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PLoS One
PLoS One
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PLOS ONE
1932-6203
Public Library of Science San Francisco, CA USA

10.1371/journal.pone.0308833
PONE-D-24-15393
Research Article
Medicine and Health Sciences
Cardiology
Heart Failure
Biology and Life Sciences
Nutrition
Diet
Medicine and Health Sciences
Nutrition
Diet
Medicine and Health Sciences
Cardiology
Myocardial Infarction
Biology and Life Sciences
Cell Biology
Cellular Structures and Organelles
Endoplasmic Reticulum
Biology and Life Sciences
Cell Biology
Cell Processes
Secretory Pathway
Endoplasmic Reticulum
Biology and Life Sciences
Biochemistry
Neurochemistry
Neurochemicals
Nitric Oxide
Biology and Life Sciences
Neuroscience
Neurochemistry
Neurochemicals
Nitric Oxide
Biology and Life Sciences
Physiology
Physiological Parameters
Body Weight
Biology and Life Sciences
Cell Biology
Cell Processes
Cell Death
Apoptosis
Biology and life sciences
Biochemistry
Proteins
DNA-binding proteins
Transcription Factors
Biology and Life Sciences
Genetics
Gene Expression
Gene Regulation
Transcription Factors
Biology and Life Sciences
Biochemistry
Proteins
Regulatory Proteins
Transcription Factors
Post-myocardial infarction heart failure and long-term high-fat diet: Cardiac endoplasmic reticulum stress and unfolded protein response in Sprague Dawley rat model
Myocardial infarction and high-fat diet: Cardiac stress response
https://orcid.org/0000-0001-8659-2948
Momot Karol Conceptualization Formal analysis Investigation Visualization Writing – original draft 1
Krauz Kamil Investigation Visualization Writing – original draft 1
Czarzasta Katarzyna Writing – review & editing 1
Tomaszewski Jakub Conceptualization 1
Dobruch Jakub Investigation 2
https://orcid.org/0000-0003-3535-715X
Żera Tymoteusz Investigation 1
https://orcid.org/0000-0002-6782-3605
Zarębiński Maciej Funding acquisition 3
Cudnoch-Jędrzejewska Agnieszka Supervision 1
https://orcid.org/0000-0003-0995-1171
Wojciechowska Małgorzata Formal analysis Writing – review & editing 1 *
1 Laboratory of Centre for Preclinical Research, Department of Experimental and Clinical Physiology, Medical University of Warsaw, Warsaw, Poland
2 Centre of Postgraduate Medical Education, Department of Urology, Warsaw, Poland
3 Department of Invasive Cardiology, Independent Public Specialist Western Hospital John Paul II, Lazarski University, Grodzisk Mazowiecki, Poland
Aguila Marcia B. Editor
Universidade do Estado do Rio de Janeiro, BRAZIL
Competing Interests: The authors have declared that no competing interests exist.

* E-mail: malgorzata.wojciechowska2@wum.edu.pl
18 9 2024
2024
19 9 e030883316 4 2024
29 7 2024
© 2024 Momot et al
2024
Momot et al
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Background

Myocardial infarction (MI) significantly contributes to the global mortality rate, often leading to heart failure (HF) due to left ventricular remodeling. Key factors in the pathomechanism of HF include nitrosative/oxidative stress, inflammation, and endoplasmic reticulum (ER) stress. Furthermore, while a high-fat diet (HFD) is known to exacerbate post-MI cardiac remodeling, its impact on these critical factors in the context of HF is not as well understood.

Aims

This study aimed to assess the impact of post-MI HF and HFD on inflammation, nitro-oxidative stress, ER stress, and unfolded protein response (UPR).

Methods

The study was performed on fragments of the left ventricle harvested from 30 male adult Sprague Dawley rats, which were divided into four groups based on diet (normal-fat vs. high-fat) and surgical procedure (sham operation vs. coronary artery ligation to induce MI). We assessed body weight, NT-proBNP levels, protein levels related to nitrosative/oxidative stress, ER stress, UPR, apoptosis, and nitric oxide synthases, through Western Blot and ELISA.

Results

HFD and MI significantly influenced body weight and NT-proBNP concentrations. HFD elevated 3-nitrotyrosine and myeloperoxidase levels and altered nitric oxide synthase levels. HFD and MI significantly affected ER stress markers and activated or inhibited UPR pathways.

Conclusions

The study demonstrates significant impacts of post-MI HF and dietary fat content on cardiac function and stress markers in a rat model. The interaction between HFD and MI on UPR activation suggests the importance of dietary management in post-MI recovery and HF prevention.

http://dx.doi.org/10.13039/501100004166 Warszawski Uniwersytet Medyczny 1MA/2/M/MG/N/23 Krauz Kamil This study was financed by a research grant from the Medical University of Warsaw (1MA/2/M/MG/N/23) to [KK] https://pnitt.wum.edu.pl/en The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data AvailabilityAll relevant data are within the manuscript.
Data Availability

All relevant data are within the manuscript.
==== Body
pmcIntroduction

Myocardial infarction (MI) is a significant cause of death worldwide [1]. Due to modern treatment options, the mortality from MI has been decreasing, resulting in a growing population of MI survivors [2]. Many of these individuals subsequently develop symptoms of heart failure (HF) [3, 4]. After the cardiomyocytes’ death due to ischemia, the development of HF is related to unfavorable left ventricular remodeling, leading to loss of function [5, 6]. Consumption of a high-fat diet (HFD) can intensify the remodeling after MI through mechanisms such ascardiac hypertrophy, cardiomyocyte apoptosis and interstitial fibrosis [7, 8].

Experimental studies have shown that HFD significantly exacerbates hypertensive heart disease in aging rats, leading to worsened atrial and ventricular remodeling and associated impairment of left ventricular systolic function [9]. Moreover, just 12 weeks of HFD can adversely affect cardiac function, as measured by the left ventricular speckle tracking imaging [10], a parameter capable of detecting subclinical left ventricular. Unfortunately, recent clinical studies have revealed that the consumption of high-fat products in the human population has been steadily increasing [11]. In the context of HF, there’s much discussion about nitrosative/oxidative stress, inflammation, and ER stress [12–15]. However, little is known about the impact of HFD on these processes in HF.

Nitrosative/oxidative stress refers to the biochemical reaction between nitric oxide (NO) and reactive oxygen species when a disorder in oxygen metabolism is present. This process results in the generation of reactive nitrogen species (such as the peroxynitrite anion), which cause nitration and damage to proteins [16]. A marker of such a damage is the 3-nitrotyrosine (3-NT) [17].

Production of NO is catalyzed by NO synthase (NOS), which has three isoforms: inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) [18]. These isoforms play crucial roles in cardiovascular health and disease. iNOS is expressed in normal heart tissue at very low levels [19]. Inflammation results in iNOS activation and overexpression, which is linked with harmful effects on the heart, whereas overexpression of nNOS and eNOS in transgenic animals improves cardiac functions following MI [20]. Myeloperoxidase (MPO) plays a vital role in the inflammatory response [21]. It is expressed mainly in neutrophils and monocytes. MPO catalyzes the production of hypochlorous acid, a potent oxidative agent [22]. Moreover, this protein can also directly contribute to forming reactive nitrogen species. Enhanced circulating levels of MPO are related to inflammation and oxidative stress [23]. What is more, a recent meta-analysis suggests that MPO can be a valuable marker for HF diagnosis [24].

ER stress occurs when misfolded or unfolded proteins overwhelm the ER, a crucial cell organelle for protein folding and lipid biosynthesis. As previously mentioned, nitrosative/oxidative stress affects the protein folding process and contributes to ER stress [25, 26]. The latter activates the unfolded protein response (UPR), a complex signaling network aiming to restore proteostasis or promote apoptosis when it is not possible. This process is crucial in the pathogenesis of HF [13]. Especially, UPR, ER stress, and iNOS overexpression have been found to influence heart failure with preserved ejection fraction (HFpEF) pathogenesis [12].

In physiological conditions, when ER stress is not exacerbated, 78-kDa glucose-regulated protein (GRP78) is attached to the ER stress sensors—inositol-requiring enzyme type 1 α (IRE1α), activating transcription factor 6 (ATF6), protein kinase R-like endoplasmic reticulum kinase (PERK) and, thus they remain inactive. When unfolded proteins excessively accumulate in the ER and ER stress exacerbates, GRP78 dissociates from these sensors, which activates them and initiates downstream signaling pathways. When ATF6 is released from GRP78, it migrates to the Golgi apparatus, where it is cleaved. Then, the cleaved ATF6 (ATF6c) is translocated to the nucleus and functions as an active transcription factor [27]. If severe ER stress persists, apoptotic pathways are activated. It has been reported that overexpression of CHOP leads to apoptosis due to ER stress [28].

This study aimed to assess the impact of post-MI HF and HFD on inflammation, nitro-oxidative stress, ER stress, and UPR.

Methods

Experimental procedures

The study was performed on fragments of the left ventricle harvested from 30 male adult Sprague Dawley rats, which underwent the following procedures (Fig 1, Table 1). This study is a continuation of previous experiments [29–31]. All experiments were approved by the Second Local Animal Research Ethics Committee of the Medical University of Warsaw and followed the European Communities Council Directive 2010/63/E.U. Rules of September 2010.

10.1371/journal.pone.0308833.g001 Fig 1 Graphical representation of the study design.

Abbreviations: HFD, high-fat diet; MI, myocardial infarction; NFD, normal-fat diet; SO, sham surgery.

10.1371/journal.pone.0308833.t001 Table 1 The experimental rat groups.

	Group 1
N = 7	Group 2
N = 9	Group 3
N = 6	Group 4
N = 8	
Diet	Normal-fat Diet (NFD)	Normal-fat Diet (NFD)	High-fat Diet (HFD)	High-fat Diet (HFD)	
Procedure	Sham operation (SO)	Ligation of the coronary artery (MI)	Sham operation (SO)	Ligation of the coronary artery (MI)	
Survival rates at the end of the study	70% (7 of 10)	60% (9 of 15)	60% (6 of 10)	50% (8 of 16)	

Starting from the fourth week of age, the animals were fed with HFD (31% fat, 17.1% protein, 35.5% carbohydrates, 0.18% sodium, and 3842 kcal/kg; Labofeed B, Kcynia, Poland) or a normal fat diet (NFD) (3.6% fat, 17.4% protein, 60% carbohydrates, 0.2% sodium, and 2864 kcal/kg; Labofeed B, Kcynia, Poland) for twelve weeks until the sixteenth week of age. The detailed composition of NFD and HFD is provided in Table 2. At the twelfth week of age, the surgical procedures were performed. During the surgeries, animals were under general anesthesia (Ketamine 10mg/100g body weight i.p., Xylazine 1mg/100g body weight i.p.). The MI model consists of a permanent ligation of the left coronary artery (LCA) with a suture thread (Ethicon 6.0). The sham surgeries (SO) were similar, but the pericardium was only touched with a needle, and the LCA was not ligated. Following surgery, the animals were administered analgesic medication (Buprenorphine chloride 3μg/100g body weight i.p.; 5.95nmol/ml, twice daily for 2–3 days) and an antibiotic (Penicillin, 10,000 IU/100g body weight i.m.; 0.047mmol/ml).

10.1371/journal.pone.0308833.t002 Table 2 Composition of diets.

	Normal-fat Diet	High-fat Diet	
Energy (kcal per 100g)	286	382	
Carbohydrates (g per 100g)	60.0	35.5	
Proteins (g per 100g)	17.4	17.1	
Fats (g per 100g)	3.2	28.0	
Saturated (%)	13	49	
Unsaturated (%)	37	44	
Polyunsaturated (%)	50	7	
Raw ash (g per 100g)	12	12	
Water (g per 100g)	12	12	

Four weeks following surgery were given to develop the post-MI HF in rats with the LCA ligation. After this period, the rats were anesthetized again. A 4 ml blood sample was taken from the right ventricle. The collected blood was immediately transferred into tubes containing Ethylenediaminetetraacetic acid (EDTA). The tubes were then centrifuged to separate the plasma from other blood components, and then levels of N-terminal prohormone of brain natriuretic peptide (NT-proBNP) were assessed using an enzyme-linked immunosorbent assay (ELISA).

At the end of the experiments, the rats were euthanized with an intraperitoneal injection of a lethal dose of Ketamine (300mg/100g body weight i.p.). The heart muscle tissue of the left ventricle was isolated, frozen in liquid nitrogen, and then stored at −80°C.

Protein analysis

Collected tissues were homogenized using the RIPA buffer, whose composition was previously described [29]. Samples containing 10μg/μl of total protein were resolved by 8% SDS-polyacrylamide gels. Isolated proteins were transferred into PVDF membranes (#1704274, Bio-Rad) or nitrocellulose membranes using the Trans-Blot® TurboTM Transfer System (Bio-Rad). Then, blots were blocked with 5% nonfat dry milk buffered solution, immunoblotted for one hour with anti-IRE1α (NB100-2324, Novus-Biologicals), anti-p-IRE1α (NB100-2323), anti-iNOS (NB300-605), anti-eNOS (NB300-500), anti-nNOS (NBP1-39681), anti-GRP78 (NBP1-06274), anti-ATF6 (NBP1-76675), anti-PERK (NBP3-12891), anti-CHOP (NBP2-13172), anti-3NT (sc-32757, Santa Cruz Biotechnology), and anti-MPO (MPO-101AP, Thermo Fisher Scientific) antibodies. As a loading control, an anti-beta actin antibody was used (ab8226, Abcam). Incubation with secondary antibodies conjugated to horseradish peroxidase (ab205718, Abcam or sc-516102, Santa Cruz) was performed for one hour. The specific bands were detected and quantified with the ChemiDoc MP Imaginating System (Bio-Rad), and then protein expressions were normalized with β-actin and expressed as a relative ratio. Each measurement was repeated three times, and the final result was calculated as the average of these repetitions. The raw data images are provided as Supporting Information (S1 Raw images).

Statistical analysis

Statistical analysis was carried out with the Statistica software, version 13.3. Normal distribution was tested using the Shapiro-Wilk test. The Levene test was used to assess the equality of variances. A two-way ANOVA was conducted to examine the effects of diet (Normal-fat Diet vs. High-fat Diet) and procedure (Sham operation vs. Myocardial infarction), as well as their interaction. Post-hoc comparisons were performed using Tukey’s HSD test to identify significant differences between groups. For all the variables measured, outliers that were 1.5 interquartile ranges (IQRs) below the first quartile or 1.5 IQRs above the third quartile were removed from the analysis. Pearson’s correlation coefficient was calculated to assess linear relationships between variables. Box plots were created using BioRender.com, and scatter plots with regression lines illustrating the relationships between variables were generated using an AI-based tool. All values presented in the text and figures are expressed as mean ± standard deviation (SD). All differences were considered significant if P < 0.05.

Results

Basic parameters

At the end of the experiment, significant differences in body weight were observed among the groups (Fig 2A). Notably, the group with post-MI HF, on an NFD, had the lowest body mass compared to other groups. A simple analysis of the main effects indicated that post-MI HF and diet significantly influenced the final body weight (P = 0.028 and P = 0.031, respectively).

10.1371/journal.pone.0308833.g002 Fig 2 Basic parameters at the end of experiments.

Abbreviations: HF, heart failure; HFD, high-fat diet; NFD, normal-fat diet; SO, sham surgery.

NT-proBNP circulating levels differed significantly between groups (Fig 2B). Notably, higher levels of NT-proBNP were observed in groups with post-MI HF–NFD-MI and HFD-MI. A simple main effects analysis revealed that post-MI HF alone had a significant effect on serum NT-proBNP levels (P < 0.0001).

Nitrosative/oxidative stress and inflammation

The levels of 3-NT in the left ventricle tissue showed significant differences between the rat groups (Fig 3A). The group with post-MI HF and an implemented HFD exhibited the highest 3-NT levels. A simple main effects analysis showed that post-MI HF and HFD had a significant effect on 3-nitrotyrosine levels (P = 0.021 and P = 0.048, respectively).

10.1371/journal.pone.0308833.g003 Fig 3 Bar graphs and representative Western blot images.

3-nitrotyrosine (A) and myeloperoxidase (B). Abbreviations: 3-NT, 3-nitrotyrosine; HF, heart failure; HFD, high-fat diet; MPO, myeloperoxidase; NFD, normal-fat diet; SO, sham surgery.

The levels of MPO in the left ventricular tissue showed significant differences among the groups (Fig 3B). The group that developed HF after MI and was subjected to HFD exhibited the highest levels of MPO. A two-way ANOVA indicated that post-MI HF and diet significantly impacted MPO levels in the left ventricular tissue (P = 0.004 and P = 0.028, respectively).

Nitric oxide synthases levels

Levels of nNOS in the left ventricular tissue significantly differed between groups (Fig 4A). Simple main effects analysis showed that post-MI HF did have a significant effect on nNOS levels (P < 0.001). It was associated with reduced levels of eNOS independently of the type of diet implemented.

10.1371/journal.pone.0308833.g004 Fig 4 Bar graphs and representative Western blot images.

nNOS (A), eNOS (B) and iNOS (C). Abbreviations: 3-NT, 3-nitrotyrosine; HF, heart failure; HFD, high-fat diet; MPO, myeloperoxidase; NFD, normal-fat diet; SO, sham surgery; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; nNOS, neuronal nitric oxide synthase.

There was a significant difference in eNOS levels within the left ventricular tissue between groups (Fig 4B). eNOS levels were significantly reduced in other groups compared to the control group–NFD-SO. A two-way ANOVA revealed a significant interaction between the effects of post-MI HF and diet (F(1,25) = 5.995, P = 0.022).

The levels of iNOS in the left ventricle tissue showed significant differences between the groups (Fig 4C). The rats with post-MI HF and an implemented HFD exhibited the highest iNOS levels, significantly higher than other groups. Simple main effects analysis showed that both post-MI HF and diet did have a significant effect on iNOS levels (P < 0.001 and P = 0.010, respectively).

Endoplasmic reticulum stress, unfolded protein response, and apoptosis

There was a significant difference in GRP78 protein levels within left ventricular tissue among the various groups (Fig 5A). GRP78 levels were significantly higher in rats subjected to HFD than those on NFD across all combinations. Furthermore, a simple main effects analysis revealed that only diet statistically impacted GRP78 levels (P < 0.0001).

10.1371/journal.pone.0308833.g005 Fig 5 Bar graphs and representative Western blot images.

GRP78 (A), p-IRE1α/IRE1α (B), ATF6c/ATF6 (C), PERK (D) and CHOP (E). Abbreviations: ATF6, activating transcription factor 6; ATF6c, activating transcription factor 6 (cleaved form); CHOP, C/EBP homologous protein; GRP78, 78-kDa glucose-regulated protein; HF, heart failure; HFD, high-fat diet; IRE1α, inositol-requiring enzyme type 1 α; NFD, normal-fat diet; PERK, protein kinase R-like endoplasmic reticulum kinase; SO, sham surgery; p-IRE1α, phosphorylated inositol-requiring enzyme type 1 α.

The activity of the IRE1α axis of the UPR, measured by the ratio of phosphorylated IRE1α to non-phosphorylated IRE1α, showed significant variation between groups (Fig 5B). The lowest activity was observed in rats subjected to HFD and after MI. A simple main effects analysis revealed that only diet affected the p-IRE1α/IRE1α ratio (P = 0.026). A two-way ANOVA indicated that the interaction between the effects of post-MI HF and diet on the activity of the IRE1α axis was significant (F(1, 22) = 7.401, P = 0.012).

The activity of the ATF6 axis of the UPR, measured by the ratio of cleaved ATF6 to non-cleaved ATF6, showed significant differences between groups (Fig 5C). The highest activity was observed in rats subjected to HFD after MI. This activity level was significantly higher than in other groups. Both post-MI HF and diet affected the activity of the ATF6 axis (P < 0.0001 and P < 0.0001). Furthermore, a two-way ANOVA revealed a significant interaction between the effects of post-MI HF and diet on the activity of the ATF6 axis (F(1, 24) = 8.707, P = 0.007).

PERK levels in the left ventricular tissue significantly differed between groups. The rats subjected to an NFD without post-MI HF exhibited the highest levels of PERK (Fig 5D). This difference was significant compared to other groups. Other groups showed similar levels of PERK, with no significant differences. A two-way ANOVA indicated a significant interaction between the effects of post-MI HF and diet on PERK levels (F(1, 25) = 7.988, P = 0.009).

CHOP levels in left ventricular tissue varied significantly between groups (Fig 5E). The highest level of CHOP was observed in the control group (NFD-SO). This difference was significant compared to other groups. Additionally, a two-way ANOVA demonstrated that post-MI HF and diet significantly affected CHOP levels in heart left ventricular tissue (P < 0.0001 and P < 0.001, respectively).

As depicted in Fig 6, the levels of 3-nitrotyrosine correlated with GRP78 levels (ρ = 0.571, P = 0.002).

10.1371/journal.pone.0308833.g006 Fig 6 Pearson linear correlation between 3-NT levels GRP78 relative expression.

Abbreviations: 3-NT, 3-nitrotyrosine; GRP78, 78-kDa glucose-regulated protein.

Discussion

This preliminary study sheds light on the pathogenesis of post-MI HF and its exacerbation by HFD. We have demonstrated that in our model of post-MI HF with an implemented HFD, we can observe disturbances similar to those seen in HFpEF—specifically ER stress, nitrosative/oxidative stress, and disturbances in the UPR. Given that HFpEF is typically not associated with MI, this finding offers a new perspective on the mechanisms underlying HF development and progression.

Our study found that HFD was associated with increased ER stress measured by GRP78 levels. Zhang et al. also revealed that GRP78 was highly expressed in the atrial myocardium of HFD mice [32]. Additionally, they demonstrated that GRP78 expression in the atrial myocardium of overweight patients was significantly higher. These data suggest that ER stress could be activated in the myocardium not only in the case of obesity but also in HFD. Interestingly, in our study, post-MI HF had no significant impact on GRP78 levels. Contrary to our findings, Mainali et al. demonstrated that after inducing MI, GRP78 was markedly upregulated in heart tissue [33]. In the study by Mainali et al., GRP78 levels were measured one week after inducing MI, which could explain why our results differed. Additionally, exercise appears to reduce GRP78 expression, suggesting that the cardioprotective effect of exercise may be mediated through the reduction of ER stress, which is involved in the intrinsic apoptosis pathway [5, 34].

Furthermore, we discovered that the intensity of nitrosative/oxidative stress, measured by 3-NT, positively correlates with increased expression of GRP78. This indicates that these two components participate in the development of post-MI heart failure (HF).The study by Dickhout et al. observed that 3-NT could colocalize with GRP78 within early atherosclerotic lesions in the walls of arteries, suggesting that nitrosative/oxidative stress and ER stress also contribute to other diseases like coronary artery disease (CAD) [35].

Our findings indicate that iNOS protein levels are increased in cardiac tissue from rats with post-MI HF and with the implementation of HFD, suggesting the occurrence of nitrosative/oxidative stress under both conditions. This observation aligns with other studies demonstrating that the iNOS expression is up-regulated in association with obesity and after MI [19, 35, 36]. Furthermore, iNOS is found to be overexpressed in cardiac tissue in cases of HFpEF [37], which has recently been recognized as a foundation of this disease’s pathomechanism [12].

Our study revealed that both nNOS and eNOS levels decrease in post-MI HF in the case of a high-fat and normal-fat diet. Furthermore, eNOS protein levels also decrease after implementing HFD, regardless of MI. Currently, there have been no studies analyzing the impact of HFD on eNOS expression in cardiac tissue. Interestingly, similar to HFD, a high-sugar diet also leads to a reduction in eNOS levels [38]. Moreover, in a model of spontaneously hypertensive rats, eNOS was downregulated explicitly in cardiomyocytes [39]. Similarly, eNOS levels are decreased in hypertrophic cardiomyopathy [40]. However, diabetes does not affect the overall eNOS protein level [41].

We report that both HFD and post-MI HF lead to the elevation of MPO levels. Moreover, studies indicate that even a single high-fat meal can elevate circulating MPO levels and contribute to oxidative stress [42], which could be detrimental if repeated frequently in the context of HF pathogenesis. Elevated MPO levels are present in CAD; also, its high levels may indicate a high risk of acute coronary syndrome (ACS) [43]. Increased level of MPO is related to oxidative stress and inflammatory state in chronic systolic HF. Elevated plasma MPO levels are also associated with an increased likelihood of more advanced HF [18, 44].

We demonstrated that both HFD and MI, as well as the combination of these two factors, significantly increase the activity of the ATF6 axis of the UPR. Interestingly, studies show that ATF6 is critical in protecting the heart after MI. Inhibiting ATF6 activity leads to dilatation of the left ventricle and depression of cardiac function [43]. ATF6 may also regulate the dynamics of CHOP induction [45]. In our study, the levels of CHOP were highest in the control group and both post-MI HF and HFD were associated with the decrease in its levels, which may suggest inhibition of apoptosis related to the ER stress. However, in other studies—HFD or cardiac injury are rather linked with elevated levels of CHOP [46, 47]. Thus, further research is needed to explore this phenomenon.

In our study, we demonstrated that the activity of the IRE1α branch of the UPR was lowest in rats subjected to both HFD and HF simultaneously. This suggests that in the event of an MI, HFD may exacerbate the accumulation of misfolded proteins in cardiomyocytes. Reduced activity of this branch plays a role in the pathogenesis of HFpEF [12]. Until now, no one has demonstrated that a similar reduction in activity can occur in cases where post-MI HF coincides with HFD.

Limitations

This study primarily relied on Western Blot assays without incorporating PCR assays, a decision driven by the limited budget and the exploratory nature of this project. Additionally, the lack of histopathological evaluation and immunohistochemical verification of the distribution of the studied markers in the left ventricle was significant limitations. The absence of echocardiographic assessment, in particular, was a major issue, as it restricted our ability to evaluate cardiac function. Given the limitations mentioned above, the findings must be interpreted with caution.

Conclusions

This preliminary study has several limitations, as mentioned above, but provides valuable insights. The findings elucidate the significant impacts of post-MI HF and dietary fat content on cardiac function and stress markers in a rat model. HFD, independently and in combination with post-MI HF, significantly influenced ER stress (as indicated by GRP78 levels), nitrosative/oxidative stress markers (3-NT, iNOS), and other forms of NOS in the heart. Notably, post-MI HF with HFD resulted in the most pronounced alterations in these markers, suggesting exacerbated cardiac dysfunction and stress responses.

The study highlights the interaction between post-MI HF and dietary factors in modulating the UPR pathways (PERK, ATF6, IRE1α) and MPO levels. What is more, HFpEF and the overlap of post-MI HF and HFD may have similarities in pathogenesis. This is a new perspective for future studies.

Supporting information

S1 Raw images (PDF)

10.1371/journal.pone.0308833.r001
Decision Letter 0
Aguila Marcia B. Academic Editor
© 2024 Marcia B. Aguila
2024
Marcia B. Aguila
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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PONE-D-24-15393Post-myocardial infarction heart failure and long-term high-fat diet: cardiac endoplasmic reticulum stress and unfolded protein response in Sprague Dawley rat modelPLOS ONE

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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: Yes

**********

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

Reviewer #1: No

Reviewer #2: 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: No

Reviewer #2: No

**********

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

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

Reviewer #1: No

Reviewer #2: 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: Review PONE-D-24-15393

The study investigated the effect of post-myocardial infarction (MI), heart failure (HF), and high-fat diet HFD on inflammation, nitro-oxidative stress, endoplasmic reticulum (ER) stress, and unfolded protein response (UPR). Twenty-eight male adult Sprague Dawley rats were divided into four groups (NFD vs. HFD) and surgical procedures (sham operation vs. coronary artery ligation to induce MI). The authors concluded that dietary fat content significantly impacts cardiac function and stress markers in a rat model of MI HF. However, several methodological weaknesses and speculative conclusions must be resolved to make the study more scientifically acceptable.

• Adherence to strict dietary standards is a cornerstone of rodent research, ensuring the reproducibility and reliability of results. The dietary pattern of rodents, as outlined in AIN93 (J Nutr 1993;123:1939-51 doi:10.1093/jn/123.11.1939), is a crucial aspect that is unfortunately not detailed in the manuscript. Different types of fat can have varying effects on animal metabolism, and the protein content of the food is also vital for post-surgical recovery. Therefore, I kindly request a table providing a comprehensive breakdown of the ingredients in the control and high-fat diets used in the study.

• What was the difference in energy between the control and HF diets?

• The names of groups should be more explicit and not confusing.

• Page 5 - Fig 1 is mentioned, but we see a table. In line 103, three references are cited to explain the experimental procedures (refs. #24-26). However, these references are a meta-analysis and two reviews that do not provide experimental details.

• Coronary artery ligation in rodents is not always lethal, but many animals die. The manuscript does not inform how many animals were operated on or the survival rate.

• When ligating the coronary artery to cause a myocardial infarction in rodents, the method produces individual lesions; some animals may have an extensive infarction area, but others may not. Evans blue and triphenyl tetrazolium chloride staining can determine infarct areas at sacrifice (a missing item that compromises the study).

• Did the operated animals eat normally? How was the animals' food intake? The information is relevant to understanding the result of body mass loss reported in the article.

• Line 139 –reference #34 used to describe the protein analysis method is a meta-analysis, so…

• I did not find an explanation for what proBNP means (which appears a few times in the text).

• Lines 206-2010 - Nitric oxide (NO) is a gas that degrades quickly. The study did not evaluate NO production and cannot discuss NO levels.

• Comparisons between groups (and markings on graphs) - there are comparisons between groups that are not comparable (e.g., unoperated ND vs. operated HFD) and should not be stated or commented on.

• The statistical analysis should be more explicit and inform where the parametric or non-parametric analysis was performed. Why was the Spearman coefficient not determined for non-parametric correlations?

• I do not understand how the authors talk about 'heart failure' (HF) if the study does not include measuring the animals' blood pressure or a cardiac functional test (ultrasound?). Analysis of left ventricular ejection fraction is essential to understand the effect of post-infarction HFD.

• There are numerous grammatical and spelling errors in the text.

Reviewer #2: This manuscript describes an exploratory study examining the influence of high fat diet on cardiac nitrosative stress and endoplasmic reticulum stress in rat model with post-myocardial infarction heart failure. Overall, the manuscript is well written. Although the novelty of this experiment is limited by the great depth of literature available using other rodent models, this manuscript reports a technically-sound experiment describing the nitrosative and endoplasmic reticulum stress markers in rats. Nonetheless, the manuscript lacks any functional metrics that could support and verify the development of heart failure in this model. The choice of single time-point also limits the information provided by this characterization study whereby it is unclear when does nitrosative stress and endoplasmic reticulum stress occurs with diet and MI-induced heart failure.

Minor comments:

Individual data points should be presented in bar graphs.

**********

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Reviewer #1: No

Reviewer #2: No

**********

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10.1371/journal.pone.0308833.r002
Author response to Decision Letter 0
Submission Version1
12 Jul 2024

Dear Editor,

On behalf of all co-authors, I would like to thank the Reviewers for their valuable comments. We have addressed each comment and revised the appropriate sections accordingly. We believe these revisions have significantly improved our manuscript.

Please find our detailed responses to the specific comments below. All modifications in the text are highlighted in yellow.

Sincerely,

Karol Momot

Małgorzata Wojciechowska

Reviewer #1:


 The study investigated the effect of post-myocardial infarction (MI), heart failure (HF), and high-fat diet HFD on inflammation, nitro-oxidative stress, endoplasmic reticulum (ER) stress, and unfolded protein response (UPR). Twenty-eight male adult Sprague Dawley rats were divided into four groups (NFD vs. HFD) and surgical procedures (sham operation vs. coronary artery ligation to induce MI). The authors concluded that dietary fat content significantly impacts cardiac function and stress markers in a rat model of MI HF. However, several methodological weaknesses and speculative conclusions must be resolved to make the study more scientifically acceptable.

⁃ Thank you for your thorough review and valuable suggestions. We have addressed your comments and made the necessary revisions, as detailed below.


• Adherence to strict dietary standards is a cornerstone of rodent research, ensuring the reproducibility and reliability of results. The dietary pattern of rodents, as outlined in AIN93 (J Nutr 1993;123:1939-51 doi:10.1093/jn/123.11.1939), is a crucial aspect that is unfortunately not detailed in the manuscript. Different types of fat can have varying effects on animal metabolism, and the protein content of the food is also vital for post-surgical recovery. Therefore, I kindly request a table providing a comprehensive breakdown of the ingredients in the control and high-fat diets used in the study.

⁃ We have prepared a table with detailed information about the ingredients in the normal-fat and high-fat diets used in the study, based on the manufacturer's specifications. We have also included the prepared table in the text at line 126. Thank you for highlighting this important aspect.


• What was the difference in energy between the control and HF diets?

⁃ We have also included information about the caloric content of both the control and high-fat diets in the table (line 126). Thank you for your attention to this detail. This type of feed, NFD and HFD, has been used in our laboratory for many years:


1. Cudnoch-Jedrzejewska A, Gomolka R, Szczepanska-Sadowska E, Czarzasta K, Wrzesien R, Koperski L, Puchalska L, Wsol A. High-fat diet and chronic stress reduce central pressor and tachycardic effects of apelin in Sprague-Dawley rats. Clin Exp Pharmacol Physiol. 2015 Jan;42(1):52-62. doi: 10.1111/1440-1681.12324. PMID: 25311903.

2. Czarzasta K, Cudnoch-Jedrzejewska A, Szczepanska-Sadowska E, Fus L, Puchalska L, Gondek A, Dobruch J, Gomolka R, Wrzesien R, Zera T, Gornicka B, Kuch M. The role of apelin in central cardiovascular regulation in rats with post-infarct heart failure maintained on a normal fat or high fat diet. Clin Exp Pharmacol Physiol. 2016 Oct;43(10):983-94. doi: 10.1111/1440-1681.12617. PMID: 27378063.

3. Czarzasta K, Koperski L, Segiet A, Janiszewski M, Kuch M, Gornicka B, Cudnoch-Jedrzejewska A. The role of high fat diet in the regulation of MAP kinases activity in left ventricular fibrosis. Acta Histochem. 2019 Apr;121(3):303-310. doi: 10.1016/j.acthis.2019.01.010. Epub 2019 Feb 4. PMID: 30733042.

4. Czarzasta K, Wojno O, Zera T, Puchalska L, Dobruch J, Cudnoch-Jedrzejewska A. The influence of post-infarct heart failure and high fat diet on the expression of apelin APJ and vasopressin V1a and V1b receptors. Neuropeptides. 2019 Dec;78:101975. doi: 10.1016/j.npep.2019.101975. Epub 2019 Oct 15. PMID: 31645268.

5. Wojno O, Czarzasta K, Puchalska L, Kowalczyk M, Cudnoch-Jedrzejewska A. Central interaction between the apelinergic and vasopressinergic systems in the regulation of the haemodynamic parameters in rats maintained on a high-fat diet. Clin Exp Pharmacol Physiol. 2020 Dec;47(12):1902-1911. doi: 10.1111/1440-1681.13381. Epub 2020 Aug 16. PMID: 32687615.

Normal-fat Diet High-fat Diet

Energy (kcal per 100g) 286 382

Carbohydrates (g per 100g) 60.0 35.5

Proteins (g per 100g) 17.4 17.1

Fats (g per 100g) 3.2 28.0

Saturated (%) 13 49

Unsaturated (%) 37 44

Polyunsaturated (%) 50 7

Raw ash (g per 100g) 12 12

Water (g per 100g) 12 12


• The names of groups should be more explicit and not confusing.

⁃ We have changed the group names from HFD HF to HFD-MI and NFD HF to NFD-MI to make them more explicit and less confusing. All the changed names have been highlighted in yellow. Thank you for your suggestion.


• Page 5 - Fig 1 is mentioned, but we see a table.

⁃ At this point in the text, both Figure 1 and Table 1 should be included. Table 1 is included in the text, and following the author guidelines, figures should not be placed within the main text. Figure 1 is included in the attachments.

• In line 103, three references are cited to explain the experimental procedures (refs. #24-26). However, these references are a meta-analysis and two reviews that do not provide experimental details.

⁃ The appropriate references are now #29-31, which were initially listed at the bottom of the reference list (Line 104). Apologies for the error.


• Coronary artery ligation in rodents is not always lethal, but many animals die. The manuscript does not inform how many animals were operated on or the survival rate.

⁃ We have added information about the number of animals operated on and the survival rates in the main text:

⁃ Sham-operated and High-fat Diet: 10 rats operated, 6 survived

⁃ Coronary artery ligation and High-fat Diet: 20 rats operated, 10 survived

⁃ Sham-operated and Normal-fat diet: 10 rats operated, 7 survived

⁃ Coronary artery ligation and Normal-fat diet: 15 rats operated, 9 survived

Please see Table 1, line 110.


• When ligating the coronary artery to cause a myocardial infarction in rodents, the method produces individual lesions; some animals may have an extensive infarction area, but others may not. Evans blue and triphenyl tetrazolium chloride staining can determine infarct areas at sacrifice (a missing item that compromises the study).

⁃ We measured the infarct area using the planimetric method: the HFD-MI group had a significantly larger infarction area than the NFD-MI group (44.57 ± 3.44% vs. 25.29 ± 0.75%, P< 0.001). We did not use Evans blue and triphenyl tetrazolium chloride staining because post-euthanasia, we collected the heart (both right and left ventricles) for gene expression analysis using RT-PCR or to estimate protein levels using Western Blot or ELISA. Staining the hearts could have affected the results of these biochemical analyses or rendered them impossible. Therefore, we opted for the planimetric method to assess the infarct scar.


• Did the operated animals eat normally? How was the animals' food intake? The information is relevant to understanding the result of body mass loss reported in the article.

⁃ We did not measure the food intake of the operated animals. However, we did assess fat tissue masses, which can be found in the articles cited in our main text:

1. Czarzasta K, Cudnoch-Jedrzejewska A, Szczepanska-Sadowska E, et al. The role of apelin in central cardiovascular regulation in rats with post-infarct heart failure maintained on a normal fat or high fat diet. Clin Exp Pharmacol Physiol. 2016;43(10):983-94. https://doi.org/10.1111/1440-1681.12617

2. Czarzasta K, Wojno O, Zera T, et al. The influence of post-infarct heart failure and high fat diet on the expression of apelin APJ and vasopressin V1a and V1b receptors. Neuropeptides. 2019;78:101975. https://doi.org/10.1016/j.npep.2019.101975


• Line 139 –reference #24 used to describe the protein analysis method is a meta-analysis, so…

⁃ The situation is the same as in line 103. We apologize for the mistake. The correct citation should be #29. Thank you for your attention to detail. Please see line 143.


• I did not find an explanation for what proBNP means (which appears a few times in the text).

⁃ We have added an explanation for the abbreviation proBNP in the text. Please see line 132-133. Thank you for pointing this out.


• Lines 206-2010 - Nitric oxide (NO) is a gas that degrades quickly. The study did not evaluate NO production and cannot discuss NO levels.

⁃ We agree with the Reviewer’s opinion. However, in our study, we analyzed the levels of synthases, which are relatively stable proteins and do not degrade as quickly as NO. These can be analyzed using Western blot.


• Comparisons between groups (and markings on graphs) - there are comparisons between groups that are not comparable (e.g., unoperated ND vs. operated HFD) and should not be stated or commented on.

⁃ Thank you for your observation. We have corrected the figures and the text to ensure only comparable groups, which differ by a single variable (either the type of procedure or the diet, but not both simultaneously), are stated and commented on..


• The statistical analysis should be more explicit and inform where the parametric or non-parametric analysis was performed. Why was the Spearman coefficient not determined for non-parametric correlations?

⁃ We did not use the Spearman coefficient because each analyzed data point followed a normal distribution and exhibited a linear relationship. Therefore, we used only the Pearson method. Additionally, upon closer analysis, we confirmed that all quantitative data were analyzed using one-way ANOVA, and none with the Kruskal-Wallis test. We have added a note about this in the methodology section. See lines 158-164. Thank you for your suggestion and apologies for the confusion.


• I do not understand how the authors talk about 'heart failure' (HF) if the study does not include measuring the animals' blood pressure or a cardiac functional test (ultrasound?). Analysis of left ventricular ejection fraction is essential to understand the effect of post-infarction HFD.

⁃ We assessed the infarct area using the planimetric method and measured NT-proBNP levels in plasma to determine if the rats had developed post-infarction heart failure. Thank you for highlighting the importance of these measurements.

⁃ In a separate study, we evaluated hemodynamic parameters, including left ventricular end-diastolic pressure (LVEDP), using invasive catheterization in both infarcted and non-infarcted rats on high-fat or standard diets:

⁃ Czarzasta K, Cudnoch-Jedrzejewska A, Szczepanska-Sadowska E, et al. The role of apelin in central cardiovascular regulation in rats with post-infarct heart failure maintained on a normal fat or high fat diet. Clin Exp Pharmacol Physiol. 2016;43(10):983-94. https://doi.org/10.1111/1440-1681.12617).


• There are numerous grammatical and spelling errors in the text.

⁃ Thank you for pointing this out. We have thoroughly reviewed the text again and corrected all grammatical and spelling errors.

Reviewer #2:

This manuscript describes an exploratory study examining the influence of high fat diet on cardiac nitrosative stress and endoplasmic reticulum stress in rat model with post-myocardial infarction heart failure. Overall, the manuscript is well written.

⁃ Thank you very much for taking the time to review our manuscript. We have addressed your comments below.

Although the novelty of this experiment is limited by the great depth of literature available using other rodent models, this manuscript reports a technically-sound experiment describing the nitrosative and endoplasmic reticulum stress markers in rats.

⁃ Thank you for your feedback. As this was a preliminary study and the results were very interesting, we decided to submit this article to the journal. To date, no one has analyzed nitrosative stress in the context of myocardial infarction and a high-fat diet. It is known that this stress is exacerbated in heart failure with preserved ejection fraction. Publishing these results will lay the foundation for further research and inspire more researchers to explore this topic.

Nonetheless, the manuscript lacks any functional metrics that could support and verify the development of heart failure in this model.

⁃ We assessed the infarct area using planimetric methods and measured NT-proBNP levels in plasma to determine if the rats had developed post-infarction heart failure. In a separate study, we also evaluated hemodynamic parameters, including LVEDP (left ventricular end-diastolic pressure) using invasive catheterization in both infarcted and non-infarcted rats on high-fat or standard diets (refer to Table in Experiment 1 from DOI: 10.1111/1440-1681.12617).

The choice of single time-point also limits the information provided by this characterization study whereby it is unclear when does nitrosative stress and endoplasmic reticulum stress occurs with diet and MI-induced heart failure.

⁃ Thank you for your insightful feedback. We would like to emphasize that these are preliminary studies, and extending the study to include additional time points is challenging due to the high mortality rate of rats following the ligation of the left coronary artery. Specifically, we observed the following survival rates in our study (See Table 1, line 110):

⁃ Sham-operated and High-fat Diet: 10 rats operated, 6 survived

⁃ Coronary artery ligation and High-fat Diet: 20 rats operated, 10 survived

⁃ Sham-operated and Normal-fat diet: 10 rats operated, 7 survived

⁃ Coronary artery ligation and Normal-fat diet: 15 rats operated, 9 survived


We chose to include one control group and three experimental groups in our study. This design allowed us to use two-way ANOVA analysis to evaluate the impact of different factors on various parameters of stress and inflammation

Attachment Submitted filename: To the editor and reviewers.docx

10.1371/journal.pone.0308833.r003
Decision Letter 1
Aguila Marcia B. Academic Editor
© 2024 Marcia B. Aguila
2024
Marcia B. Aguila
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
18 Jul 2024

PONE-D-24-15393R1Post-myocardial infarction heart failure and long-term high-fat diet: cardiac endoplasmic reticulum stress and unfolded protein response in Sprague Dawley rat modelPLOS ONE

Dear Dr. Wojciechowska,

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 reviewer #2 approved the authors' responses but requested a comment in the conclusion indicating that this study is preliminary. However, the reviewer #1 did not accept the authors' responses and insists on them being addressed adequately. As the academic editor, I will grant the authors another opportunity, and the responses will be forwarded to reviewer #1 for evaluation.

Please submit your revised manuscript by Sep 01 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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.

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We look forward to receiving your revised manuscript.

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

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 #1: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: No

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 #1: Yes

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 #1: Yes

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 #1: Review PONE-D-24-15393R1

I thank the authors for reviewing the manuscript and considering previous comments. However, there are still some issues that need to be resolved:

Lines 137-138 "The infarction surface was measured planimetrically". Planimetry is a method for estimating areas (just like a scale measures mass). The issue with evaluating the infarcted area in the left ventricle is that the manuscript does not have any information on how this was done. As mentioned in the first review, Evans blue and triphenyl tetrazolium chloride staining is generally used to determine infarct areas at sacrifice. However, the authors responded that they did not do it this way but by planimetry (?). OK, but how did the authors select the region of infarction? Were histological sections made from the left ventricle and a dye used? How many cuts, and at what levels of the left ventricle? How did the authors perform the fractionation of the ventricle to estimate the infarction area? Does planimetry use point counting, image analysis, or other techniques? It is essential to highlight that infarction is a three-dimensional issue; planimetry alone (which determines the areas of infarction in sections) does not provide information on the volume of the injured myocardium. Unfortunately, this result should be removed from the article because it does not make sense scientifically.

Page 8 (statistical analysis) – It is problematic that such a small sample was normally distributed for all parameters analyzed (perhaps the authors should review this). It is not understood to do a one-way ANOVA and then a two-way ANOVA. What software was used in the study? Bio-render and AI-based tools are not acceptable software for statistics.

Finally, the lack of a functional study of the post-infarct heart and estimation of left ventricular ejection fraction significantly reduces the contribution that could be made by the experiment and is a significant limitation of the study.

Reviewer #2: Authors have addressed all my previous comments. They have acknowledge that this is a preliminary study and it would be ideal that they mention this in the discussion and conclusion.

**********

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

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

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

Reviewer #1: No

Reviewer #2: No

**********

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10.1371/journal.pone.0308833.r004
Author response to Decision Letter 1
Submission Version2
24 Jul 2024

TO REVIEWER 1:

Dear Reviewer,

Thank you for your feedback and suggestions. We have carefully considered your comments. Below, we provide our responses point by point:

1. As requested, we have decided to remove the section on planimetry from the manuscript.

However, to provide a clear understanding of our methodology, we would like to include the following detailed explanation only in this correspondence:

After euthanasia, the heart was excised from the thorax, and the left ventricle, including the septum, was separated from the right ventricle. Both ventricles were weighed. Infarct size was determined planimetrically as previously described [Leenen et al. 1999; Brain “ouabain” and angiotensin II contribute to cardiac dysfunction after myocardial infarction; doi.org/10.1152/ajpheart.1999.277.5.H1786], with some modifications [Dobruch et al. 2009; Enhanced involvement of brain vasopressin V1 receptors in cardiovascular responses to stress in rats with myocardial infarction, doi.org/10.1080/10253890500456287]. The left ventricle was cut along the longitudinal axis and placed flat on a transparent plastic sheet. The infarcted areas on both the inner (endocardial) and outer (epicardial) surfaces of the ventricle were outlined. The average of these two measurements was then calculated and expressed as a percentage of the total left ventricle wall size.

2. We acknowledge your point that one-way ANOVA is inappropriate. In response, we have removed all references to it from the methodology and results sections of the manuscript.

We have retained the two-way ANOVA analysis to examine the effects of diet (Normal-fat Diet vs. High-fat Diet) and procedure (Sham operation vs. Myocardial infarction) and their interaction. The revised methodology section now reads:

"A two-way ANOVA was conducted to examine the effects of diet (Normal-fat Diet vs. High-fat Diet) and procedure (Sham operation vs. Myocardial infarction), as well as their interaction. Post-hoc comparisons were performed using Tukey’s HSD test to identify significant differences between groups."

3. Regarding your concerns about the normality of data distribution, we conducted Shapiro-Wilk tests during statistical analysis; the results are provided in the table below. It is worth noting that each Western blot measurement was repeated three times, and the final result was calculated as the average of these repetitions.

Despite a single instance where the Shapiro-Wilk test indicated non-normality (p < 0.05) for "HFD MI," the use of a two-way ANOVA is justified. Levene's test for homogeneity of variances yielded a p-value of 0.09195, indicating no significant difference in variances between groups. ANOVA is robust to minor deviations from normality, especially with similar sample sizes. Therefore, acknowledging the limitation, two-way ANOVA remains appropriate for this analysis.

4. Regarding your question about the software used in the study, we have clarified this point in the manuscript. Statistical analysis was carried out using Statistica software, version 13.3. This information has been added to the "Statistical Analysis" section of the main text.

5. We acknowledge your point regarding the lack of a functional study of the post-infarct heart and the estimation of left ventricular ejection fraction.

Unfortunately, the study has already been conducted, and we cannot address this limitation.

However, we believe that our study, as preliminary research, provides valuable insights and perspectives for future investigations. We have emphasized this limitation once more in the "Limitations" section at the end of the article to ensure that readers interpret the findings with caution.

6. We have made the necessary revisions to the text in response to your comments and have emphasized that this study is preliminary research both in the discussion and conclusion sections.

For your review, the changes have been made using the track changes mode.

We sincerely thank both reviewers for the valuable and scientific discussion and appreciate all the effort put into the thorough review. We hope this clarification addresses your concerns.

Sincerely,

Karol Momot and colleagues

Diet Procedure Shapiro Wilk P-value (GRP/actin)

HFD MI 0.823293

HFD SO 0.590984

NFD MI 0.565416

NFD SO 0.368853

Diet Procedure Shapiro Wilk P-value (mean ATF6c/ATF6)

HFD MI 0.046620

HFD SO 0.612515

NFD MI 0.720524

NFD SO 0.590896

Diet Procedure Shapiro Wilk P-value (PERK/actin)

HFD MI 0.094379

HFD SO 0.566535

NFD MI 0.784923

NFD SO 0.820542

Diet Procedure Shapiro Wilk P-value (CHOP/actin)

HFD MI 0.419551

HFD SO 0.408671

NFD MI 0.258975

NFD SO 0.460599

Diet Procedure Shapiro Wilk P-value (nNOS/actin)

HFD MI 0.782037

HFD SO 0.589341

NFD MI 0.758002

NFD SO 0.746428

Diet Procedure Shapiro Wilk P-value (eNOS/actin)

HFD MI 0.464793

HFD SO 0.696505

NFD MI 0.573740

NFD SO 0.181428

Diet Procedure Shapiro Wilk P-value (NT/actin)

HFD MI 0.288189

HFD SO 0.166167

NFD MI 0.364409

NFD SO 0.909621

Diet Procedure Shapiro Wilk P-value (MPO/actin)

HFD MI 0.200157

HFD SO 0.861766

NFD MI 0.108960

NFD SO 0.918455

Diet Procedure Shapiro Wilk P-value (iNOS/actin)

HFD MI 0.118528

HFD SO 0.884183

NFD MI 0.923192

NFD SO 0.117495

Diet Procedure Shapiro Wilk P-value (pIRE/IRE)

HFD MI 0.253621

HFD SO 0.416927

NFD MI 0.060167

NFD SO 0.238902

Diet Procedure Shapiro Wilk P-value (NT-proBNP)

HFD MI 0.656257

HFD SO 0.133989

NFD MI 0.427857

NFD SO 0.194803

Diet Procedure Shapiro Wilk P-value (body mass)

HFD MI 0.624347

HFD SO 0.255299

NFD MI 0.371023

NFD SO 0.560306

TO REVIEWER 2:

Dear Reviewer,

Thank you for your feedback and suggestions. We have made the necessary revisions to the text in response to reviews and have emphasized that this study is preliminary research in both the discussion and conclusion sections.

For your review, the changes have been made using the track changes mode.

We sincerely thank both reviewers for the valuable and scientific discussion. Your input has been greatly appreciated and has significantly contributed to the development of our study. We hope this clarification addresses your concerns.

Sincerely,

Karol Momot and colleagues

Attachment Submitted filename: To the reviewer 2.docx

10.1371/journal.pone.0308833.r005
Decision Letter 2
Aguila Marcia B. Academic Editor
© 2024 Marcia B. Aguila
2024
Marcia B. Aguila
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version2
30 Jul 2024

Post-myocardial infarction heart failure and long-term high-fat diet: cardiac endoplasmic reticulum stress and unfolded protein response in Sprague Dawley rat model

PONE-D-24-15393R2

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10.1371/journal.pone.0308833.r006
Acceptance letter
Aguila Marcia B. Academic Editor
© 2024 Marcia B. Aguila
2024
Marcia B. Aguila
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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==== Refs
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