When right ventricular pressure meets volume: The impact of arrival time of reflected waves on right ventricle load in pulmonary arterial hypertension

Abstract Right ventricular (RV) wall tension in pulmonary arterial hypertension (PAH) is determined not only by pressure, but also by RV volume. A larger volume at a given pressure generates more wall tension. Return of reflected waves early after the onset of contraction, when RV volume is larger, may augment RV load. We aimed to elucidate: (1) the distribution of arrival times of peak reflected waves in treatment‐naïve PAH patients; (2) the relationship between time of arrival of reflected waves and RV morphology; and (3) the effect of PAH treatment on the arrival time of reflected waves. Wave separation analysis was conducted in 68 treatment‐naïve PAH patients. In the treatment‐naïve condition, 54% of patients had mid‐systolic return of reflected waves (defined as 34–66% of systole). Despite similar pulmonary vascular resistance (PVR), patients with mid‐systolic return had more pronounced RV hypertrophy compared to those with late‐systolic or diastolic return (RV mass/body surface area; mid‐systolic return 54.6 ± 12.6 g m–2, late‐systolic return 44.4 ± 10.1 g m–2, diastolic return 42.8 ± 13.1 g m–2). Out of 68 patients, 43 patients were further examined after initial treatment. At follow‐up, the stiffness of the proximal arteries, given as characteristic impedance, decreased from 0.12 to 0.08 mmHg s mL–1. Wave speed was attenuated from 13.3 to 9.1 m s–1, and the return of reflected waves was delayed from 64% to 71% of systole. In conclusion, reflected waves arrive at variable times in PAH. Early return of reflected waves was associated with more RV hypertrophy. PAH treatment not only decreased PVR, but also delayed the timing of reflected waves. Key points Right ventricular (RV) wall tension in pulmonary arterial hypertension (PAH) is determined not only by pressure, but also by RV volume. Larger volume at a given pressure causes larger RV wall tension. Early return of reflected waves adds RV pressure in early systole, when RV volume is relatively large. Thus, early return of reflected waves may increase RV wall tension. Wave reflection can provide a description of RV load. In PAH, reflected waves arrive back at variable times. In over half of PAH patients, the RV is exposed to mid‐systolic return of reflected waves. Mid‐systolic return of reflected waves is related to RV hypertrophy. PAH treatment acts favourably on the RV not only by reducing resistance, but also by delaying the return of reflected waves. Arrival timing of reflected waves is an important parameter for understanding the relationship between RV load and its function in PAH.

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----------------EDITOR COMMENTS Reviewing Editor: This manuscript describes wave separation analysis in a fairly large PAH patient cohort. Wave reflections returning at different time points were associated with differences in impedance and right ventricular characteristics. This type of analysis is underutilized, and while the current study may not provide major mechanistic insights, it does lay a foundation for future work.
The reviewers were in agreement that the methods and conclusions are sound and that this study adds new insights to the field. However, both reviewers also felt that there are important methodological details, further analysis (i.e., separation by sex) and data presentations that should be included to strengthen the impact and interpretation of the data. These requested changes/additions are clearly delineated by the reviewers in their comments and should be easily achievable with the dataset in hand. In particular, since the journal does not have page/figure limitations, I would recommend moving the material in the supplement to the main article document.
For a revision, the authors will need to provide the raw datasets and complete the statistical summary. Please also consider converting the two existing bar graphs (Fig 6C and Fig 4 RV mass) to box and whisker or scatter plots. While it is assumed that "n" refers to the number of subjects for each measurement, it would also be helpful to explicitly define the "n" within the legends or Statistical Analysis section.

Methods Details:
There are a few points that need additional descriptions, as outlined by the reviewers.
Ethics Concerns: While the study appears to have used previously collected data and was judged not to fall into Human Subjects research by the Institution, it seems likely that informed consent must have been provided at some point for use of the data for research purposes. Please see the comments of the Ethics Editor below.
Senior Editor: Please ensure that the graphs adhere to JP statistics and data policy. Referee #2: In the manuscript, "When right ventricular pressure meets volume", Fukumitsu M et al. used wave separation analysis to investigate the prevalence of mid-and late-systolic return of reflected waves in treatment-naïve patients with PAH. They found that the peak reflected wave returned during mid-systole in over half of the cohort. These participants had more pronounced RV hypertrophy. At follow-up there was decreased stiffness in the proximal arteries accompanied by decreased wave speed and a delay in the arrival of the peak reflected wave.

Major comments:
At baseline where the CMR and RHC performed under treatment naïve conditions? The methods state that the CMR was within 28 days before or after RHC but was treatment started after both the baseline RHC and CMR?
What is time % of peak reflected waves, % of systole? Based on supplemental methods, the time zero is defined as onset of RV systole and then the time forward and reflected were then normalized to the duration of the RV systole? How much variability is there in the length of systole between patients? Did the heart rate of the patient play a factor in the time of return? What is the difference in Delayed and not-delayed waves in Figure 6C and Supplemental table 3? Figure 5. Did the authors perform linear regression analysis on log-transformed data and then plot it based on non-log transformed data? It might be more appropriate to either show the log-transformed data that was used in the analysis and/or include the equation of the regression line and additional information.
Page 8 and figure 6C. What is the rationale for defining the change in RV mass/BSA from baseline to follow-up as -(followup -baseline)/follow-up*100? I would have expected that (follow-up -baseline)/baseline*100 to demonstrate the percent change from baseline.
Supplemental methods: ZC was calculated based on the average modulus between the 3rd and 10th harmonics of the PA input impedance but the PA pressures were measured using fluid-filled catheters. Flow is measured from PA phase contrast velocity quantifications that were sampled at 45Hz and then interpolated to increase sampling rate to 1000Hz. Is there sufficient fidelity to have accurate Impedance measurements at higher harmonics?  Table 2 -What is the meaning of lower estimated wave speed in the mid-systolic returners at 9 m/sec compared to the diastolic returners at 21 m/sec? I would have expected a higher wave speed would mean an earlier return of the wave. Is there a disconnect between this measurement of wave speed and sites of reflected waves? It seems that they could be coupled/factors in this measurement.

26-Oct-2021
Minor Comments: Figure 4. It would be helpful to also see the points in addition to the boxplots to better see the distribution of the data and the number of subjects in each group. Figure 6 A and B -It would also be helpful to overlay the median or mean with distribution for the time points. It is difficult to appreciate the change from baseline to follow-up.

Referee #3 (ethics review):
Thank you for submitting your manuscript to The Journal of Physiology. There are some issues pertaining to ethics that must be clarified.
It appears that the assessments were made as part of "routine" clinical assessment and that the authors have performed a retrospective analysis based upon the data to hand. If this is the case then this point should be made explicitly in the manuscript. The study was approved by the local ethics committee, and the authors highlight that: "This study complied with the Declaration of Helsinki. This study did not fall within the scope of the Medical Research Involving Human Subjects Act, as confirmed by the Medical Ethics Review Committee of the VU University Medical Centre (approval No. 2017.025)". I read this as approval by the ethics committee to use historical clinical data for research purposes. The authors do not include a statement on the issue of informed consent. Is it that the ethics board determined that no informed consent was required, that is, consent that the data collected for clinical purposes could be used for research purpose? I appreciate that this would not have been done prospectively, since the data were not collected as part of a research study (or trial), but rather are now included in a retrospective study. It is understandable given the protracted period covered by the authors (1996-2018) that the requirement for consent might be waived by the committee. This point needs to be clarified by the authors, to include revision of the text in the manuscript, and if so will need to be further considered by The Journal.

Confidential Review
Summary: Fukumitsu et al. postulate a relationship between right ventricular (RV) wall tension and wave reflections, and use wave separation analysis to examine differences in PAH severity and wave reflection times. The article uses pulmonary artery pressure and flow data from 68 patients with PAH, with follow-up data from 43 of these patients. The authors show that patients with mid-systolic wave reflection times have a significant decrease in characteristic impedance compared to those with reflections returning during diastole, and observe a larger normalized RV mass in patients with mid-systolic wave reflection times vs those with diastolic reflected wave arrival times. The manuscript is well written and draws novel conclusions.
However, there are several details that should be included in the manuscript to increase the overall impact. Major and minor comments for revision are provided below.
Major comments: 1. Abstract, page 4: "Wave speed was attenuated from 13.3 to 9.1 m/sec …" Wave speed values are minimally discussed in this work (results presented in Table 2 and supplement), yet the estimated wave speed plays a critical role in determining characteristic impedance and wave separation. The authors should document differences in wave speed in the main text and include at least one sentence regarding changes in wave speed magnitude for the different wave reflection times.
2. Introduction, page 5: "Furthermore, reflected waves returning during diastole … have negligible effects on RV pressure and load." This comment is linked to a prior publication by the group (Fukumitsu et al. 2020) on a proximal and distal CTEPH cohort. On this basis, the conclusion that reflected waves during diastole have "negligible effects" is unwarranted. Either more evidence should be provided or the claim should be deleted. so details on how that is derived would be beneficial in this section. 5. Page 10: "mid-systolic returners were characterized higher pulse pressure and lower total arterial compliance than late-systolic returners, and by a lower than diastolic returners." Correlations between pulse pressure, total arterial compliance, and for all groups could help the reader visualize these relationships better than bar graphs. Or, better yet, a surface map of the dependence of on these parameters color coded for the different groups (early, mid, and late returners). Overall, the figures accurately present the data but do not provide mechanistic insights.
6. Page 10. It is not clear how compliance of the main PA was assessed. Details should be provided in methods. 7. Figure 4: A boxplot with the wave speed for each reflection type with any statistically significant differences noted should be included. 8. Figure 6 A: It is difficult to interpret these results. Consider plotting a single line from pre-to post-treatment with error bars in addition to each patient trajectory. 9. Page 11: "PAH treatment attenuated the stiffness of the proximal arteries and decreased wave speed…" Can the authors be more specific? Is this based on wave speed data? If so, these quantitative values and test statistics should be provided in the results.
10. Page 11. The authors claim that PVR can be approximated as arterial elastance but in fact arterial elastance is sensitive to both PVR and compliance so this statement should be removed.
11. Page 12. The authors state "Simply, larger reflected waves can increase RV pressure greatly" but this is an oversimplification. Indeed, isn't the point of this article that the timing of the reflected wave is key to RV pressure and ultimately RV function? 12. Page 13: "A possible explanation is that the time course of development … may be different between PAH and CTEPH." The study by Su et. al 14. Page 14: "lower may represent larger area but not higher stiffness…" Please include this discussion in the main text rather than the supplement; perhaps the authors could consider distal vascular stiffening common in PAH, and link that to these possibly conflicting results in and compliance? 15. Page 14: "In this study, PAH treatment was associated with attenuated proximal arterial stiffness and wave speed, which delayed the arrival of peak reflected waves." Are these differences in peak reflected wave arrival times solely the effect of proximal stiffness or could this be due to distal vascular vasodilation? We would like to thank all reviewers and ethical reviewers, and the editorial team for your very stimulating comments that helped improving our manuscript. All authors have read the comments carefully and discussed the plan for revision. Our point-by-point responses to all comments from the reviewers are provided.
As the reviewers suggested, we described detailed method of wave reflection, for example, how pressure waves were separated into forward and reflected waves, and how time% of reflected waves was derived, using the specific equations. Furthermore, we provided more details of the ethical considerations in relation to the present study according to the ethical reviewer. A redlined version and a clean version of our revised manuscript are provided.
We hope that the reviewers and the editorial team find the revision satisfactory.

Response to Reviewing Editor:
Thank you for your constructive comments and suggestions. We revised our manuscript according to your comments and suggestions.

Comments
This manuscript describes wave separation analysis in a fairly large PAH patient cohort. Wave reflections returning at different time points were associated with differences in impedance and right ventricular characteristics. This type of analysis is underutilized, and while the current study may not provide major mechanistic insights, it does lay a foundation for future work.
The reviewers were in agreement that the methods and conclusions are sound and that this study adds new insights to the field. However, both reviewers also felt that there are important methodological details, further analysis (i.e., separation by sex) and data presentations that should be included to strengthen the impact and interpretation of the data. These requested changes/additions are clearly delineated by the reviewers in their comments and should be easily achievable with the dataset in hand.

Response
We deeply appreciate your comments and suggestions. According to both reviewers' suggestions, we provided the details of method, for example how time% of peak reflected waves was derived in the method section. To clarify this point, we revised the manuscript adding some equations.
Please kindly see the sections of "Cardiac MRI: RV volume, mass and wall tension", "Cardiac MRI: Compliance of the main PA", and "Wave separation analysis".

Response (continued)
We performed further analysis (for example; differences in hemodynamic and wave reflection between female and male, correlations of pulse pressure and total arterial compliance with Zc in each group in treatment-naïve condition, difference in RV systolic duration among each group). In mid-systolic returners, higher Zc was associated with lower total arterial compliance and higher pulse pressure, but not in late-systolic or diastolic returners ( Figure 5). RV systolic duration was prolonged more in mid-systolic returners than diastolic returners (Table 1).

Response (further continued)
According to the reviewers' suggestions, we re-created the Figure, for example; by adding the box-plots in Figure 4, providing the correlations of pulse pressure and total arterial compliance with Zc in each group in Figure 5, adding plots of the log-transformed data in Figure 6, and providing the box-lots in Figure 7.
Please kindly see Figure 4 to 7.
In particular, since the journal does not have page/figure limitations, I would recommend moving the material in the supplement to the main article document.

Response
Thank you very much for your recommendation. We moved the data and discussion in the supplement to the main article document.
For a revision, the authors will need to provide the raw datasets and complete the statistical summary.
Please also consider converting the two existing bar graphs [ Fig 6C (New Fig 8)

and Fig 4 RV mass]
to box and whisker or scatter plots. While it is assumed that "n" refers to the number of subjects for each measurement, it would also be helpful to explicitly define the "n" within the legends or Statistical Analysis section.

Response
According to your suggestions, we will submit the raw datasets and the statistical summary. Since data of RV mass/BSA was distributed normally, we created the new Figure with box-plot (mean ± standard deviations). According to your suggestion, we added the scatter plots in Figure 4 and Figure 8.

Methods Details:
There are a few points that need additional descriptions, as outlined by the reviewers.

Response
Thank you very much for your comments. We revised the manuscript according to the reviewers' suggestions.

Ethics Concerns:
While the study appears to have used previously collected data and was judged not to fall into Human Subjects research by the Institution, it seems likely that informed consent must have been provided at some point for use of the data for research purposes. Please see the comments of the Ethics Editor below.

Response to Senior Editor
Thank you for your constructive comments and suggestions. We revised our manuscript according to your comments and suggestions.

Comments
Please ensure that the graphs adhere to JP statistics and data policy.

Response
Thank you very much. We will submit the raw data and statistical summary according to the policy of the Journal of Physiology.
I concur with the reviewing editor's suggestions. Please ensure that the MS in compliance with the JP ethics and statistics policies.

Response
We appreciate your suggestions. According to the reviewing editor and the ethical editor, we revised the manuscript regarding ethics as follows.

Response to Referee #1
Thank you for your constructive comments and suggestions. We revised our manuscript according to your comments and suggestions.

Response
Thank you for the comments. We agree with you that estimated wave speed is the critical role in considering the timing of returned wave reflection. According to your suggestion, we described the result of estimated wave speed in the main results and the new Table. <Page 12 Lines 280 -282> PAH treatment decreased Zc from 0.12 to 0.08 mmHg/sec/ml, increased compliance of the main PA from 6.8 to 9.1 ml/mmHg, and delayed wave speed from 13.3 to 9.1 m/sec (Table 4, Figure 7).

Response (continued)
The data of changes in estimated wave speed was included in Table 4 and Figure 7B.
Please kindly see the Table 4 and Figure 7B.

Response
We appreciate your suggestion very much. We revised this sentence and added a new reference as follows.

Response
We appreciate your suggestions. Based on the journal policy in which the journal of physiology does not have page/figure limitations, we described the following mathematical methods in the main text. where area is the mean area of main PA and ρ is blood viscosity given as 1.04 g/cm 3 .

Response (continued)
Furthermore, we clarified the method how the reflection index was derived as follows. Specifically, time% of peak reflected waves was given by the following equation.

% = × 100
Eq. 6 5. Page 10: "mid-systolic returners were characterized higher pulse pressure and lower total arterial compliance than late-systolic returners, and by a lower than diastolic returners." Correlations between pulse pressure, total arterial compliance, and for all groups could help the reader visualize these relationships better than bar graphs. Or, better yet, a surface map of the dependence of on these parameters color coded for the different groups (early, mid, and late returners).
Overall, the figures accurately present the data but do not provide mechanistic insights.

Response
Thank you very much. According to your comments, we described the correlations of pulse pressure and total arterial compliance with Zc for all groups (mid-systolic, late-systolic and diastolic returners) in the main text and Figure 5.
<Page 11, Lines 254 -257> In mid-systolic returners, higher Zc was associated with lower total arterial compliance and higher pulse pressure, but not in late-systolic or diastolic returners ( Figure 5) Please kindly see Figure 5.

Response
As the reviewer points out, the details of how the compliance of the main PA was calculated were descried in the method section as follows.
<Page 8, Lines 187 -189> By multiplying ∆area with 2.0 cm, which is assumed as the length of the main PA for all patients, local compliance of the main PA was given as follows (Saouti N et al. 2009).

Response
Thank you for your suggestion. We created the new Figure 4 with box plots and statistical significance, where appropriate, of the estimated wave speed in each group (mid-systolic, late-systolic and diastolic returners).
Please kindly see the Figure 4. Figure 7A): It is difficult to interpret these results. Consider plotting a single line from pre-to post-treatment with error bars in addition to each patient trajectory.

Response
According to your suggestion, we added the box plot for each parameter in Figure 7A.
Please see the Figure 7A.

Response
As the reviewer pointed out, this description is based on the quantitative analysis of estimated wave speed.
We clarified the specific numbers as follows.

Page 11. The authors claim that PVR can be approximated as arterial elastance but in fact
arterial elastance is sensitive to both PVR and compliance so this statement should be removed.

Response
We greatly appreciate your comment. We understand that PVR and arterial elastance should be discussed more carefully. With some assumptions (for example, 1; when end-systolic RV pressure is approximated as mean PAP, 2; mean PAWP is negligible in a disease such as PAH, 3; heart rate remains constant), PVR can be approximated as arterial elastance. To clarify this point, we revised the manuscript as follows. 14. Page 14: "lower may represent larger area but not higher stiffness…" Please include this discussion in the main text rather than the supplement;

Response
Thank you very much. The section "lower total compliance but also lower characteristic impedance of mid-systolic returners" was included in the main text.
Please kindly see the section "Lower total compliance but also lower characteristic impedance of mid-systolic returners" from lines 406 to 427 <Page 17, Lines 385 -388>

Here, lower Zc may represent larger area but not higher stiffness of the proximal arteries (see the details in the section "Lower total compliance but also lower characteristic impedance of mid-systolic returners")
perhaps the authors could consider distal vascular stiffening common in PAH, and link that to these possibly conflicting results in and compliance?

Response
As the reviewer pointed out, the distal part of elastic arteries may explain low Zc as well as low total arterial compliance. As described, less tapering but stiffer vessels at the distal part of the elastic arteries can induce both lower total arterial compliance and lower Zc. The following explanation was provided in the main discussion.

Page 14: "In this study, PAH treatment was associated with attenuated proximal arterial stiffness
and wave speed, which delayed the arrival of peak reflected waves." Are these differences in peak reflected wave arrival times solely the effect of proximal stiffness or could this be due to distal vascular vasodilation?
Response PAH treatment can decrease PVR and distal vascular vasodilatation. Therefore, not only decreasing Zc and wave speed, but also moving the reflection sites to more distal location can delay the arrival of reflected waves after treatment. This point was clarified as follows.

Response
We greatly appreciate your comments. As you mentioned, there are several methods to analyze wave transmission, including wave intensity analysis. However, regardless of the methodology to separate wave reflection, early return of reflected waves may be the determinant of RV stress in PAH, because mid-systolic notch of flow waves in echocardiography, which might be caused but mid-systolic return of reflected waves, was associated with poor RV function. According to your suggestions, the limitation was expanded as follows. are key to the impact of the study, they must be reported in the body of the text.

Response
Thank you very much. All data in the conclusion was provided in the main text/Table/ Figure. Minor comments: 1. Page 8. "PA flow waves during a cardiac cycle was obtained" should be "PA flow waves during a cardiac cycle were obtained"

Response
According to your suggestion, I revised the manuscript as follows.

Response
Thank you. I revised the manuscript as follows.
<Page 11, Lines 246 -249> When comparing mid-systolic, late-systolic and diastolic returners in the treatment-naïve condition, no differences were found in clinical background (similar age, sex, type of PAH and diffusing capacity of the lungs for carbon monoxide; Table 1).
3. Page 10: Please include description of the non-significant differences between groups in the text (e.g., does not show a statistically significant difference between mid-systolic and late-systolic groups).

Response
We added the description of non-significance of pulse pressure and total arterial compliance between mid-systolic and diastolic returners, and that of Zc between mid-systolic and late-systolic returners.
<Page 11, Lines 252 -253> There were no differences in pulse pressure and total arterial compliance between mid-systolic and diastolic returners. Also, no difference was also found in Zc between mid-systolic and late-systolic returners.

Page 10:
For the second to last sentence, should it be "mean PAP" instead of "mean PA"

Response
Thank you. We revise the manuscript as follows.

Response to Referee #2
Thank you for your constructive comments and suggestions. We revised our manuscript according to your comments and suggestions. In

Response
To clarify the method how time% of peak reflected waves is calculated, we added the description and the equation as follows.
<Page 9, Lines 209 -213> Therefore, time of peak forward and reflected pressure were normalized as the time over the duration of RV systole (% in systole: referred to as time% of peak forward and reflected waves) [Fukumitsu M. et al. 2020].
Specifically, time% of peak reflected waves was given by the following equation.

How much variability is there in the length of systole between patients?
Response RV systolic duration was prolonged more in mid-systolic returners than diastolic returners. We described this difference in the main manuscript and Table as follows. <Page 11, Lines 256 -257> RV systolic duration was prolonged in mid-systolic returners more than diastolic returners (Table 1).
Please kindly see the Table 1.
Did the heart rate of the patient play a factor in the time of return?

Response
Since the cardiac cycle, especially duration of RV systole, can affect an interpretation of timing of peak forward and reflected waves, we added the explanation in the manuscript as follows.
<Page 9, Lines 208 -212> The cardiac cycle, especially duration of RV systole, can affect an interpretation of timing of peak forward and reflected waves. Therefore, time of peak forward and reflected pressure were normalized as the time over the duration of RV systole (% in systole: referred to as time% of peak forward and reflected waves) [Fukumitsu M. et al. 2020]. Figure 6C (new Figure 8) and

What is the difference in Delayed and not-delayed waves in
Supplemental

Response
We agree with you thoroughly. As we replied to the reviewer #1, this sentence was mistyped. Correctly, "a change in RV mass/BSA from baseline to follow-up after treatment was determined as harmonics of the PA input impedance but the PA pressures were measured using fluid-filled catheters.
Flow is measured from PA phase contrast velocity quantifications that were sampled at 45Hz and then interpolated to increase sampling rate to 1000Hz. Is there sufficient fidelity to have accurate Impedance measurements at higher harmonics?

Response
We appreciate your comment. Sampling rates of both RHC and CMRI were sufficient to calculate the impedance at higher harmonics, because for example, when HR is 90 bpm, the 3 rd to 10 th harmonics corresponds to 4.5 to 15 Hz. For helping the readers' understanding, we added the explanation in the manuscript as follows.

Response
Thank you for your comments. A symbol * means P<0.05 between mid-systolic and late-systolic returners.
A symbol # means P<0.05 between mid-systolic and diastolic returners. There was no statistical difference among groups when a Kruskal-Wallis test with Dunn's multiple comparisons.
Please kindly see the caption of Table 1. Table 2 -What is the meaning of lower estimated wave speed in the mid-systolic returners at 9 m/sec compared to the diastolic returners at 21 m/sec? I would have expected a higher wave speed would mean an earlier return of the wave. Is there a disconnect between this measurement of wave speed and sites of reflected waves? It seems that they could be coupled/factors in this measurement.

Response
As the reviewer pointed out, the difference in wave speed between mid-systolic and diastolic returners should be discussed more carefully. The wave speed should be considered to be independent of the distance to the reflection sites, therefore our result implies the possibility that the location of the reflection sites is more important to induce early return of reflected waves, rather than wave speed. Based on low Zc and low total arterial compliance, a wider proximal PA with abrupt narrowing may induce early return of reflected waves at a low wave speed. To clarify these points, we added the explanation as follows.
<Page 16, Lines 377 -380> Early return of reflected waves can occur 1) when the speed of wave transmission is high or 2) when the distance from the RV to the reflection sites is short [Naeije R. and Huez S. 2007]. In terms of mechanics, the speed of wave transmission should be considered to be independent of the distance to the reflection sites.

Response
According to the reviewer's suggestion, we created the new Figure 7 with box-plot.
Please kindly see the Figure 7.

Response to Referee #3 (ethics review)
Thank you for your constructive comments and suggestions. We revised our manuscript according to your comments and suggestions.
Thank you for submitting your manuscript to The Journal of Physiology. There are some issues pertaining to ethics that must be clarified.
It appears that the assessments were made as part of "routine" clinical assessment and that the

The authors do not include a statement on the issue of informed consent. Is it that the ethics board
determined that no informed consent was required, that is, consent that the data collected for clinical purposes could be used for research purpose? I appreciate that this would not have been done prospectively, since the data were not collected as part of a research study (or trial), but rather are now included in a retrospective study. It is understandable given the protracted period covered by the authors (1996-2018) that the requirement for consent might be waived by the committee. This point needs to be clarified by the authors, to include revision of the text in the manuscript, and if so will need to be further considered by The Journal.

Response
We greatly appreciate your comments and suggestions. The present study was retrospectively conducted from a registry of patients with diagnosed pulmonary arterial hypertension who routinely underwent right-sided heart catheterization and cardiac MRI. Informed consent was given by all patients after 2010 to use their data for scientific purposes. Before 2010, the medical ethical committee waived the requirement of consenting the patients in retrospect and allowed using the clinical data collected in that period or scientific purposes (approval No. 2017.025). To clarify these points, we revised the manuscript as follows.

04-Mar-2022 1st Revision -Editorial Decision
Dear Dr Westerhof, Re: JP-RP-2021-282422R1 "When right ventricular pressure meets volume: the impact of arrival time of reflected waves on right ventricle load in pulmonary arterial hypertension" by Masafumi Fukumitsu, Joanne A Groeneveldt, Natalia J. Braams, Ahmed A Bayoumy, J. Tim Marcus, Lilian J. Meijboom, Frances S de Man, Harm-Jan Bogaard, Anton Vonk Noordegraaf, and Berend E. Westerhof Thank you for submitting your revised Research Article to The Journal of Physiology. It has been assessed by the original Reviewing Editor and Referees and has been well received. Some final revisions have been requested.
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