Implication of RAS in Postnatal Cardiac Remodeling, Fibrosis and Dysfunction Induced by Fetal Undernutrition

Round 1

Reviewer 1 Report

The study by Rodriguez aims to investigate the effects of undernutrition during gestation (MUN) in the pathophysiology of cardiac dysfunction in male litters. It is an interesting study and results are quite clear about the harmful effects that the undernutrition during gestation can have for the next generation. It is a question that the same research group has been pursuing for a while.  

The study is a phenotypic characterization of the cardiac function in male rats exposed to MUN. These animals exhibit cardiac dysfunction associated with collagen deposition in tissues and vessels from the heart, effects that were associated with changes in receptors AT1, AT2, Mas and Mrg. It is a bit disappointing that the study does show mechanisms, but it is totally understandable because the nature of the publication.

Regarding to the data, I have just on major question:

1. Animals that were submitted to MUN showed a very significant reduction in E/A ration which is a cardiac dysfunction associated with inflammatory response. Is there increased inflammatory infiltration and proinflammatory cytokine in the heart of these animals? These results would help to understand about some of the mechanisms whereby the cardiac dysfunction is so severe.        

Author Response

We would like to thank the reviewer for the revision of the manuscript. Below please find our answer to the question raised. We have modified the text accordingly and the changes are marked in the new version of the manuscript.

Regarding to the data, I have just on major question:

1.Animals that were submitted to MUN showed a very significant reduction in E/A ration which is a cardiac dysfunction associated with inflammatory response. Is there increased inflammatory infiltration and proinflammatory cytokine in the heart of these animals? These results would help to understand about some of the mechanisms whereby the cardiac dysfunction is so severe.  

ANSWER. We agree with the reviewer on the interest to evaluate inflammation in this animal model of fetal programming. However, it was beyond the scope of this manuscript to evaluate this aspect. Regarding the mechanisms responsible for the observed hypertrophy, we only investigated the possible implication of TGF-b. Given the possible implication of inflammation as underlying mechanism, we have added some discussion (lines 540-555).

Reviewer 2 Report

Dear authors,

In this original manuscript, Rodríguez- Rodríguez et al. examine the effect of fetal undernutrition on later development of cardiac dysfunction during ageing. The authors rationalize that fetal undernutrition causes a disbalance in RAS that is associated with cardiac remodeling during lactation leading to cardiac hypertrophy and remodeling of intracardiac arteries. To examine this, they exposed pregnant female rats to reduced daily intake of chow starting from day 11 to the end of pregnancy. Control pregnant female rats received ad libitum chow during the whole course of pregnancy. During the lactation phase, both groups of female rats received ad libitum diet. Only male newborn pups were used for the study.

They demonstrate that maternal undernutrition during pregnancy leads to changes in the heart function, increases the cardiac area and the media/lumen ratio of intracardiac arteries and induces cardiac fibrosis. Furthermore, they investigated the expression pattern of receptors associated with RAS.

Major comments:

• Line 68 – 69: Please revise the sentences. Ang I is degraded to Ang (1-9) by ACE, not by ACE2. Ang A degradation by ACE2 leads to alamandine, but Ang (1-7) is degraded by another enzyme.
• Please explain why you focused your experimental model on male pups only. Which findings would you assume for female pups, especially in regards of the RAS receptor expression?
• Figure 2a: Please provide photos from the myocardial area. In the methods part, there is no explanation for the quantification of the myocardial area. Explain how you quantified the area. Why do you have differences in the group sizes (controls n=7, MUN n=5)? I think to underline your information about cardiac remodeling you need to provide more data characterizing the changes in the cardiac tissue. Do you see changes in the size of individual cardiac myocytes as well? Can you please provide data on hypertrophic markers by qPCR?
• Figure 2b: Please explain how you calculated the artery media:lumen ratio.
• Figure 3: Please provide data for the expression of fibrotic markers by qPCR.

Minor comments:

• Line 57: Please rephrase “…, which is hypertrophied…”. Vessels, arteries, aorta are not hypertrophied, they are dilated or enlarged. Same line 339.
• Misspelling in line 119; line 188; line 246; line 293; line 310; line 314.
• Figure 4: Some of the scale bars are almost cut out of the photo. In Figure 4 a, the unit of the provided scale bar is wrong (should be μm). Misspelling in all y-axes (area). The “D” of “MrgD” is in another line.
• Figure 5: Misspelling in all y-axes (area). The “D” of “MrgD” is in another line.
• Line 313: I am assuming you mean “…quantitative analysis of MUN and C arteries…” and not “myocardium”.
• Line 313: “percentage of total tissue area”.
• Edit sentence in lines 365-366.
• Edit sentence in lines 369-372.
• Edit coma in line 299; line 386.
• Edit sentence in lines 415-416.
• Figure 6: The “g” of “ageing” is in another line.

Dear authors, I recommend that you revise your whole manuscript for misspellings, language, comas, etc.

Author Response

We would like to thank the reviewer for the thorough revision of the manuscript. Below please find our point-by-point answer to the questions raised. We have modified the text accordingly; all changes are marked in the new version of the manuscript.

Major comments:

Line 68 – 69: Please revise the sentences. Ang I is degraded to Ang (1-9) by ACE, not by ACE2. Ang A degradation by ACE2 leads to alamandine, but Ang (1-7) is degraded by another enzyme.

ANSWER. We have corrected this sentence regarding the different enzymatic pathways and peptides (lines 68-74).

Please explain why you focused your experimental model on male pups only. Which findings would you assume for female pups, especially in regards of the RAS receptor expression?

ANSWER. We focused in male pups since we have several lines of evidence showing that females have a better response to fetal undernutrition regarding cardiac alterations in early life. In particular, 1) in fetus cardiac growth is normal (males exhibit a deficient cardiac growth), 2) during perinatal life females do not exhibit cardiac hypertrophy (only males show increased heart/body weight) or signs of oxidative stress; females have a larger circulating antioxidants and do not have alterations in cardiac NADPH Oxidase expression (males show overexpression). Besides, we have preliminary echocardiographic data showing that females do not have altered cardiac function at the age of 21 days. Since, Ang II is implicated in oxidative damage, fibrosis and hypertrophy through NADPH oxidase and RAS receptors, the aim of this manuscript was to test the implication of RAS alterations in male hearts.

Figure 2a: Please provide photos from the myocardial area. In the methods part, there is no explanation for the quantification of the myocardial area. Explain how you quantified the area. Why do you have differences in the group sizes (controls n=7, MUN n=5)? I think to underline your information about cardiac remodeling you need to provide more data characterizing the changes in the cardiac tissue. Do you see changes in the size of individual cardiac myocytes as well? Can you please provide data on hypertrophic markers by qPCR?

ANSWER. We have now included photos in the figure, and explanation of the quantification of the myocardial area in methods section as requested (lines 176-179).

Regarding the different number of data in MUN and Control, it was a mistake. In the graph of control rats, we did not average data from rats of one of the litters. We have now amended figure 2, also providing representative images of myocardial area.

We understand the importance to evaluate possible mechanisms implicated in the observed morphological findings with a molecular marker. It was not possible to conduct q-PCR experiments. However, we have performed additional protein expression experiments by Western Blot in hearts from rats from the same litters, which were stored. We used TGF-b1, since this factor has been shown to be a marker of hypertrophy and fibrosis mediated by Ang II. This has now been included in the manuscript in methods (lines 237-248), results (lines 355-382) and discussion (lines 536-555).

Figure 2b: Please explain how you calculated the artery media:lumen ratio.

ANSWER. This is now explained in the methods section (lines 180-183).

Figure 3: Please provide data for the expression of fibrotic markers by qPCR.

ANSWER. We have included TGF-b1 protein expression data.

Minor comments:

Line 57: Please rephrase “…, which is hypertrophied…”. Vessels, arteries, aorta are not hypertrophied, they are dilated or enlarged. Same line 339.

ANSWER. Hypertrophied has been changed to “enlarged” in line 57 and “enlargement”

Misspelling in line 119; line 188; line 246; line 293; line 310; line 314.

ANSWER. We hope that misspelled words are now corrected and marked in the text.

Figure 4: Some of the scale bars are almost cut out of the photo. In Figure 4 a, the unit of the provided scale bar is wrong (should be μm). Misspelling in all y-axes (area). The “D” of “MrgD” is in another line.

Figure 5: Misspelling in all y-axes (area). The “D” of “MrgD” is in another line.

ANSWER. You are right, it was not possible to see the scale bars in some of the figures and there were some other errors in the figures. All the figures have been revised and we hope all the errors have been corrected

Line 313: I am assuming you mean “…quantitative analysis of MUN and C arteries…” and not “myocardium”.

Line 313: “percentage of total tissue area”.

ANSWER. The correct sentence is …”quantitative analysis of MUN and C intramyocardial artery stained fractional areas (percentage of total tissue area)”. This has been corrected.

Edit sentence in lines 365-366.

Edit sentence in lines 369-372.

Edit coma in line 299; line 386.

Edit sentence in lines 415-416.

Figure 6: The “g” of “ageing” is in another line.

Dear authors, I recommend that you revise your whole manuscript for misspellings, language, comas, etc.

ANSWER. Thank you for the thorough revision. We have corrected the detected errors and have revised the entire manuscript. We hope it has no typos.

Round 2

Reviewer 1 Report

 Authors answered my questions and added some new data based on the concerns observed during the first review.

  I have no additional questions

Reviewer 2 Report

Line 544: cardiomyocyte

 

Otherwise, no further questions.