sugar diet - (HS; n=14); high-fat diet - (HF; n=13), and high-fat and high-sugar diet (HFHS; n=13). RV: right ventricle. (A) Lung weight/body weight ratio. (B) Right lung weight/body weight ratio. (C) RV weight/body weight. (D) Lung water content (C = 5; HS = 8; HF = 5, and HFHS = 6). One-way analysis of variance (ANOVA) followed by the Tukey post hoc test.

Figure 4. Effect of different diet composition on contractile function and calcium transients of left ventricular cardiomyocytes from control diet (C; n= 5; cells= 71), high-sugar diet (HS; n=6, cells= 115), high-fat diet (HF; n= 6, cells= 106) and high-fat and high-sugar diet (HFHS; n=5, cells= 81). Data are shown as mean ± SEM. (A) Representative contraction traces obtained from the cardiomyocytes of rats: (B) Cell shortening expressed as % of resting cell length. (C) Time to 50% of contraction. (D) Time to 50% of relaxation. (E) Amplitude of transients. (F) Time to peak. (G) Time to from peak transient to half resting value p < 0.05 vs. * C; # HS vs. HF; & HFHS vs. HS. One-way analysis of variance (ANOVA) followed by the Tukey post hoc test.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

23 Jan 2020

PONE-D-19-28274R2

Hypercaloric diet models do not develop heart failure, but the excess sucrose promotes contractility dysfunction

PLOS ONE

Dear Mr Leopoldo,

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.

Although the authors have addressed some of the questions raised by the reviewer, the reviewer still has two additional comments below. The authors need to give point-by-point responses to the reviewer's comments before the manuscript is considered for publication.

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.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Tohru Minamino, M.D., Ph.D.

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 #2: (No Response)

**********

2. 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 #2: Partly

**********

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

Reviewer #2: Yes

**********

4. 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 #2: No

**********

5. 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 #2: Yes

**********

6. 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 #2: The authors have reviewed the manuscript entitled “Hypercaloric diet models do not develop heart failure, but the excess sucrose promotes contractility dysfunction” that aimed to develop a hypercaloric animal model of cardiac remodeling and predisposition to heart failure. The authors have answered adequately to some of the questions raised however I have 2 additional comments:

1- I really don’t understand how the animals HFHS that have a decreased caloric intake in comparison with the controls gain weight and have an increased body fat and adiposity index compared with controls. (table 2)

2- Please include in methods section, the methods to analyze insulin and glucose.

**********

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 #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

24 Jan 2020

Manuscript Number: PONE-D-19-28274R2

Title: Hypercaloric diet models do not develop heart failure, but the excess sucrose promotes contractility dysfunction

Review Comments to the Author

Reviewer #2: The authors have reviewed the manuscript entitled “Hypercaloric diet models do not develop heart failure, but the excess sucrose promotes contractility dysfunction” that aimed to develop a hypercaloric animal model of cardiac remodeling and predisposition to heart failure. The authors have answered adequately to some of the questions raised however I have 2 additional comments:

1- I really don’t understand how the animals HFHS that have a decreased caloric intake in comparison with the controls gain weight and have an increased body fat and adiposity index compared with controls. (table 2)

Dear Reviewer, we appreciate the commentary and as requested, the explanation about how the animals HFHS have a decreased caloric intake, but higher body fat and adiposity index is related to feed efficiency, which it is the ability to transform consumed calories into body weight, was determined by following the formula: mean body weight gain (g)/total calorie intake (kcal). In this sense, the feed efficiency (%) was higher in the HF (14.3%) and HFHS groups (20.8%) than in C (data included in the Table 2) as demonstrated (HFHS: 4.64 ± 0.09 and HF: 4.39 ± 0.09 vs. C: 3.84 ± 0.08; p < 0.05). The elevation in body weight and adiposity in the hyperlipidic experimental models (HF) increased with sugar, refers to caloric intake and food efficiency which, generally, lead to a greater availability of calories and hyperphagia [1,2]. Even without an increase in caloric intake, it is possible to develop obesity, since the change in nutrient composition influences the efficiency of food utilization, thus leading to an increase in fat storage per calorie consumed. The elevation in body fat may be due to an increase in the caloric density of the diet, which may lead to a higher total caloric intake or an increase in the intake of a given macronutrient [3]. Although they had a similar total caloric intake, the HF and HFHS groups consumed 61% and 43% more calories from fat, respectively, than the C animals did. Therefore, the elevation of feed efficiency in HFHS could explain how the animals HFHS had an increased body fat and adiposity index compared with C rats, despite the absence of alterations in caloric intake among HFHS and HF in relation to C group (Table 2).

1. Dourmashkin J, Chang G, Gayles E, Hill J, Fried S, Julien C, Leibowitz S. Different forms of obesity as a function of diet composition. Int. J. Obes. 2005;29:1368–1378.

2. White S, Cercato LM, Araujo D, Souza LA, Soares AF, Barbosa APO, et al. Modelo de obesidade induzida por dieta hiperlipidica e associada à resistência à ação da insulina e intolerância a glicose. Arq Bras Endocrinol Metab 2013;57:339–345.

3. Pereira-Lancha LO, Campos-Ferraz PL, Lancha AH. Obesity: Considerations about etiology, metabolism, and the use of experimental models. Diabetes Metab Syndr Obes. 2012;5:75–87.

The added sentences and Table 2 were detailed below:

In order to analyze if dietary-induced obesity was associated with alterations in the nutritional behavior, the food consumption (FC) was measured daily and the calorie intake (CI) was calculated weekly by the average weekly FC × dietary energetic density, as well as the feed efficiency to show the higher body fat and adiposity index in HF and HFHS in comparison to C group (Table 2). These information and description of results were added in the Table 2 and results section. (Results, page 9, lines 1-11)

The added sentence about the nutritional behavior was “The nutritional profile of rats is summarized in Table 2. The C rats had an approximately 17.9%, 29.6% and 37.6% greater daily food consumption (g) than the HF, HFHS and HS groups, respectively, but the daily caloric intake was higher in the HS group in relation to the C and HFHS groups (HS: 92.1 ± 2.1 vs. C: 79.2 ± 2.6 and HFHS: 77.3 ± 1.8 kcal/day, p < 0.05). In addition, there was a difference in FC between rats fed the HF diet compared with the HS group since rats fed the HS diet consumed significantly less food (HG: 16.2 ± 0.5 vs. HF:18.9 ± 0.7; p < 0.05). Furthermore, HF presented a 12.4% increase in caloric intake over HFHS (p < 0.05). There was no difference in the caloric intake of C compared to HF and HFHS (p > 0.05). While the feed efficiency (%) was higher in the HF (14.3%) and HFHS groups (20.8%) than in C (Table 2), there was a lower feed efficiency in HS rats than C (HS: 3.32 ± 0.05 vs. C: 3.84 ± 0.08; p < 0.05).” (Results, pages 9 and 10, lines 1-11)

Table 2. General Characteristics

Variables Experimental Groups

C HS HF HFHS

IBW (g) 107 ± 3 110 ± 3 111 ± 3 110 ± 4

FBW (g) 533 ± 17 538 ± 13# 649 ± 34* 616 ± 22

BW gain (g) 426 ± 17 428 ± 12# 538 ± 32* 506 ± 20

Epididymal fat pad (g) 11.2 ± 0.6 10.5 ± 0.7 13.1 ± 0.9 13.5 ± 1.0

Visceral fat pad (g) 11.4 ± 0.6 10.6 ± 0.6#& 18.5 ± 1.4* 15.9 ± 1.4*

Retroperitoneal fat pad (g) 21.9 ± 1.0 21.8 ± 2.0#& 40.4 ± 4.5* 34.4 ± 2.2*

Body fat (g) 44.5 ± 1.6 42.9 ± 0.6#& 72.0 ± 6.3* 63.8 ± 4.3*

Adiposity index (%) 8.3 ± 0.2 7.9 ± 0.3#& 10.9 ± 0.5* 10.2 ± 0.3*

Food consumption (g/day) 22.3 ± 0.7 16.2 ± 0.5*# 18.9 ± 0.7* 17.2 ± 0.4*

Caloric intake (kcal/day) 79.2 ± 2.6 92.1 ± 2.1*& 86.9 ± 3.5 77.3 ± 1.8α

Feed efficiency (%) 3.84 ± 0.08 3.32 ± 0.05* 4.39 ± 0.09*# 4.64 ± 0.09*&

Glucose (mg/dL) 108 ± 2 112 ± 3 115 ± 4 115 ± 3

Insulin (ng/mL) 1.86 ± 0.13 1.77 ± 0.15 2.19 ± 0.16 2.39 ± 0.23

Data are presented as the mean ± SEM. Control diet - (C; n=12); high-sugar diet - (HS; n=14); high-fat diet - (HF; n=13), and high-fat and high-sugar diet (HFHS; n=13). IBW: initial body weight; FBW: final body weight; BW: body weight. One-way ANOVA for independent samples followed by Tukey post hoc test. p < 0.05 vs. * C; # HF vs. HS; & HFHS vs. HS, α HF vs. HFHS.

Dear Reviewer, the information about how was the analyze of feed efficiency is mentioned below and added in the text.

All animals had free access to water and chow (40 g/day). To analyze whether dietary-induced obesity was associated with alterations in nutritional behavior, food consumption (FC) was measured daily. Calorie intake (CI) was calculated weekly by the average weekly FC × dietary energetic density. Feed efficiency (FE), the ability to transform consumed calories into body weight, was determined by following the formula: mean body weight gain (g)/total calorie intake (kcal). The HS group had water supplemented with sugar (300 g/l) in alternate weeks. For the calculation of the caloric intake of the HS group, the caloric energy from the water supplemented with sugar was also quantified (1.2 kcal/mL consumed). (Material and Methods, Animal care and Experimental design, page 4, line 2-10)

2- Please include in methods section, the methods to analyze insulin and glucose.

Dear Reviewer, we appreciate the comment and as requested the methods to analyze insulin and glucose was added in the methods section.

The added sentence is described below:

Metabolic and Hormonal Measurements

After 20 weeks, the animals were subjected to 12–15 h of fasting, and blood samples were collected in dry tubes. The serum was separated by centrifugation at 10,000 rpm for 10 min. (Heraeus Megafuge 16R Centrifuge, Thermo Scientific, Massachusetts, USA) and stored at −80 °C for subsequent analysis (Coldlab Ultra Freezer CL374-86V, Piracicaba, São Paulo, Brazil). Serum glucose concentration was measured using specific kit (Bioclin Bioquímica®, Belo Horizonte, Minas Gerais, Brazil and Synermed do Brasil Ltda., São Paulo, Brazil) and analyzed by automated biochemical equipment BS-200 (Mindray do Brasil-Comércio and Distribuição de Equipamentos Médicos Ltda., São Paulo, Brazil). Insulin was determined using an enzyme-linked immunosorbent assay (ELISA) using specific kit (Linco Research Inc., St. Louis, MO, USA). The reading was carried out using a microplate reader (Asys Expert Plus Microplate Reader, Cambourne, Cambridge, UK). (Material and Methods, Metabolic and Hormonal Measurements, page 6, lines 1-11)

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

27 Jan 2020

Hypercaloric diet models do not develop heart failure, but the excess sucrose promotes contractility dysfunction

PONE-D-19-28274R3

Dear Dr. Leopoldo,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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.

With kind regards,

Tohru Minamino, M.D., Ph.D.

Academic Editor

PLOS ONE

31 Jan 2020

PONE-D-19-28274R3

Hypercaloric diet models do not develop heart failure, but the excess sucrose promotes contractility dysfunction

Dear Dr. Soares Leopoldo:

I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Tohru Minamino

Academic Editor

PLOS ONE