Cardiomyocyte orientation recovery at micrometer scale reveals long‐axis fiber continuum in heart walls

Abstract Coordinated cardiomyocyte contraction drives the mammalian heart to beat and circulate blood. No consensus model of cardiomyocyte geometrical arrangement exists, due to the limited spatial resolution of whole heart imaging methods and the piecemeal nature of studies based on histological sections. By combining microscopy and computer vision, we produced the first‐ever three‐dimensional cardiomyocyte orientation reconstruction across mouse ventricular walls at the micrometer scale, representing a gain of three orders of magnitude in spatial resolution. We recovered a cardiomyocyte arrangement aligned to the long‐axis direction of the outer ventricular walls. This cellular network lies in a thin shell and forms a continuum with longitudinally arranged cardiomyocytes in the inner walls, with a complex geometry at the apex. Our reconstruction methods can be applied at fine spatial scales to further understanding of heart wall electrical function and mechanics, and set the stage for the study of micron‐scale fiber remodeling in heart disease.

24th Feb 2023 1st Editorial Decision Dear Dr. Sirajuddin, Thank you for submitting your manuscript for consideration by the EMBO Journal.It has now been seen by three referees whose comments are shown below.As you will see, the referees have raised concerns that the biological insight, specifically the newly identified longitudinal cardiomyocyte tracts, is not sufficiently supported by the data.That said, the referees are overall positive on the importance of the method provided.Therefore, I would like to invite you to submit a revised version of the manuscript, addressing the comments of all three reviewers.Unless you are prepared to provide more evidence to support your biological insights as suggested by the referees, we would ask that you readjust the language related to these findings and rather reframe this manuscript to be more protocol-focused.It is EMBO Journal policy to allow only a single round of revision, and acceptance of your manuscript will therefore depend on the completeness of your responses in this revised version.It would be good to discuss your plan to address the referee concerns and I am available to do so by email or zoom in the coming weeks.I have also attached a guide to revisions for your convenience.
When preparing your letter of response to the referees' comments, please bear in mind that this will form part of the Review Process File, and will therefore be available online to the community.For more details on our Transparent Editorial Process, please visit our website: https://www.embopress.org/page/journal/14602075/authorguide#transparentprocess We generally allow three months as standard revision time.As a matter of policy, competing manuscripts published during this period will not negatively impact on our assessment of the conceptual advance presented by your study.However, we request that you contact the editor as soon as possible upon publication of any related work, to discuss how to proceed.Should you foresee a problem in meeting this three-month deadline, please let us know in advance and we may be able to grant an extension.
Thank you for the opportunity to consider your work for publication.I look forward to your revision.

Yours sincerely, Kelly
Kelly M Anderson, PhD Editor The EMBO Journal k.anderson@embojournal.orgFurther information is available in our Guide For Authors: https://www.embopress.org/page/journal/14602075/authorguideWe realize that it is difficult to revise to a specific deadline.In the interest of protecting the conceptual advance provided by the work, we recommend a revision within 3 months (25th May 2023).Please discuss the revision progress ahead of this time with the editor if you require more time to complete the revisions.Use the link below to submit your revision: https://emboj.msubmit.net/cgi-bin/main.plex------------------------------------------------Referee #1: In the manuscript of EMBOJ-2022-113288 entitled "Myofiber reconstruction at micron scale reveals long-axis fiber continuum in heart ventricular walls", Dr. Dileep and colleagues reported a prominent long-axis fiber system in the outer walls of the heart, distinct from the well-known helical myofibers and the longitudinal myofibers in the inner heart walls, by using microscopy and computer vision with 3D reconstruction of myofibers at the micron scale.This paper is well written and may address an important topic.Also, the quality of figure and video is good; however, it will be somewhat hard to understand, and there are some concern to be addressed.1.The authors emphasized better understanding of myofiber geometry at the micron scale, compared with DT-MRI studies.Please discuss about it and clarify the clinical impact, rather than mentioning the association between pathological heart conditions and fiber organization.2. This study is based on histological examination.In human study, only small samples from endomyocardial biopsy are obtained, while DT-MRI can provide noninvasively whole-heart assessment even as less spatial resolution.Minor comments 1.Can they show the images without deconvolution and denosing?Is there any difference in results if not using deconvolution or denosing?2. Page 10, line 33 What do they mean as Figure 1a?Please modify the figure legend.3. Page 3, line 26 Words such as PSAX-PML, HLA-4C need to be spelled out.
Referee #2: Summary: In this manuscript, Dileep et al apply the CLARITY tissue clearing process to mouse hearts (and one rat heart), apply a stain that allows visualization of cardiomyocyte boundaries (WGA).Sections of these hearts are imaged using confocal microscopy, and then processed using structure tensor analysis.While this manuscript has some nice imaging data, in this reviewer's opinion importance of the novel "long-axis myofiber layer" is not supported by the data in the manuscript or the supplemental data.Overall, I don't recommend this manuscript for publication in its current form.Larger sample sizes and additional imaging or other data -separate from the structure-tensor processed data -would be needed in order for it to be considered a genuine anatomical discovery.In this reviewer's opinion this manuscript would be better suited to focus on its excellent image processing and visualization.
Major concerns: You refer to the "long-axis myofiber layer" "long-axis bands of fibers" "distinct from the known helical myofibers" "separate longaxis outer wall fiber system".Is there some sort of anatomic separation of this set of cardiomyocytes?Some sort of collagen structure or membrane?It seems that the distinctness of these cardiomyocytes from the rest of the myocardium is over-stated.The authors refer to "myofibers" throughout.The heart doesn't contain myofibers, and in this reviewer's opinion the term should no longer be accepted for anatomic descriptions of the heart.Within a large voxel (say 1x1x1 mm) there can be considered to be a "net cardiomyocyte orientation" or "aggregate cardiomyocyte orientation".If you're referring to structure tensor data, refer to the eigenvector directions.If you're referring to tract/streamline, refer to tract/streamline orientation.The text in the results section provides an insufficient summary of the experimental data.The majority of the text in the "results" section looks like it belongs in the methods or discussion section.Please refer to actual numerical results as much as possible, and avoid interpreting color maps.For me to consider a genuine "layer" of cells based on transmural helix angle I would want to see a clear discontinuity, which I don't see in your data.The abstract should be much more specific.What breed of mouse?How many mice?What tissue clearing process?How were the images processed?What is the most important helix angle data?pg 2 line 30-34 & pg 3 line 1-5 This paragraph is essentially wrong because of the Garcia-Canadilla et al 2019 paper (doi:10.1111/joa.13058),which you later misreport as using DT-MRI (pg10 line 12).Also Pope et al 2008(doi:10.1152/ajpheart.00484.2008), Teh et al 2017(doi:10.1186/s12968-017-0342-x).pg 5 line 19-23 This would likely look different depending which colormap you used.You should stick to reporting angle numbers rather than colors from color maps.pg 6 line 12-14 "entirely new discovery" To me the epicardial structures you're reporting look the same as those reported by Teh et al 2016Teh et al (doi:10.1016Teh et al /j.pbiomolbio.2016.06.06.001) Figures 3 and 7, and probably others.Why do you think the steep transition in epicardial helix angle is not observed in Pope et al 2008Pope et al (doi:10.1152Pope et al /ajpheart.00484.2008) ) which also as micron scale data?pg 6 18-21 You need to be much more descriptive in what Figure 3b is showing, and what resolutions the different colored lines are referring to, dotted versus solid etc. pg 6 line 26-29 How confident are you that these are not just papillary muscles?pg 8 line 9-11 "a resolution gain of about 3 orders of magnitude".I don't think this is correct.Please re-check Garcia-Canadilla et al 2019(doi:10.1111/joa.13058), Pope et al 2008(doi:10.1152/ajpheart.00484.2008), Teh et al 2017 (doi:10.1186/s12968-017-0342-x).pg 9 line 1-17 You suggest that your new "long-axis myofiber layer" is important for electrical propagation in the heart, but you spend only a single sentence including the cardiac conduction system and the Purkinje fibers.Please indicate your long-axis layer would work with the cardiac conduction system, and whether your layer are specialized conduction cells.pg 12 line 22-25 It is not clear which angles you are referring to, and unfortunately the diagram in Figure 2 is also not clear.Are you using projected helical angle, transverse angle and helical angle as in Agger et al 2020 Figure 2 (doi:10.1002/nbm.4205)?If so please refer to them using these names.For theta, when the cell long axis is projected onto the short-axis plane, are you calculating the angle from the radial or the circumferential direction?
Minor concerns: pg 1 line 17 Cardiomyocytes often have a branching structure and do not form long exclusively end-to-end myofibers as in skeletal muscle.pg 2 line 3 Cardiomyocytes often have a branching structure and do not form long exclusively end-to-end myofibers as in skeletal muscle.pg 4 line 6 This is entirely expected of cutting cylinder-shaped cells at different angles -it seems strange to call it a "striking feature" pg 4 line 13-14 "strong agreement between the two" I'd like to see a numerical result here.What was the mean/median difference in orientation?What it centered on zero?Was there any bias?pg line 21-23 Why use three angles?Only two are needed.Helix angle and transverse angle OR helical angle and intrusion angle (Agger et al 2019(Agger et al , doi:10.1002/nbm.4205) /nbm.4205)pg line 24 Please use circumferential, longitudinal, radial (C, L, R) coordinates -the reader isn't particularly interested in the orientation you imaged the samples, more in the anatomical directions.pg line 1-3 Cardiomyocytes often have a branching structure and do not form long exclusively end-to-end myofibers as in skeletal muscle.pg line 24 "distinct bands of long-axis fibers" In my opinion you would need histology data that shows a clear membrane or separation of these epicardial cardiomyocytes to make this claim.pg line 15-16 I'm not convinced that there are three separate fiber/tract systems here.The apical section clearly has a triangular shape that may or may not be giving the impression of three groups.Even numerical analysis of these tracts will be impacted by the apparent size of the tracts and the kernel size used for the structure tensor analysis.pg line 24-25 "revealed an entirely new long-axis fiber system" Even Streeter et al 1969(doi:10.1161/01.RES.24.3.339)showed a sharp transition of longitudinally oriented to circumferentially oriented cardiomyocytes.How is your finding different from this? pg line 27 "ventricular walls of four-chambered hearts" 6 mouse hearts and 1 rat heart is not sufficient data to make this claim.Figure 1 It is very difficult to see the purple cylinders, perhaps because the yellow cylinders are so much longer than the purple ones.Figure 2 I know the angle diagrams are difficult to construct, but the angle diagram is not clear, apart from the helix angle.Figure 3 In b, make clear which sections each of the three solid lines represent from the 2 hearts.Also in the pseudo low resolution the main thing missing is the sharp but somewhat random transitions (green -65 to -90, blue -95 to -85, red -85 to -90), are you saying that these variable transitions indicate your long-axis fiber layer?I'm not sure what this pseudo low resolution data is showing me.Edits: pg line 23 Referencing error -presumably they shouldn't be the same reference (18).They're also a different referencing style.pg line 23 Italicize "ex vivo" pg line 22-30 This belongs in the methods section, not the results section.pg line 32-34 This belongs in the methods section, not the results section.pg line 1-5 This belongs in the methods section, not the results section.pg line 9-11 This belongs in the methods section, not the results section.pg line 14-17 This belongs in the discussion section, not the results section.pg line 21-27 This belongs in the methods section, not the results section.pg line 27-32 This belongs in the discussion section, not the results section.pg line 32-33 This belongs in the methods section, not the results section.pg line 3-5 This belongs in the methods section, not the results section.pg line 5-7 This belongs in the discussion section, not the results section.pg line 10-12 This belongs in the methods section, not the results section.pg line 15-18 This belongs in the discussion section, not the results section.pg line 30-32 This belongs in the discussion section, not the results section.pg line 32-33 This belongs in the methods section, not the results section.pg line 1-2 This belongs in the methods section, not the results section.pg line 9-12 This belongs in the discussion section, not the results section.pg line 14-15 This belongs in the intro section, not the results section.pg line 15-18 This belongs in the methods section, not the results section.pg line 2 Please use "wedge sector" or simply "sector" rather than "pie-shaped sector".pg line 4 Please use anatomic directions such as anterior/posterior/septal/lateral/RV instead of "3 O'clock position" Referee #3: The paper describes a method to reconstruct myocardial fibers at micron-scale resolution.The topic is of interest for the clinical cardiac imaging community, in particular DT-MRI, since microscopy can provide ground truth orientation measurements.The proposed experimental protocol included tissue clearing to prepare the fresh excised mice hearts, slicing of short-axis and longaxis slabs, and confocal microscopy imaging.Image processing methods are used to register and assemble the image stacks, to mitigate blurring and noise, and to reconstruct fiber orientation using the structure tensor.The overall patterns of myofiber organization agree with the literature, however it is claimed that a new distinct fiber structure was found in the outer wall.The experiments and methods are clearly described.The image processing methods are sound and use state-of-the-art algorithms.I have some questions though, listed below.Major comments 1) About the claim of a new fiber system found in the outer wall : isn't is the pericardium?It seems to me that some layers of pericardium contain fibrous structures.Would it be useful to indicate the different cell types on the raw images to make it clear?2) Validation of the structure tensor calculations: 70 myocytes where manually selected for comparison, how where they selected?Was it randomly, did the authors check on difficult cases, i.e. when the structure tensor is very different from the expected elongated cell type? 3) Other ways to visualize the orientation results could also be considered, inspired from DT-MRI: color-coded fractional anisotropy map is a nice way to summarize the fiber orientation in a single image.Fractional anisotropy may also help to check where the rank-1 structure tensor is not a good fit to the actual structure.4) Figure 3 is difficult to read: it is not clear what the top graph is showing (missing legends in b, not obvious which region the zoomed plot corresponds to).Penetration depth should rather be call transmural depth (to avoid confusion with the light penetration depth problem mentioned in another part of the paper).In b, c, d, it could be indicated where is the inner/outer edge of the wall, i.e. left or right in the abscise axis?Also, I found the sharp changes indicated by the authors are clearly visible in the alphaH images, but not so clear on the transmural plots (maybe indicate them with an arrow).5) I found the application of the patch-based denoising interesting, i.e. training the dictionary on the high-SNR data to denoise the lower-SNR data.Since the SNR varies with the z location in the slab, there might be a risk of a slight bias in the HA maps, due to residual noise being interpreted as structural gradient by the structure tensor.Did the authors consider that?As an alternative, would it be possible to acquire the images twice with flipped orientation of the sample?Minor comments 6) Averaging of the structure tensor (section 3.6 of Supplementary material): why did the authors apply another filtering here, wouldn't it be simpler to increase the Gaussian kernel width/sigma already present in the structure tensor definition?7) Maybe a point of discussion: apart from DT-MRI, phase-contrast CT has also been reported to achieve 3D myofiber reconstruction, and its resolution may be on the order of 10 microns.Since anyway, we are talking about cell orientation, going below the cell size is not strictly necessary.Actually with the Gaussian filter in the structure tensor definition (sigma=3 voxels) and the further averaging, the present study is working roughly at the same resolution.I think the main advantage is not so much the gain in resolution, but the more direct and clear visualization of cell boundaries which make the estimate more trustable and which can be validated by the manual contouring (this seems to me the most important part in the results).8) Helix angle, in all color-coded images, is scaled between 0 and 90{degree sign}, whereas it should be between -90 and 90{degree sign} (as it is in the transmural plots).Could the authors make it consistent throughout the figures?9) Fig 2e : there seems to be a problem with the aplhaH computation here, could the authors comment (although theta and phi maps seem correct)?Also it seems to be a slightly different slice and mask compared to c and d.

Detailed Response to Referee comments (EMBOJ-2022-113288R):
Referee #1 (Remarks to the Author): In the manuscript of EMBOJ-2022-113288 entitled "Myofiber reconstruction at micron scale reveals long-axis fiber continuum in heart ventricular walls", Dr. Dileep and colleagues reported a prominent long-axis fiber system in the outer walls of the heart, distinct from the well-known helical myofibers and the longitudinal myofibers in the inner heart walls, by using microscopy and computer vision with 3D reconstruction of myofibers at the micron scale.This paper is well written and may address an important topic.Also, the quality of figure and video is good; however, it will be somewhat hard to understand, and there are some concern to be addressed.
1.The authors emphasized better understanding of myofiber geometry at the micron scale, compared with DT-MRI studies.Please discuss about it and clarify the clinical impact, rather than mentioning the association between pathological heart conditions and fiber organization.
2. This study is based on histological examination.In human study, only small samples from endomyocardial biopsy are obtained, while DT-MRI can provide noninvasively whole-heart assessment even as less spatial resolution.

Author Response:
We thank the reviewer for this summary of our methodology and findings and for appreciating our work.In the Discussion we have included mention of the clinical impact of our work and have also addressed the tradeoff between assessing myofiber geometry at the cellular scale versus at the wholeheart (typically millimeter) scale of other studies.Please refer to page 8, lines 31-33 and page 9, lines 1-15.

Minor comments
1.Can they show the images without deconvolution and denoising?Is there any difference in results if not using deconvolution or denoising?
Author Response: We computed the orientation estimates and the corresponding helix angle for SAS3 data and show the comparison between raw data and the pre-processed images in Supplementary Figure 4c.The comparison shows that pre-processing (deconvolution + denoising) significantly improves the orientation estimation and calculation of angles.
2. Page 10, line 33 What do they mean as Figure 1a?Please modify the figure legend Author Response: We thank the reviewer for pointing out this mistake.We have corrected this error in the revised manuscript.

Author Response:
We have provided the expanded form of each of these acronyms in the revised manuscript.Please see Results,page 3, 5th Jun 2023 1st Authors' Response to Reviewers Referee #2 (Remarks to the Author): Summary: In this manuscript, Dileep et al apply the CLARITY tissue clearing process to mouse hearts (and one rat heart), apply a stain that allows visualization of cardiomyocyte boundaries (WGA).Sections of these hearts are imaged using confocal microscopy, and then processed using structure tensor analysis.While this manuscript has some nice imaging data, in this reviewer's opinion importance of the novel "long-axis myofiber layer" is not supported by the data in the manuscript or the supplemental data.Overall, I don't recommend this manuscript for publication in its current form.Larger sample sizes and additional imaging or other data -separate from the structure-tensor processed data -would be needed in order for it to be considered a genuine anatomical discovery.In this reviewer's opinion this manuscript would be better suited to focus on its excellent image processing and visualization.

Author Response:
We appreciate the concerns raised by the reviewer.We acknowledge the limitations of our work, and as the reviewer suggests, we now refer to the long-axis fibers as a geometrically distinct long-axis system, which is what our data and 3D reconstructions support.We appreciate the reviewer's positive comments about the image processing and visualization methods we have introduced.
Major concerns: You refer to the "long-axis myofiber layer" "long-axis bands of fibers" "distinct from the known helical myofibers" "separate long-axis outer wall fiber system".Is there some sort of anatomic separation of this set of cardiomyocytes?Some sort of collagen structure or membrane?It seems that the distinctness of these cardiomyocytes from the rest of the myocardium is over-stated.

Author Response:
We appreciate this concern.From our imaging data and analyses we have not observed a physical or anatomical separation between the long-axis fibers and the circumferential (helical) ones.However, Pope et. al. 2008(doi:10.1152/ajpheart.00484.2008)have found evidence of a distinct organization of collagen in the outer wall region, suggesting that some level of anatomical separation in terms of the collagen matrix could occur.As stated above, we now refer to cardiomyocyte orientation and the recovered streamlines as a geometrically distinct system, throughout the revised manuscript.Please see page 1, lines 23-24, page 3, lines 16-23, page 6, lines 1-4, page 8, lines 12-15.The authors refer to "myofibers" throughout.The heart doesn't contain myofibers, and in this reviewer's opinion the term should no longer be accepted for anatomic descriptions of the heart.Within a large voxel (say 1x1x1 mm) there can be considered to be a "net cardiomyocyte orientation" or "aggregate cardiomyocyte orientation".If you're referring to structure tensor data, refer to the eigenvector directions.If you're referring to tract/streamline, refer to tract/streamline orientation.

Author Response:
We agree and thank the reviewer for this comment.Indeed, although the term `myofiber' has been widely used in the literature, fibers do not actually exist in a structural sense in the heart wall.We have modified our manuscript to explain, at the beginning, our use of the term 'fibers' to mean 'cardiomyocyte aggregates', and have also discussed the relationship to streamlines generated from cardiomyocyte orientation estimates.We have followed the reviewer's constructive suggestion to refer to eigenvector based orientations or tract/streamline orientations or to use the term aggregate, when appropriate.
The text in the results section provides an insufficient summary of the experimental data.The majority of the text in the "results" section looks like it belongs in the methods or discussion section.Please refer to actual numerical results as much as possible, and avoid interpreting color maps.For me to consider a genuine "layer" of cells based on transmural helix angle I would want to see a clear discontinuity, which I don't see in your data.
Author Response: The epicardial structures reported in Teh et al. 2016 are at the millimetre voxel resolution.Figure 3 of Teh et al. 2016 shows orientation glyphs in a short-axis view, where a sharp change in aH at the outer wall is not evident.Figure 8  Author Response: Garcia-Canadilla et al. 2019 use high resolution episcopic microscopy to image a thin section of the postnatal mouse heart, infiltrated with methacrylate resin.They report an XY resolution of ~5.5 um and 3 um Z slice thickness.The voxel size used by Garcia-Canadilla is therefore ~164 um^3.Thus their study is at a coarser resolution than ours.Pope et al. 2008 focuses on the organization of collagen in the extracellular matrix in wild type rat hearts at an isotropic resolution of 1 um.Further, they only image a limited heart wall region and not the entire heart section.Teh et al. 2017 use an isotropic voxel of size 7.2 um.Both these studies do not use staining specific to cell membranes in the heart and thus cannot provide data to infer cardiomyocyte boundaries or fiber orientation.We have included this point in the Discussion (page 9, lines 4-12).pg 9 line 1-17 You suggest that your new "long-axis myofiber layer" is important for electrical propagation in the heart, but you spend only a single sentence including the cardiac conduction system and the Purkinje fibers.Please indicate your long-axis layer would work with the cardiac conduction system, and whether your layer are specialized conduction cells.

Author Response:
This was a speculative discussion point, drawing a connection to past work (e.g., the connection to conduction in the long-axis epicardial system in the Circulation paper by Vetter et al.).We have not determined whether the cells in the long-axis system are specialized conduction cells.Thus, the question of the role of a geometrically distinct long axis system for conduction remains a topic for future investigation.We have expanded this point in the Discussion (page 10, lines 22-33).
pg 12 line 22-25 It is not clear which angles you are referring to, and unfortunately the diagram in Figure 2 is also not clear.Are you using projected helical angle, transverse angle and helical angle as in Agger et al 2020 Figure 2 (doi:10.1002/nbm.4205)?If so please refer to them using these names.For theta, when the cell long axis is projected onto the short-axis plane, are you calculating the angle from the radial or the circumferential direction?
Author Response: We thank the reviewer for pointing this out.We have improved the illustration in Figure 2a.We have also cited Agger et al. 2020 for the helix angle we are referring to.
Minor concerns: pg 1 line 17 Cardiomyocytes often have a branching structure and do not form long exclusively endto-end myofibers as in skeletal muscle.pg 2 line 3 Cardiomyocytes often have a branching structure and do not form long exclusively endto-end myofibers as in skeletal muscle.

Author Response:
In the revised manuscript we have addressed this by referring to the aggregate cardiomyocyte orientation, following the reviewer's earlier suggestion.pg 4 line 6 This is entirely expected of cutting cylinder-shaped cells at different angles -it seems strange to call it a "striking feature"

Author Response:
We have modified this sentence to clarify what we meant (page 4, lines 12-13).pg 4 line 13-14 "strong agreement between the two" I'd like to see a numerical result here.What was the mean/median difference in orientation?What it centered on zero?Was there any bias?
Author Response: We thank the reviewer for this comment and the opportunity to clarify this point.We have examined the absolute value of the difference between hand segmented and quantitatively estimated cardiomyocyte orientations.We have included the numerical mean/median absolute difference in orientation in the revised manuscript (page 4, lines 19-24).This is true, but it is not straightforward to robustly compute the helix angle aH in the long-axis sections since an estimate of the transmural penetration direction is required for it.We therefore chose the magnitude of the  angle to highlight the long-axis orientation of outer wall cells in a short-axis section (Figure 2d).We have also improved our presentation of definitions of angles and their illustration in Figure 2a.pg 4 line 24 Please use circumferential, longitudinal, radial (C, L, R) coordinates -the reader isn't particularly interested in the orientation you imaged the samples, more in the anatomical directions.

Author Response:
We have included the C, L, R coordinate frame in our illustration of the helix angle.Please see Figure 2a.pg 5 line 24 "distinct bands of long-axis fibers" In my opinion you would need histology data that shows a clear membrane or separation of these epicardial cardiomyocytes to make this claim.
Author Response: This concern is similar to that raised by the reviewer earlier.We have addressed this point and have revised the manuscript accordingly to refer to a geometrically distinct long-axis system.Please see page 1, line 23-24, page 3, lines 16-23, page 6, lines 1-4, page 8, lines 12-17.pg 7 line 15-16 I'm not convinced that there are three separate fiber/tract systems here.The apical section clearly has a triangular shape that may or may not be giving the impression of three groups.Even numerical analysis of these tracts will be impacted by the apparent size of the tracts and the kernel size used for the structure tensor analysis.pg 7 line 24-25 "revealed an entirely new long-axis fiber system" Even Streeter et al 1969 (doi:10.1161/01.RES.24.3.339)showed a sharp transition of longitudinally oriented to circumferentially oriented cardiomyocytes.How is your finding different from this? Author Response: The Streeter et al. 1969 article shows the presence of longitudinal fibers in the endocardium and epicardium, similar to Green et al. 1981.However, both these reports do not comment on the fiber continuum and neither do several follow up studies.This marks the major difference between our work and previous studies.We describe a long-axis fiber continuum in the outer and inner ventricular walls.In Figure 6, we have shown different apical sections from the apex to the base of the heart.At the bottommost apex section we see three geometrically distinct aggregate cardiomyocyte orientations converge at what appears to be a singularity.We agree that these are qualitative observations.In the above sections (towards the base), these aggregate orientation bands emerge as separate units in the LV wall.We have revised Figure 6 to include the apex data as a series of sections from apex to base, which should clarify our claims.pg 7 line 27 "ventricular walls of four-chambered hearts" 6 mouse hearts and 1 rat heart is not sufficient data to make this claim.

Author Response:
We have changed our wording and have softened claims that are not substantiated by data.Please see page 8, lines 15-17.
Figure 1 It is very difficult to see the purple cylinders, perhaps because the yellow cylinders are so much longer than the purple ones.

Author Response:
We thank the reviewer for pointing this out.We have provided an improved version of Figure 1 in the revised manuscript.We thank the reviewer for pointing out this.We have provided an improved version of Figure 2, illustrating the angles we have used, in the revised manuscript.

Author Response:
We have modified Figure 3 thoroughly to accommodate the suggested changes.We also have included the description and findings from the pseudo low resolution analysis in the Results section (page 6, lines 28-33).

Edits:
Author Response: We thank the reviewer for all the suggested edits listed below.pg 2 line 23 Referencing error -presumably they shouldn't be the same reference (18).They're also a different referencing style.We have fixed this referencing issue in the revised article.pg 3 line 23 Italicize "ex vivo" We have italicized this word in the revised article.Please see page 3, line 28.pg 3 line 22-30 This belongs in the methods section, not the results section.pg 3 line 32-34 This belongs in the methods section, not the results section.pg 4 line 1-5 This belongs in the methods section, not the results section.pg 4 line 9-11 This belongs in the methods section, not the results section.pg 4 line 14-17 This belongs in the discussion section, not the results section.pg 4 line 21-27 This belongs in the methods section, not the results section.pg 4 line 27-32 This belongs in the discussion section, not the results section.pg 4 line 32-33 This belongs in the methods section, not the results section.pg 5 line 3-5 This belongs in the methods section, not the results section.pg 5 line 5-7 This belongs in the discussion section, not the results section.pg 5 line 10-12 This belongs in the methods section, not the results section.pg 5 line 15-18 This belongs in the discussion section, not the results section.pg 5 line 30-32 This belongs in the discussion section, not the results section.pg 5 line 32-33 This belongs in the methods section, not the results section.pg 6 line 1-2 This belongs in the methods section, not the results section.pg 6 line 9-12 This belongs in the discussion section, not the results section.pg 6 line 14-15 This belongs in the intro section, not the results section.pg 6 line 15-18 This belongs in the methods section, not the results section.pg 6 line 2 Please use "wedge sector" or simply "sector" rather than "pie-shaped sector".pg 6 line 4 Please use anatomic directions such as anterior/posterior/septal/lateral/RV instead of "3 O'clock position" Author Response: We have changed the wording to" wedge shaped sector" or "sector" and have included the anatomic directions both in Figure 3 and the Results.Please refer to page 6, lines 13-20.Similarly we have modified the manuscript to accommodate all the reviewers' editorial suggestions and The EMBO Journal requirements.

Referee #3 (Remarks to the Author):
The paper describes a method to reconstruct myocardial fibers at micron-scale resolution.The topic is of interest for the clinical cardiac imaging community, in particular DT-MRI, since microscopy can provide ground truth orientation measurements.The proposed experimental protocol included tissue clearing to prepare the fresh excised mice hearts, slicing of short-axis and long-axis slabs, and confocal microscopy imaging.Image processing methods are used to register and assemble the image stacks, to mitigate blurring and noise, and to reconstruct fiber orientation using the structure tensor.The overall patterns of myofiber organization agree with the literature, however it is claimed that a new distinct fiber structure was found in the outer wall.The experiments and methods are clearly described.The image processing methods are sound and use state-of-the-art algorithms.I have some questions though, listed below.
Major comments 1) About the claim of a new fiber system found in the outer wall:isn't it the pericardium?It seems to me that some layers of pericardium contain fibrous structures.Would it be useful to indicate the different cell types on the raw images to make it clear?
Author Response: We thank the reviewer for the opportunity to elaborate on this point.The outer wall long-axis fibers described in the manuscript are not merely fibrous structures but rather are stacked end-on-end cardiomyocytes forming an entire distinct geometrical system (seen in our short-and long-axis heart sections).The dimensions of the cardiomyocytes (diameter in a short-axis-section and length in a long-axis-section) in the outer wall are within a range that is expected of a typical cardiomyocyte.Our confocal images are also undersampled so that we acquire isotropic data, at 2-micron voxel resolution, which cannot resolve other small non-cardiomyocyte cells (please see Supplementary Figure 1f).We have added text about this in the Results section (page 4, lines 4-6).
In addition, in the figure below, we provide evidence for the reviewer that the outer wall cells we refer to are indeed cardiomyocytes, as shown by the presence of sarcomeric structures with the help of alpha-actinin staining.We are confident that the outer wall cells we report are indeed cardiomyocytes that form a long-axis system.
A representative image highlighting the cardiomyocytes stained using WGA, DAPI and alpha actinin to mark the cell membranes, nuclei and Z-disc regions of sarcomeres, respectively.The images were acquired with a 60X objective using a confocal microscope.The LV-wall region marked with a black box in the schematic representation of the short axis section (bottom middle) is shown in the top panel.A zoomed in region (yellow box) showing the middle wall to outer wall area is shown in the bottom right panel.The cardiomyocytes in the outer wall pick up staining for alpha actinin, confirming the presence of cardiac muscle cells.The scale bars are as indicated.
2) Validation of the structure tensor calculations: 70 myocytes where manually selected for comparison, how where they selected?Was it randomly, did the authors check on difficult cases, i.e. when the structure tensor is very different from the expected elongated cell type?
Author Response: We thank the reviewer for this query and for the opportunity to clarify this point.For validation we handpicked 70 myocytes from a randomly selected 3D image stack (field of view) but we did not bias the selection of cell types in any particular way.We have elaborated on this in the Detailed Methods (Section 3.7).In our study, we have data from only cardiomyocytes, which are elongated cells.To validate our structure tensor calculations, we have now also calculated the Fractional Anisotropy (FA) scores based on structure tensor eigenvalues, for the entire short-axis section and have found that the majority of the voxels have high FA scores.We have illustrated this using a colormap and an FA distribution plot in Supplementary Figure 3a.These analyses reveal that the structure tensor indeed consistently reveals an elongated cell type.
3) Other ways to visualize the orientation results could also be considered, inspired from DT-MRI: color-coded fractional anisotropy map is a nice way to summarize the fiber orientation in a single image.Fractional anisotropy may also help to check where the rank-1 structure tensor is not a good fit to the actual structure.

6th Aug 2023 2nd Revision -Editorial Decision
Dear Minhaj, Congratulations on an excellent manuscript, I am pleased to inform you that your manuscript has been accepted for publication in The EMBO Journal.Thank you for your comprehensive response to the referee concerns, it has been a pleasure to work with you to get this to the acceptance stage.
I will begin the final checks on your manuscript before submitting to the publisher next week.Once at the publisher, it will take about 3 weeks for your manuscript to be published online.As a reminder, the entire review process, including referee concerns and your point-by-point response, will be available to readers.I will be in touch throughout the final editorial process until publication.In the meantime, I hope you find time to celebrate!Kind regards, Kelly Kelly M Anderson, PhD Editor, The EMBO Journal k.anderson@embojournal.orgPlease note that it is EMBO Journal policy for the transcript of the editorial process (containing referee reports and your response letter) to be published as an online supplement to each paper.If you do NOT want this, you will need to inform the Editorial Office via email immediately.More information is available here: https://www.embopress.org/page/journal/14602075/authorguide#transparentprocessYour manuscript will be processed for publication in the journal by EMBO Press.Manuscripts in the PDF and electronic editions of The EMBO Journal will be copy edited, and you will be provided with page proofs prior to publication.Please note that supplementary information is not included in the proofs.
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in this article shows aH plots, where again no sharp changes are seen.The article does not describe a geometrically distinct long-axis epicardial system.ThePope et al. 2008  article reports collagen organization across rat ventricular walls, but does not show aH analyses.However, this article has reported a distinct collagen orientation in the subepicardial regions.This finding provides indirect support for our findings of geometrically distinct cardiomyocyte arrangements in the outer walls.We have added this point in the Discussion (page 9, lines 8-12).pg 6 18-21 You need to be much more descriptive in what Figure 3b is showing, and what resolutions the different colored lines are referring to, dotted versus solid etc.Author Response:We thank the reviewer for this comment.We have improved the presentation and discussion of Figure3.pg 6 line 26-29 How confident are you that these are not just papillary muscles?Author Response: Papillary muscles are seen in the cavity of the ventricles and are attached to their walls.The geometrically distinct long-axis cardiomyocyte layer we refer to is observed in the outer ventricular walls and is quite distinct from these.pg 8 line 9-11 "a resolution gain of about 3 orders of magnitude".I don't think this is correct.Please re-checkGarcia-Canadilla et al 2019 (doi:10.1111/joa.13058),Pope et al 2008  (doi:10.1152/ajpheart.00484.2008),Teh et al 2017 (doi:10.1186/s12968-017-0342-x).
pg 4 line 21-23 Why use three angles?Only two are needed.Helix angle and transverse angle OR helical angle and intrusion angle (Agger et al 2019, doi:10.1002/nbm.4205)Author Response:

Figure 2 I
Figure 2 I know the angle diagrams are difficult to construct, but the angle diagram is not clear, apart from the helix angle.

Figure 3
Figure 3 In b, make clear which sections each of the three solid lines represent from the 2 hearts.Also in the pseudo low resolution the main thing missing is the sharp but somewhat random transitions (green -65 to -90, blue -95 to -85, red -85 to -90), are you saying that these variable transitions indicate your long-axis fiber layer?I'm not sure what this pseudo low resolution data is showing me.

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