The ABA/LANCL1-2 Hormone/Receptors System Controls ROS Production in Cardiomyocytes through ERRα

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript is very interesting however some points needs to be resolve:

1. Is there any reason why use rat Cardiomyocytes? Are these cell lines? Which is the context of using this cells?

2. In the introduction there is a lot of information, a diagram would be recomendable.

3. The aims of this study were two-fold: Please correct! line 117.

4. In results figure 1: why LANCL expression y SCR and PLV is so different? is there any explanation?

5.  In figure 2 B why there si no result for  NOX 4?

6. Please include how the ABA/LANCL1-2 system is regulated.

7. Please include the MW in the western blots.

8. In the discussion could you please elaborate an idea  how this system could help reducing ROS on other cells/tissues?

9. Could you please inlude more information concerning the last paragraph, I think it is important and very interesting.

 

Author Response

Reviewer 1

Manuscript is very interesting however some points needs to be resolve:

1. Is there any reason why use rat Cardiomyocytes? Are these cell lines? Which is the context of using this cells?

Thank you for your comments. We recently published two articles in which we studied the role of ABA/LANCL1-2 system in LANCL1/2-overexpressing H9c2 cardiomyocytes compared with double-silenced cells and we observed that LANCL1/2-overexpressing H9c2 cells show: (i) increased mitochondrial respiration, with higher basal and maximal respiration rates, a doubling of the spare respiratory capacity and a steeper proton gradient (∆Ψ); (ii) increased fatty acid-fueled respiration rate; (iii) increased NO generation; (iv) increased transcription and expression of contractile and ion channel proteins; (v) improved resistance to hypoxia/reoxygenation; (vi) increased proliferation rates. In short, LANCL1/2 overexpression, and their targeted stimulation by treatment of the cells with ABA, transforms H9c2 cardiomyoblasts into “super-cells”, particularly regarding mitochondrial function, arguably the “power station” of the cell and the site of potential ROS production as a by-product of increased mitochondrial respiration. For this reason, we believed that H9c2 cells were a good starting model for studies on the role, if any, of the ABA/LANCL system in protecting from oxidative stress. A sentence has been added at the end of the introduction to clarify the choice of H9c2 as the cell model for this study.

 

1. Spinelli S, Guida L, Vigliarolo T, Passalacqua M, Begani G, Magnone M, Sturla L, Benzi A, Ameri P, Lazzarini E, Bearzi C, Rizzi R and Zocchi E. The ABA-LANCL1/2 Hormone-Receptors System Protects H9c2 Cardiomyocytes from Hypoxia-Induced Mitochondrial Injury via an AMPK- and NO-Mediated Mechanism Cells, 11, 2888 (2022).
2. Spinelli S, Guida L, Passalacqua M, Magnone M, Cossu V, Sambuceti G, Marini C, Sturla L*, Zocchi, E. Abscisic Acid and Its Receptors LANCL1 and LANCL2 Control Cardiomyocyte Mitochondrial Function, Expression of Contractile, Cytoskeletal and Ion Channel Proteins and Cell Proliferation via ERRα Antioxidants, 12:1692 (2023).

 

2. In the introduction there is a lot of information, a diagram would be recomendable.

We added the following diagram at the end of introduction: 

 

In addition, a graphical abstract summarizes the results obtained in this study.

 

3. The aims of this study were two-fold: Please correct! line 117.

We corrected and deleted “two-fold” from the text

4. In results figure 1: why LANCL expression y SCR and PLV is so different? is there any explanation?

As we wrote in the legend, “The exposure time of the Western Blot panel in Figure 1A and Figure 1B is different and higher in the silenced H9c2 cells.” We needed to use longer exposure times in silenced H9c2 cells, in order to detect the very low expression levels of LANCL1/2; conversely, in the LANCL1/2 overexpressing cells, the exposure time of the blots were much shorter, due to the high protein expression levels. We added the exposure times to the legend of Figure 1 (30 sec for Figure 1A and 180 sec for Figure1B).

5. In figure 2 B why there is no result for NOX 4?

In fact, we performed Western blots for Nox4, but the primary antibody we purchased did not yield good technical results; therefore, we only included results of the qRT-PCR for Nox4.

Please include how the ABA/LANCL1-2 system is regulated.

Thank you for comment, we added this sentence in introduction:

ABA regulates the expression of both receptors, and the two receptors LANCL1 and LANCL2 also exhibit reciprocal regulation. In previous studies, we found that LANCL1 expression was significantly higher in the quadriceps muscles of LANCL2 knockout mice compared with wild-type controls mice. Chronic oral ABA treatment further elevated LANCL1 expression at both the protein and mRNA levels [10]. Similarly, silencing of LANCL1 induced a marked increase of LANCL2 expression in L6 muscle cells [10]. Both LANCL1 and LANCL2 receptors and chronic ABA treatment modulate the AMPK/PGC-1α/Sirt1 axis and GLUT4 expression in muscle cells, cardiomyocytes and skeletal muscles of wild-type mice. These findings, along with the redundancy of ABA receptors, underscore the physiological importance of the ABA/LANCL hormone/receptor system in mammals.

7. Please include the MW in the western blots.

We added molecular weight to all Western Blot panels.

6. In the discussion could you please elaborate an idea how this system could help reducing ROS on other cells/tissues?

We inserted the following sentences at the end of the penultimate paragraph to discuss recent findings that highlight the role of LANCL11 and LANCL2 as proteins involved in redox homeostasis [refs 40-41]:

Specifically, a consistent negative correlation was observed between LANCL1 expression and ROS levels, as well as ROS-responsive gene expression, in hepatocellular carcinoma (HCC) cell lines [40]. Additionally, LANCL2 plays a crucial role in maintaining the redox balance in round spermatids, which is essential for acrosomal development and sperm quality. Overexpression of LANCL2 in NCCIT cells significantly improved cell viability when cells were treated with H2O2, and LANCL2 knockout testes showed reduced GSH and increased GSSG levels, decreased testicular SOD activity and elevated levels of malondialdehyde (MDA) and protein carbonyl (PCO), indicating a disruption in testicular redox balance following LANCL2 deletion [41]. These findings indicate that LANCL1 and LANCL2 proteins play an important role in maintaining redox balance in cell types as different as hepatocytes, cardiomyocytes and spermatids.

 

9. Could you please include more information concerning the last paragraph, I think it is important and very interesting.

Thank you for your interest, a final paragraph has been added to the discussion, summarizing considerations that were originally made in the discussion of a previous paper (ref. 25 of the present manuscript), but which also apply to the findings described here.

In the discussion in ref. 25 of the present manuscript we reasoned: “To sum up, both a proton “leak” from the intermembrane space and an electron “leak” from the respiratory chain flow (enabled by the retrograde electron flux at some of the respiratory complexes) are mechanisms of OXPHOS fine tuning, which can be activated in mitochondria to adapt to quantitative changes in the flow of charges in order to maximize energy production and limit ROS generation. The ABA/LANCL system, via ERRα, appears to take advantage of, or to regulate, some of these OXPHOS fine-tuning mechanisms. Indeed, despite a two-fold increase in the proton leak in overexpressing cells, the spare respiratory capacity of these cells is approximately twice that of double-silenced cells.”

We added a concluding remark in the discussion of the present manuscript summarizing this concept.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled (The ABA/LANCL1-2 hormone receptors system controls ROS production in cardiomyocytes through ERRα) is well written and the context is a major of interest in the related field. However, the following concerns need to be addressed to confirm the conclusion of this work.

Major comment:

The impact of the ABA/LANCL1-2 system needs to be validated with cellular functional assays affected by ROS levels, such as cell viability, or other relevant assays.

 

Minor concerns:

M&M section.

Line 124: Additional information is required, such as the passage number used for the experiments....Alternatively, refer to a relevant work for these details.

 

In Lentiviral Cell Transduction

Please provide a brief description of how this procedure was conducted.

 

Line 140: Clarify what is meant by "After 18 hr of starvation." Does this refer to a growth medium without FBS or other substances? Please specify.

Line 142:  "Quantitative Real-time PCR" should be abbreviated as "qRT-PCR."

Line 170:  (H2DCFDA) is already defined in line 162.

Results

Lines 193-194: This content should be moved to the discussion section; only the obtained results can be explained here.

Figure 1, please specify and add the molecular weight for each protein (kDa).

Statistical asterisks: Using t-test analysis, the significant differences are expressed as follows: * refers to 0.01, ** refers to 0.001, and *** refers to 0.0001. However, here * refers to 0.001. Please correct this.

Lines 216-217: The exposure times of the Western Blot panels in Figures 1A and 1B are different and higher in silenced H9c2 cells. Please specify the exposure time.

Line 219: Use "Unpaired Student's t-test" instead of "Unpaired t-test."

 

 Figure 6.B: Clarify how the relative fluorescence intensity is measured and please specify the software used.

Author Response

Reviewer 2

The manuscript entitled (The ABA/LANCL1-2 hormone receptors system controls ROS production in cardiomyocytes through ERRα) is well written and the context is a major of interest in the related field. However, the following concerns need to be addressed to confirm the conclusion of this work.

Major comment:

The impact of the ABA/LANCL1-2 system needs to be validated with cellular functional assays affected by ROS levels, such as cell viability, or other relevant assays.

We performed cell viability assays and lipid hydroperoxide quantifications after H₂0₂ treatment of LANCL1/2-double silenced vs. -overexpressing cells: results obtained are shown in the new Figure 2A and B

Minor concerns:

M&M section.

Line 124: Additional information is required, such as the passage number used for the experiments. Alternatively, refer to a relevant work for these details.

We have added a sentence about cell culture and passages: “The cells were subcultured upon reaching 70–80% confluence. Experiments were done using cells between passage 5 and 8”.

 

In Lentiviral Cell Transduction

Please provide a brief description of how this procedure was conducted.

We have included two additional paragraphs in the "Lentiviral and Retroviral Transduction" section, providing a brief description of the procedure.

Line 140: Clarify what is meant by "After 18 hr of starvation." Does this refer to a growth medium without FBS or other substances? Please specify.

We clarified that “starvation” refers to incubation with growth medium DMEM low glucose without serum

Line 142:  "Quantitative Real-time PCR" should be abbreviated as "qRT-PCR."

We have replaced qPCR in qRT-PCR.

Line 170:  (H2DCFDA) is already defined in line 162.

Thank you for your comment. We cancelled the extensive name at line 170.

Results

Lines 193-194: This content should be moved to the discussion section; only the obtained results can be explained here.

We deleted the first sentence of the paragraph entitled “Overexpression and silencing of LANCL1 or LANCL2 in H9c2 cells”

Figure 1, please specify and add the molecular weight for each protein (kDa).

We added molecular weight to all Western Blot panels.

Statistical asterisks: Using t-test analysis, the significant differences are expressed as follows: * refers to 0.01, ** refers to 0.001, and *** refers to 0.0001. However, here * refers to 0.001. Please correct this.

We corrected all statistical asterisks (* p<0.01, ** p<0.001). We left $ symbol for internal statistical referring to ABA treatment.

Lines 216-217: The exposure times of the Western Blot panels in Figures 1A and 1B are different and higher in silenced H9c2 cells. Please specify the exposure time.

We added the exposure times in the legend of Figure 1 (30 sec for Figure 1A and 180 sec for Figure1B)

Line 219: Use "Unpaired Student's t-test" instead of "Unpaired t-test."

We replaced "Unpaired t-test." with "Unpaired Student's t-test"

Figure 6.B: Clarify how the relative fluorescence intensity is measured and please specify the software used.

We added the following sentence in the method paragraph titled "ROS detection assays": “Fluorescence intensity was analyzed after background subtraction with FIJI ImageJ software (version 2.14.0/1.54f), using a quantitative analysis based on the intensity measurement of specific selected ROIs”.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed all the issues and suggestions. The revisions enhanced the conclusion and overall quality.

I have no further comments and recommend that the manuscript be considered for publication.

Author Response

Dear Reviewer,

thank you for your comment. I'm sending you in attached file the legend of first diagram.

Best regards

Sturla

Author Response File: Author Response.pdf