Quantitative Analysis of the Cardiac Phosphoproteome in Response to Acute β-Adrenergic Receptor Stimulation In Vivo

Round 1

Reviewer 1 Report

Title: Unbiased quantitative analysis of the cardiac phosphoproteome in response to acute beta-adrenergic receptor stimulation in-vivo

Summary:

The authors main objective was to characterize beta-adrenergic receptor stimulation by using mass spec and phosphoproteome analysis. They found that there are a multitude of rapid changes in phosphorylated proteins following direct beta-adrenergic receptor stimulation, as expected. The reviewer warmly welcomes this manuscript, as it was pleasing to read and well-written. The reviewer also finds this review paper suitable for this journal. However, there are several concerns that must be addressed prior to consideration for publication. Please see the concerns below.

Abstract:

• No comments

Introduction:

• Although the introduction includes pertinent information, it must be revisited. It is too long. Address the prevalence CVD/obesity/metabolic syndrome, risk factors, and the impacts of exercise on these risk factors. Please reduce the intro to 2 pages.
• In addition, cite the other meta-analyses on the topic that are claimed to produce short fallings in the literature. It is not made entirely clear why this meta-analysis is better than the others, please make it more evident.
• There is no mention regarding the potential differential mechanisms of these types of exercise training. Please address.

Methods

• Why were male mice used? And what is the rationale for the age?

Statistical analysis

• No comments

Results

• Please report the acute increase in heart rate in response to ISO in-text in the results section in addition to the figure. Were there any other physiological measures in response to the stimulation to confirm it?
• Please report the average dose of ISO according to the mg/kg dose.
• What was the reported half life of the ISO administration? Metabolism?

Discussion

• What may be the physiological and/or potential clinical impacts of these findings?
• How could the age of the mice impact the signaling cascade? Adrenergic receptor sensitivity depreciates with age, please discuss this.
• Not using differing ages/sex is a limitation to the study. Please discuss.
• How would this cascade potentially change in disease states?
• Again, physiological implications should at least be addressed.

Figures/tables

• The study protocol figure is clear and well-designed.
• Was there any splicing made to Supplementary figure 5? GAPDH in 5A looks like different background coloring in VEH vs. OA. If this is the case, it must be clear that the gel was spliced.

Author Response

Summary:

The authors’ main objective was to characterize beta-adrenergic receptor stimulation by using mass spec and phosphoproteome analysis. They found that there are a multitude of rapid changes in phosphorylated proteins following direct beta-adrenergic receptor stimulation, as expected. The reviewer warmly welcomes this manuscript, as it was pleasing to read and well-written. The reviewer also finds this review paper suitable for this journal.

Authors' Reply: We thank the reviewer for the overall positive feedback on our manuscript and appreciate the suggestions for improvement.

Introduction:

Although the introduction includes pertinent information, it must be revisited. It is too long. Address the prevalence CVD/obesity/metabolic syndrome, risk factors, and the impacts of exercise on these risk factors. Please reduce the intro to 2 pages.

In addition, cite the other meta-analyses on the topic that are claimed to produce short fallings in the literature. It is not made entirely clear why this meta-analysis is better than the others, please make it more evident.

There is no mention regarding the potential differential mechanisms of these types of exercise training. Please address.

Authors' Reply: We fear that there has been an error such that the comments above refer to a different study and not to our current manuscript. The introduction section of our manuscript is only 1 page long, our manuscript is not a meta-analysis but describes an original research study, which is not on CVD/obesity/metabolic syndrome or risk factors and did not employ any form of exercise training.

Methods:

Why were male mice used? And what is the rationale for the age?

Authors' Reply: These issues are now addressed in a new “Study limitations” section that has been added at the request of Reviewer 2 (please see page 10 of the revised manuscript).

Results:

Please report the acute increase in heart rate in response to ISO in-text in the results section in addition to the figure. Were there any other physiological measures in response to the stimulation to confirm it?

Authors' Reply:  We report the ISO-induced acute increase in heart rate in-text, in the very first paragraph of the Results section (page 3, lines 3-6 of the revised manuscript). In the present study, we monitored heart rate, which is readily accomplished through ECG recording, only as a means of ensuring successful administration of ISO. In a recent publication (1), we have described broader physiological effects of ISO at an identical dose and route of administration, as assessed by ultrasound echocardiography.

(1) Lewis, H., Eminaga, S., Gautel, M. and Avkiran, M. Phosphorylation at serines 157 and 161 is necessary for preserving cardiac expression level and functions of sarcomeric Z-disc protein telethonin. Frontiers in Physiology, 2021 Sep 8;12:732020. doi: 10.3389/fphys.2021.732020.

Please report the average dose of ISO according to the mg/kg dose. What was the reported half life of the ISO administration? Metabolism?

Authors' Reply:  We report the dose of ISO used in section 4.1 (“Preparation of Mouse Tissue Samples”) of the Materials and Methods section (page X, lines X-X of the revised manuscript): “After baseline stabilization of heart rate, 0.1 mg/kg body weight dose of ISO dissolved in sterile saline (0.9% NaCl)…”

 As also described under Materials and Methods, hearts were explanted and cryopreserved 2 min after ISO administration, at the peak of the heart rate response (illustrated in Figure 2A) that suggests maximum agonist bioavailability. Detailed pharmacokinetic analysis of ISO is outside the scope of our study.

Discussion:

What may be the physiological and/or potential clinical impacts of these findings?

Authors' Reply:  This issue is now addressed in a new “Concluding comments” subsection in the Discussion. (please see page 11 of the revised manuscript).

How could the age of the mice impact the signaling cascade? Adrenergic receptor sensitivity depreciates with age, please discuss this. Not using differing ages/sex is a limitation to the study. Please discuss.

Authors' Reply: These issues are now addressed in a new “Study limitations” section that has been added at the request of Reviewer 2 (please see page 10 of the revised manuscript). To examine the impact of sex, ageing and other confounders on the principle effects of stimulation that our study describes would require significant additional experimentation, which is well beyond the scope of our study. As our data are not suited to examine the influence of e.g. ageing, detailed discussion of this would be largely speculative. We have rather added an additional “Concluding comments” section in which we clarify that our results provide a key resource for further experimentation-

How would this cascade potentially change in disease states? Again, physiological implications should at least be addressed.

Authors' Reply: Please see our comment to the reviewer’s question above. These issues are now addressed in a new “Study limitations” section that has been added at the request of Reviewer 2 (please see page 10 of the revised manuscript) and a new “Concluding comments” section (please see page 11 of the revised manuscript).

Figures/Tables:

Was there any splicing made to Supplementary figure 5? GAPDH in 5A looks like different background coloring in VEH vs. OA. If this is the case, it must be clear that the gel was spliced.

Authors' Reply: No splicing was made in any of the figures illustrating immunoblot analyses. Apparent differences in background in figure 5A reflect how individual samples resolved in the pertinent SDS-PAGE gel.

Author Response File: Author Response.pdf

Reviewer 2 Report

Authors employed mass spectrometry-based proteome and phosphoproteome analysis using SuperSILAC standardization to generate a comprehensive map of acute phosphoproteome changes in mice upon administration of isoprenaline (ISO), a synthetic βAR agonist that targets both β1-AR and β2-AR subtypes.

1. I would suggest to remove the „unbiased” term from the whole manuscript, especially from the title.
2. In the Abstract, please add some values, e.g. how much the expression was increased, etc.
3. In Figure 2 a-actinin load is unequal, especially in samples where JP1 had high results. I would suggest to repeat WB.
4. I would suggest to add Limitation section.

Author Response

Authors employed mass spectrometry-based proteome and phosphoproteome analysis using SuperSILAC standardization to generate a comprehensive map of acute phosphoproteome changes in mice upon administration of isoprenaline (ISO), a synthetic βAR agonist that targets both β1-AR and β2-AR subtypes.

I would suggest to remove the „unbiased” term from the whole manuscript, especially from the title.

Authors' Reply: We agree with the reviewer that the term ‘unbiased’ is not necessary to describe and present the findings of our study. We have therefore removed it from the manuscript, as suggested.

In the Abstract, please add some values, e.g. how much the expression was increased, etc.

Authors' Reply: We haved added the values of detected phosphopeptides, phospho-events (both total and regulated) and phosphoproteins to the abstract as suggested.

In Figure 2 a-actinin load is unequal, especially in samples where JP1 had high results. I would suggest to repeat WB.

Authors' Reply: The aim of this experiment was to provide a qualitative confirmation of JP1 expression in cardiac tissue.  We used a-actinin to account for differences in protein loading in individual sample lanes, however determining relative expression between vehicle and ISO was not the objective of this experiment.

Data shown here qualitatively confirms that JP1 is indeed expressed, as suggested by the proteomics data. In addition, the appearance of the low molecular weight band in the ISO treated samples and its absence in the vehicle treated samples indicate the ISO-induced fragmentation of JP1 in cardiac tissue.

I would suggest to add Limitation section.

As suggested by the reviewer, we have added ‘Study limitations’ section (please see page 10 of the revised manuscript).

Author Response File: Author Response.pdf