Isoproterenol Induces Cardiac Injury and Senescence in Sprague–Dawley Rats: A Cost-Effective Pharmacological Model

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Cellular senescence is a contributor to cardiovascular dysfunction in the aging population.  However, to study cellular senescence in cardiac injury models typically requires a skilled surgeon and doesn’t necessarily model what happens in the human condition. The authors sought to explore whether isoproterenol could serve as a pharmacological-based method of inducing cellular senescence and cardiac dysfunction.

Major Concerns:

1. Authors note that ISO was able to induce cellular senescence through SA-B-gal staining and cardiac dysfunction through increased collagen deposition, cardiomyocyte hypertrophy, however, the physiological impact on cardiac function was not assessed via echocardiography. Please justify why these experiments were not performed in the discussion as this would have strengthened the ISO-induced cardiovascular dysfunction connection.
2. It would help readers to know what markers are being used in each immunofluorescence panel, especially Figure 3. They are noted in the figure legend but would provide clarity if written off the side of each panel.

Minor Concerns:

1. Authors should note why only male rats were used in this study and note whether they suspect females would have a similar ISO response or if there would be a significant hormonal component.
2. Concerning the fibrosis/collagen staining in Figure 1. Please note whether the authors observed differences between interstitial or perivascular signal?
3. In the results when listing whether an effect was significant, please specify the exact ‘p value’ instead of just listing that the significance was less than 0.05. This will provide cohesion between the body of the text and the figures.

Author Response

We sincerely thank the reviewer for his valuable comments and suggestions, which have improved the manuscript. We appreciate your time and input. Changes in the text are highlighted

Major Concerns:

1. Authors note that ISO was able to induce cellular senescence through SA-B-gal staining and cardiac dysfunction through increased collagen deposition, cardiomyocyte hypertrophy, however, the physiological impact on cardiac function was not assessed via echocardiography. Please justify why these experiments were not performed in the discussion as this would have strengthened the ISO-induced cardiovascular dysfunction connection.

Response:

Thank you for this important comment. We agree that echocardiographic assessment would have strengthened the study. However, echocardiography facilities were not available at the institution where the study was conducted. Therefore, our study focused on established histological and morphometric indicators of cardiac injury and remodeling, including inflammatory cell infiltration, collagen deposition, cardiomyocyte hypertrophy, and increased heart weight-to-body weight ratio. These parameters are widely used to characterize pathological cardiac remodeling following isoproterenol administration. We acknowledge that the absence of functional assessment limits conclusions regarding cardiac injury and have highlighted this limitation in the Discussion section (lines 388-391).

1. It would help readers to know what markers are being used in each immunofluorescence panel, especially Figure 3. They are noted in the figure legend but would provide clarity if written off the side of each panel.

Response:

Thank you for this valuable comment. We agree that labeling the immunofluorescence markers will improve the figure's clarity. Therefore, we have updated the figures accordingly.

Minor Concerns:

1. Authors should note why only male rats were used in this study and note whether they suspect females would have a similar ISO response or if there would be a significant hormonal component.

Response:

Thank you for this important comment. We acknowledge the importance of including both male and female animals in preclinical studies. Male rats were selected to reduce biological variability associated with the estrous cycle and to establish an initial characterization of this exploratory model. Previous studies have reported sex-dependent differences in response to isoproterenol. We have acknowledged this limitation and added a paragraph in the Discussion (Lines 402–412).

1. Concerning the fibrosis/collagen staining in Figure 1. Please note whether the authors observed differences between interstitial or perivascular signal?

Response:

Thank you for this comment. Unfortunately, we did not separately quantify interstitial and perivascular fibrosis in the present study. Therefore, we cannot draw definitive conclusions regarding potential differences between these patterns of collagen deposition. However, based on visual assessment of the sections, we did not notice obvious differences in the perivascular regions between groups.

1. In the results when listing whether an effect was significant, please specify the exact ‘p value’ instead of just listing that the significance was less than 0.05. This will provide cohesion between the body of the text and the figures.

Response:

Thank you for this suggestion. We agree that reporting exact p-values will provide cohesion. However, because several sections of the Results summarize multiple related comparisons in one sentence, including all exact p-values in the text would substantially reduce readability. Therefore, we have retained the current format in the Results section, while the exact p-values are provided in the corresponding figures.

For example: ISO treatment significantly increased p16 (Day 10, p = 0.003; Day 28, p = 0.011), p21 (Day 10, p = 0.001; Day 28, p = 0.023), and SA-β-gal (Day 10, p = 0.0004; Day 28, p = 0.017).

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

This manuscript investigates the ability of isoproterenol (ISO) to induce cardiac damage and cellular senescence in Sprague-Dawley rats, proposing a simple and cost-effective model for studying cardiac senescence and testing senolytic therapies. The topic is relevant, and the study is generally well-designed. However, several issues should be addressed prior to publication.

Key Observations
1) Lack of assessment of cardiac function. The study relies exclusively on histological and molecular parameters. Functional data (e.g., echocardiography) would significantly enhance the translational value of the model.
2) Limited characterization of senescence. Senescence is inferred from SA-β-gal, p16, p21, and γH2AX. Adding markers and a more detailed discussion of their limitations would strengthen the conclusions.
3) Cell type identification. It is still unclear which cardiac cell populations (cardiomyocytes, fibroblasts, endothelial cells, etc.) undergo senescence. This should be recognized as a limitation.
4) SASP assessment. Only mRNA expression was measured. Validation at the protein level (ELISA or multiplex assays) would provide more robust evidence of SASP activation.

Minor Comments
1) The rationale behind choosing days 10 and 28 should be explained more clearly in the Methods section.
2) The title of Figure 1 should be revised, as the figure primarily shows inflammation and fibrosis rather than hypertrophy.
3) Some statements in the Discussion and Conclusions are slightly exaggerated and should be revised, particularly regarding the utility of the model for developing senescence-targeted therapies.

Kind regards.

Author Response

We sincerely thank the reviewer for his valuable comments and suggestions, which have improved the manuscript. We appreciate your time and input. Changes in the text are highlighted

Key Observations
1) Lack of assessment of cardiac function. The study relies exclusively on histological and molecular parameters. Functional data (e.g., echocardiography) would significantly enhance the translational value of the model.

Response:

Thank you for this important comment. We agree that echocardiographic assessment would have strengthened the study. However, echocardiography facilities were not available at the institution where the study was conducted. Therefore, our study focused on established histological and morphometric indicators of cardiac injury and remodeling, including inflammatory cell infiltration, collagen deposition, cardiomyocyte hypertrophy, and increased heart weight-to-body weight ratio. These parameters are widely used to characterize pathological cardiac remodeling following isoproterenol administration. We acknowledge that the absence of functional assessment limits conclusions regarding cardiac performance and have highlighted this limitation in the Discussion section (lines 387-391).

2) Limited characterization of senescence. Senescence is inferred from SA-β-gal, p16, p21, and γH2AX. Adding markers and a more detailed discussion of their limitations would strengthen the conclusions.

Response:

Thank you for this important comment. Cellular senescence does not have a single gold-standard marker that is both fully sensitive and specific. In addition, there are currently no universally accepted guidelines for senescence identification. Therefore, we evaluated multiple established senescence-associated markers, including SA-β-gal activity, p16, p21, γH2AX, and several SASP-related factors. These markers are consistent with previous studies investigating cardiac senescence, which similarly used combinations of SA-β-gal, p16, p21, DNA damage markers, and SASP factors to characterize senescent cell accumulation.

Example of papers that used similar approaches

• DOI: 10.1111/acel.13249
• DOI: 10.1097/FJC.0000000000000878
• DOI: 10.1016/j.actbio.2021.08.028
• DOI: 10.1371/journal.pone.0182668

 

Additional markers such as Lamin B1 loss, telomere-associated DNA damage foci (TAFs), or expanded SASP profiling could provide further characterization. We believe that the combined evidence presented in this study provides a generally accepted assessment of senescence in this cardiac injury model and is consistent with current in vivo studies in the field. However, we have added this point in discussion (Lines 390-393) .

3) Cell type identification. It is still unclear which cardiac cell populations (cardiomyocytes, fibroblasts, endothelial cells, etc.) undergo senescence. This should be recognized as a limitation.

Response:

Thank you for this important comment. We agree that identifying the specific cardiac cell populations undergoing senescence would provide additional mechanistic insight into the model. In the present study, our primary objective was to establish whether ISO induces cardiac senescence and remodeling at the tissue level rather than to determine the contribution of individual cardiac cell types. Therefore, the senescence markers were assessed across cardiac tissue sections without cell-type-specific co-localization. Future studies combining senescence markers with cell-specific markers for cardiomyocytes, fibroblasts, endothelial cells, and immune cells would help identify the cellular sources of senescence in this model. We have now acknowledged this limitation in the Discussion (Lines 397-401).

4) SASP assessment. Only mRNA expression was measured. Validation at the protein level (ELISA or multiplex assays) would provide more robust evidence of SASP activation.

Response:

Thank you for this valuable comment. We agree that measuring SASP factors at the protein level using ELISA or multiplex cytokine assays would further strengthen the assessment of SASP activation. However, in the present study, we assessed several SASP-related transcripts alongside established senescence markers, including SA-β-gal, p16, p21, and γH2AX. We have acknowledged this limitation in the Discussion (393-395).

Minor Comments
1) The rationale behind choosing days 10 and 28 should be explained more clearly in the Methods section.

Response:

Thank you for this important comment. We agree that the rationale for selecting days 10 and 28 was not clear in the Methods . Therefore, we have expanded the description and clarified the reasons for selecting these time points. (Lines 96–102).

2) The title of Figure 1 should be revised, as the figure primarily shows inflammation and fibrosis rather than hypertrophy.

Response:

Thank you for this important comment. The title of Figure 1 was mistakenly written as referring to hypertrophy. The title has now been updated.

3) Some statements in the Discussion and Conclusions are slightly exaggerated and should be revised, particularly regarding the utility of the model for developing senescence-targeted therapies.

Response:

Thank you for this important comment. We agree that some statements were slightly exaggerated. Therefore, we have modified the language in the abstract (lines 26-28) and conclusion (Lines 415-420).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors have sufficiently addressed reviewer comments. Great work!