Application of Digital Holographic Imaging to Monitor Real-Time Cardiomyocyte Hypertrophy Dynamics in Response to Norepinephrine Stimulation

Round 1

Reviewer 1 Report

Wahida Akter, et al. presented an article using Holographic monitor to observe the dynamic change of cardiomyocytes after the treat of norepinephrine. The followings are some comments to be addressed.

comments

1.         The study itself is very interesting and the utility of Holographic monitor to observe the dynamic change of cardiomyocyte is appealing. This study can be the basis of further application of studies about dynamic cardiomyocyte changes.

2.         About the use of norepinephrine and cardiomyocyte hypertrophy, I think the link is not so strong, especially in the study design of this presented article. Hypertrophic change may take months to be happened. Besides, there are some other peptides contributing to hypertrophy. The value of this article is to provide an evidence that the dynamic change can be recorded by the Holographic monitor. Therefore, I don’t think the authors need to connect norepinephrine to hypertrophy. Just showing the cell change by the treatment of norepinephrine and the ability to record it by the Holographic monitor is enough to contribute to scientific value.

3.         I do have a concern about the smaller optical volume of cells treated by norepinephrine. However, the authors have had a comprehensive explanation in the discussion section. I appreciate with that. 

Author Response

We thank the reviewer for their comments and suggestions. Our point-by-point response to each of the reviewer’s comments is provided below in blue.

1. The study itself is very interesting and the utility of Holographic monitor to observe the dynamic change of cardiomyocyte is appealing. This study can be the basis of further application of studies about dynamic cardiomyocyte changes.

We thank the reviewer for their enthusiasm for the approach and we agree that the technology could empower future studies looking at cardiomyocyte hypertrophy dynamics in vitro.

2. About the use of norepinephrine and cardiomyocyte hypertrophy, I think the link is not so strong, especially in the study design of this presented article. Hypertrophic change may take months to be happened. Besides, there are some other peptides contributing to hypertrophy. The value of this article is to provide an evidence that the dynamic change can be recorded by the Holographic monitor. Therefore, I don’t think the authors need to connect norepinephrine to hypertrophy. Just showing the cell change by the treatment of norepinephrine and the ability to record it by the Holographic monitor is enough to contribute to scientific value.

We thank the reviewer for this comment. To clarify, our study aims to provide a proof-of-principle demonstration of the capabilities of the Holomonitor M4 to record cardiomyocyte hypertrophy dynamics in vitro. In vivo, cardiac hypertrophy can indeed take months to manifest involving the complex interactions of numerous signaling pathways. In vitro, however, norepinephrine is sufficient to induce hypertrophic growth in cultured rat cardiomyocytes in serum-free culture, specifically through activation of alpha-adrenergic signaling. Therefore, the goal of this manuscript is not to link norepinephrine to cardiomyocyte hypertrophy since this is already well established, but rather use norepinephrine as a tool to demonstrate the Holomonitor M4’s ability to record and track dynamic changes in cardiomyocyte surface area and volume in response to this stimulus. 

3. I do have a concern about the smaller optical volume of cells treated by norepinephrine. However, the authors have had a comprehensive explanation in the discussion section. I appreciate with that.

Yes, we shared the reviewer’s concern as well as it is unlikely that the initial absolute volumes of cardiomyocytes treated with norepinephrine are actually less than that seen in controls at the beginning of the experiment. In our experiments, it was evident that norepinephrine induces a rapid “spreading” effect in cardiomyocytes. We feel that it is important to also highlight the limitations of the Holomonitor M4 imaging approach and provide transparency with regard to this observation in the discussion section. Therefore, there may be a limitation in the Holomonitor M4’s analysis software to distinguish signal from noise in cells that are thinly spread.

Reviewer 2 Report

Authors have submitted a manuscript regarding the use of Holomonitor M4 in monitoring Cardiomyocyte hypertrophy dynamics after norepinephrine stimulation. Despite the fact that the topic is quite interesting, some inherent caveats need to be highlighted. The manuscript structure needs to be formed according to guidelines : Introduction, Material-Methods, Results, Discussion, Conclusions. This manuscript does not include conclusions and the other parts do not follow the appropriate sequence. Please form the manuscript in accordance to these guidelines. Also, references need to be modified by adding more recent and contemporary literature and be formed according to guidelines of the journal e.g…….(1). In the results authors have to support statistical significance by adding p value. Images and graphs included are thought to be a strong positive point of the manuscript making the topic more clear and easy to understand for the reader. However , it would be favourable if a clinical message and extension is added in the manuscript in order to link basic science with clinical applications. 

Author Response

We thank the reviewer for their comments and suggestions. Our point-by-point response to each of the reviewer’s comments is provided below in blue.

Authors have submitted a manuscript regarding the use of Holomonitor M4 in monitoring Cardiomyocyte hypertrophy dynamics after norepinephrine stimulation. Despite the fact that the topic is quite interesting, some inherent caveats need to be highlighted.

We thank the reviewer for their enthusiasm in the approach and agree that it is equally important to highlight the approach’s limitations in addition to its strengths. In the discussion section, we highlight two significant caveats in the methodology. First, we point out a possible source of measurement error in the analysis software to distinguish signal from background noise, which could underestimate the raw surface area and volume measurements made, especially in thinly spread cells. Second, we highlight the limitation of the approach to accurately monitor cardiomyocytes found in aggregate cell clusters. Therefore, this technique seems to be most appropriately applied to the well-isolated cardiomyocytes that could be tracked with high fidelity. 

The manuscript structure needs to be formed according to guidelines : Introduction, Material-Methods, Results, Discussion, Conclusions. This manuscript does not include conclusions and the other parts do not follow the appropriate sequence. Please form the manuscript in accordance to these guidelines.

We thank the reviewer for pointing this out. We’ve reformatted the document accordingly and have now included a conclusions section. The references are in the ACS style as recommended by the “Reference List and Citations Style Guide for MDPI Journals”.

Also, references need to be modified by adding more recent and contemporary literature and be formed according to guidelines of the journal e.g…….(1). 

We thank the reviewers for this suggestion. We now cite recent papers demonstrating the use of digital holography to monitor human-induced pluripotent stem cell-derived cardiomyocyte contraction dynamics. To the best of our knowledge, we did not identify any papers using the digital holography to monitor the dynamics of cardiomyocyte hypertrophic growth, which is the focus of this paper.

In the results authors have to support statistical significance by adding p value. 

We thank the reviewer for this comment. To clarify, the p-values for all statistically significant differences noted by asterisks are noted within the associated figure legends.

Images and graphs included are thought to be a strong positive point of the manuscript making the topic more clear and easy to understand for the reader. However , it would be favourable if a clinical message and extension is added in the manuscript in order to link basic science with clinical applications.

We thank the reviewer for this suggestion. We’ve now further discussed the significance of pathological cardiac hypertrophic responses in the introduction to solidify the clinical relevance of this basic science question.

Reviewer 3 Report

Dear Authors,

Firstly, I want to express my appreciation for the contribution of your paper titled "Application of Digital Holographic Imaging to Monitor Real-Time Cardiomyocyte Hypertrophy Dynamics in Response to Norepinephrine Stimulation" to the field of applied science. Your work presents an intriguing application of digital holographic microscopy to study the hypertrophic response of neonatal rat ventricular myocytes, which undoubtedly adds valuable insights to the understanding of cardiomyocyte dynamics.

However, I believe there are areas in which the paper can be strengthened to better emphasize its scientific relevance and application potential. Specifically, the introduction lacks sufficient arguments to highlight the scientific relevance of holographic microscopy, and a more comprehensive discussion could contrast its benefits with other techniques. Moreover, the comparison limited to untreated cells overlooks the opportunity to showcase differences among various hypertrophic inducers, thereby underscoring the versatility of your method.

I also noticed that the paper refers to the cells as rat cardiomyocytes and depicts them as adult cardiomyocytes in Figure 1A, which may lead to confusion. It's important to clarify that neonatal rat ventricular myocytes are utilized in the study, and further discussion regarding the translatability of findings to other types of cardiomyocytes, such as adult cardiomyocytes or IPS-derived cardiomyocytes, would be beneficial.

Additionally, statistical analysis information should be included in the method section rather than in the figure captions. Finally, in Figure 1F, error marks are missing in the columns of the graph, which must be rectified.

Author Response

We thank the reviewer for their comments and suggestions. Our point-by-point response to each of the reviewer’s comments is provided below in blue.

Dear Authors,

Firstly, I want to express my appreciation for the contribution of your paper titled "Application of Digital Holographic Imaging to Monitor Real-Time Cardiomyocyte Hypertrophy Dynamics in Response to Norepinephrine Stimulation" to the field of applied science. Your work presents an intriguing application of digital holographic microscopy to study the hypertrophic response of neonatal rat ventricular myocytes, which undoubtedly adds valuable insights to the understanding of cardiomyocyte dynamics.

We thank the reviewer for their enthusiasm in the approach. We agree with the reviewer in the potential for this methodology to yield novel future insights into the dynamic of cardiomyocyte hypertrophic responses in vitro.

However, I believe there are areas in which the paper can be strengthened to better emphasize its scientific relevance and application potential. Specifically, the introduction lacks sufficient arguments to highlight the scientific relevance of holographic microscopy, and a more comprehensive discussion could contrast its benefits with other techniques. 

We thank the reviewer for this suggestion. We’ve now included additional discussion regarding the limitations of existing techniques to measure cardiomyocyte size in the introduction. More specifically, we include discussion regarding traditional surface area measurements, Coulter counters, flow cytometry, and confocal microscopy.

Moreover, the comparison limited to untreated cells overlooks the opportunity to showcase differences among various hypertrophic inducers, thereby underscoring the versatility of your method.

We thank the reviewer for this comment. Our main comparisons in this manuscript are between untreated and norepinephrine-treated cardiomyocytes. We used norepinephrine solely as a model hypertrophic stimulus to demonstrate the utility of the Holomonitor M4 to monitor cardiomyocyte hypertrophy dynamics. We do believe that using this methodology to characterize similarities, differences, and synergies between various hypertrophic inducers would be quite interesting, but also think these comparisons would be best pursued as follow-up future studies. We now mention the potential for these future applications in the discussion section.

I also noticed that the paper refers to the cells as rat cardiomyocytes and depicts them as adult cardiomyocytes in Figure 1A, which may lead to confusion. It's important to clarify that neonatal rat ventricular myocytes are utilized in the study, 

We acknowledge the reviewer's point that adult rodent cardiomyocytes are thick and rod-shaped. In our experience, freshly isolated neonatal rodent cardiomyocytes are also rod-shaped, skinnier, and elongated. The diagram in Figure 1A is now edited to demonstrate this difference. Additionally, we include the word “neonatal” in the labels to further clarify any confusion. 

and further discussion regarding the translatability of findings to other types of cardiomyocytes, such as adult cardiomyocytes or IPS-derived cardiomyocytes, would be beneficial.

We thank the reviewer for this suggestion and now include the broader potential of this approach to investigate hypertrophic responses in other cardiomyocytes as well including adult and iPS-derived cardiomyocytes in the discussion.

Additionally, statistical analysis information should be included in the method section rather than in the figure captions. 

We thank the reviewer for this suggestion. Further details on the statistical analyses performed are now mentioned in the methods section.

Finally, in Figure 1F, error marks are missing in the columns of the graph, which must be rectified.

We thank the reviewer for pointing this out. The purpose of the original Figure 1F was to show the approximate fold-change in cardiomyocyte optical volume increase after norepinephrine treatment. To improve clarity, we now revised the organization of Figures 1D and 1E. Figure 1D now focuses exclusively on the raw optical volumes that we measured in cardiomyocytes. Figure 1E now focuses on the raw optical volume measurements in non-cardiomyocytes. Error bars depicting the standard deviation of individual measurements per cell are now shown. Since the revised Figure 1D and Figure 1E are sufficient to show the statistically significant increase in cardiomyocyte optical volume after norepinephrine treatment, we’ve omitted Figure 1F. These changes are made in the figures and are now reflected in the manuscript as well.

Reviewer 4 Report

In this paper, the authors proposed a study that utilized the Holomonitor M4 digital holographic imaging system to perform real-time imaging and single-cell tracking of neonatal rat cardiomyocytes, both untreated and stimulated with norepinephrine. This approach allowed for the measurement and analysis of cellular parameters such as surface area and optical volume, revealing dynamic changes in cardiomyocyte morphology and volume over a 24-hour period. Although this study successfully validated the reliability of the technique in exploring cardiomyocyte hypertrophic responses with enhanced temporal resolution. Nevertheless, there are a few points worth mentioning. Firstly, technical constraints may still pose challenges in the long-term monitoring of cardiomyocyte changes. Prolonged imaging and cell tracking could potentially lead to altered cellular states or cell death, thereby compromising the reliability of the proposed results. Secondly, the lack of certain control groups or insufficient consideration of all possible variables might affect the outcomes. For instance, Subfigures C) and E) in Figure 2 article appear highly similar, thus necessitating more scenarios for long-term observations. Thirdly, while leveraging the unique advantages of the Holomonitor M4 system has demonstrated promise in understanding cardiomyocyte hypertrophy, further improvements and validations are crucial to fully harness its potential. Additionally, the use of neonatal rat primary cardiomyocytes for in vitro experiments generally does not raise significant ethical concerns. However, obtaining official ethical approval would be advisable to address this issue.

English is okay for publication. Minor revisions will improve the reabability.

Author Response

We thank the reviewer for their comments and suggestions. Our point-by-point response to each of the reviewer’s comments is provided below in blue.

In this paper, the authors proposed a study that utilized the Holomonitor M4 digital holographic imaging system to perform real-time imaging and single-cell tracking of neonatal rat cardiomyocytes, both untreated and stimulated with norepinephrine. This approach allowed for the measurement and analysis of cellular parameters such as surface area and optical volume, revealing dynamic changes in cardiomyocyte morphology and volume over a 24-hour period. Although this study successfully validated the reliability of the technique in exploring cardiomyocyte hypertrophic responses with enhanced temporal resolution. Nevertheless, there are a few points worth mentioning. 

Firstly, technical constraints may still pose challenges in the long-term monitoring of cardiomyocyte changes. Prolonged imaging and cell tracking could potentially lead to altered cellular states or cell death, thereby compromising the reliability of the proposed results. 

We acknowledge the concern raised by the reviewer. The Holomonitor M4 utilizes a low-powered 635 nm laser to generate holograms for three-dimensional image reconstruction. This laser wavelength has been reported to minimize phototoxicity and promotes live-cell imaging over extended durations. We now add these references to our manuscript. While it still remains possible that 635 nm light may cause subtle changes in cardiomyocyte behavior, our experiments still validate the applicability of this approach to observe dynamic changes in norepinephrine-induced hypertrophic growth in comparison to untreated controls under the conditions described. However, if phototoxicity is observed, it may be possible to acquire timelapse images at longer intervals to further minimize the risk. These points are now added to the discussion.

Secondly, the lack of certain control groups or insufficient consideration of all possible variables might affect the outcomes. For instance, Subfigures C) and E) in Figure 2 article appear highly similar, thus necessitating more scenarios for long-term observations. 

We thank the reviewer for this comment. We clarify that the data presented in panel C represents raw surface area measurements, while panel E depicts normalized surface area measurements. We normalized the data in panel E by dividing the measurement for each cell at each time point by the starting surface area measurement for each cell at time 0 hour. The raw surface area measurements in Panel C look similar to the normalized measurements in Panel E since the starting baseline values for untreated and norepinephrine-treated cells were very similar. However, as acknowledged in the discussion section, there may be some error in characterizing the absolute surface or volume measurements due to the limitations of the analysis software to detect signals from background noise, particularly in thinly spread cells.

Thirdly, while leveraging the unique advantages of the Holomonitor M4 system has demonstrated promise in understanding cardiomyocyte hypertrophy, further improvements, and validations are crucial to fully harness its potential. 

We agree with the reviewer. Further refinement of imaging techniques and experimental methodologies will be essential to fully capitalize on the advantages of digital holographic imaging in advancing our understanding of hypertrophic processes and their implications for cardiovascular health. We note the limitations and future applications of this approach in the discussion section.

Additionally, the use of neonatal rat primary cardiomyocytes for in vitro experiments generally does not raise significant ethical concerns. However, obtaining official ethical approval would be advisable to address this issue.

We agree with the reviewer on the importance of obtaining ethical approval for the use of animals in research. All studies reported here are approved under IACUC protocol 1072 at San Jose State University, which is mentioned in the methods section.