Frequency-Dependent Properties of the Hyperpolarization-Activated Cation Current, If, in Adult Mouse Heart Primary Pacemaker Myocytes
Round 1
Reviewer 1 Report
The paper by Hu et al. aims at exploring If (f-current) of mouse SAN cells at 35 degrees in detail, and then implement the gained data in a mathematical model to explore the role of If. While I find the topic very interesting, I have had serious problems to read the paper. There were many inconsistencies between text, figures and legends. I will list them below (mixed up with some minor points) in the order they appear in the paper. A general, and a quite critical point, is that the new data are not very strong and convincing, and therefore the conclusions drawn in the paper are not at all clear. I hope my comments below will substantiate this. If I have missed the point, I am happy to be informed.
Page 2, first paragraph, line 5: I do not think that five classical reviews (1-5) need to be cited.
Page 3, last para, lines 8-9: “reduced data” is, in my mind, an unorthodox expression. I understand what you mean bur I suggest to spell out it out.
Figures in general: It took me some time to figure out what was figure legends and what was text. I do not know if this I because the form of the submission system or not. A large part of the figure legends were incorporated in the main text with similar font size as the main text, while the first part of the figure legend was in another format. But I solved this. A comment to the authors is that the legends were quite fluffy, with much repetition of the main text. I suggest to condense these legends substantially. It I also difficult to find information about the panel. I suggest to write clearly “A)…” and so on.
Fig. 1: There are too many digits in the mean, SD and SEM data. You do not have that precision in your recordings. I think the relation between SD and SEM is incorrect if N = 33. The expression “overshoot potential” is not needed. Why not just “potential”? Overshoot was a surprise in 1939, but not now.
Page 4, line 6 from bottom: msec is ued throughout this paper. Shouldn’t the abbreviation in the SI system be used? (ms).
Page 4, lines 3-5 from the bottom: “This prepulse was applied to inactivate the Na+ current and also to activate L-type Ca2+ channels thus ensuring that each preparation that was included in this study generated robust ICaL, IKr and If records.” I do not fully understand this. In the recordings it looks as if the Ca current inactivate during the 200 ms prepulse. This is mentioned in the legend. How does this match the cited sentence?
Fig. 2: Which voltage do you use for the tail, at 1.2 s? I cannot find the information
Page 5, last two paragraphs: The language is sometimes unnecessary cumbersome. These are two sections. I suggest that you are more to the point. For instance “These results can be used for inspection of the regions of interest” can be shortened. There are many similar sentences in the text.
Fig. 3:
-You claim that ISO increases the current at -20 to -50 and that this is even more pronounced at more negative voltages. This is one of the major findings of the paper. This is definitely not easy to see, and my question is if this increase is statistically significant? There is no discussion about statistical tests at all in the paper. This is a very important point. If it is not statistically significant increase, there is not increase.
-What does “Col 15 vs Col 16” mean?
-It is said that A is from a pulse protocol with 200 ms pulses and that B is from one with 1 s pulses. Is this true? Personally I would have measured 200 ms after 200 ms in the 1 s pulse protocols. It would be good if this can be clarified. And if you had different pulse protocols, why?
-In the legend you write that you have used pulses down to -110 mV, but no such data are presented. What is correct?
Page 6, second para, line 4: You use the expression “quasi instantaneous current change” (and sometimes “quasi steady-state”) in many place in the text. What is meant? Why quasi? Isn´t is a real instantaneous current?
Page 6, 5-6 lines from the bottom: What is ‘paired pulse” protocol? I cannot find any information about this in the legend to fig 4, where it is said to be described.
Page 7, lines 1-2: “It is apparent from the results in the right hand panel that these types of recordings of If do indeed, exhibit a quasi-instantaneous current change…” I am not sure I understand. I cannot see any instantaneous current in the right panel. (Maybe, maybe in the -135 mV trace). But the following sentence points at no instantaneous current in the right panel.
Page 7, second para, lines 2-5: “Inspection of the current changes that take place during the 500 msec. depolarizing clamp step to membrane potentials in the range -35 to +45 mV reveal that under baseline conditions the reversal potential is near -15 mV +/- 10 mV.” I have no idea how I can figure out that the reversal potential is -15 mV.
Fig. 4: Are panel A and C from the same cell? If so, why is Cm so different? Which voltage I used for the tail (2.1-2.6 s). Why is the outward current so different in shape in the two panels? I cannot detect any difference in current size between B and D. Didn’t Ba and Ni remove the instantaneous current, and should we not expect different currents in panels B and D. Have I missed something?
Legend to Fig 4: If there is 10 mV between each step how is then possible that you start at -50 and end at -135 mV? The last sentence states that “The resulting conductance-voltage relation was fitted to a Boltzmann function (defined as: G = Gmax/(1 + exp[(Vm – Vh)/s])) and plotted as shown in Panel C.” This does not fit anything in Fig 4. I am confused.
Page 8, last line: “not shown” is not OK for me. If you have the data, they must be shown.
Fig. 5: Why is the trace for -100 mV missing?
Legend to Fig 5: It is said that the pulse length is 10 s, but as far as I can see it is 20 s in the figure. It is said that you use pulses down to -135 mV, but I can just find down to -120 mV. It is stated that “For deactivation, a 10 sec step to -120 mV was utilized to fully activate If; and then its deactivation kinetics were studied by applying 10 sec. steps to membrane potentials in the range -50 to -110 mV.” This is not seen at all.
Page 9, Eq 1: This cannot be correct. At the voltage Vm=V0 the equation is undetermined.
Page 9, lines 5-9 from bottom: “Moreover, when the SAN beating rate increases approximately 2.5-fold in the presence of ISO the timedependent changes in If during the shortened diastolic intervals will be even smaller. In fact, the activation of If that occurs during each action potential does not fully deactivate within the following diastolic interval.” How do you know this? You do not present any data for If at voltage more positive to -50 mV. In addition, do you have any data on time constants for If under ISO exposure?
Fig. 6: Time constants from a double exponential fit without amplitudes are quite meaningless. You should also present the fraction of amplitudes at different voltages. Furthermore, you have pooled data from deactivation and activation. I definitely think that data should be presented for the separately. In the analysis, they can be pooled.
Legend to Fig. 6: This is, in my ind, one example of a too wordy legend. It must be stated which equation is used to get the parameter values.
Page 10, lines 2-5 (after the legend): “A decision was made to attempt to record the raw data that is required for deriving this information by applying voltage clamp protocols that utilized ramp, as opposed to rectangular, command waveforms.” Unnecessary wordy.
Page 10, para 2, line 4-6: “A typical estimated leakage current, assuming a seal resistance in the 10 Gigaohm range is shown in red in Figure 7A (assuming that this current is linear over the voltage range of interest).” I do not understand why you have to assume a seal resistance. Can’t you measure it?
Fig. 7B: It is a little bit confusing that you have switched colors. In Fig. 5 the fit was red and data in black. Now it is the opposite. I thought the black curve was a Boltzmann fit, but there is a slight shakiness of the curve. Why?
Legend to Fig 7: Again too wordy for my taste. I think the way voltage ramps are a little bit confusing. Sometimes they are called ramps, sometimes they are linear (but even square-like voltage clamp steps are linear), sometimes they are triangular. I suggest to use a consistent language. The used equation should have a number.
Page 11, lines 6-8 from bottom: “A typical steady-state current trajectory in this range of potentials is shown in Figure 8A and the conductance voltage relationship derived from this data is shown in Panel B.” In the figure it is the opposite.
Fig. 8: Why is the conductance in A given in nS/pF while the current in B is given only in pA? Why I there no noise in all the panels of this figure. We would expect massive noise in C. I think this is a very critical point in my review. These are the data that are important.
Legend to Fig. 8: The gain is not 300-fold. It is 20-fold.
Page 13, line 2: 600 ms should be 60 ms and 1 s should be 100 ms if I interpret the figure correctly.
Page 13 and Fig. 9: I have problems with this figure and associated text: The colors (blue and black) are extremely difficult to differentiate in my printed version. If I correctly dissect the colors I have problem to get the text in line with the figures. For instance, from 0.04 to 0.06 ISO makes the current more negative in panel A(i). This time interval corresponds to a voltage of about -40 to -60 mV. But in Panel B, the ISO induced current is positive. I am confused. From 0.06 and onwards, the ISO induced current in A(i) switches from a negative to a positive. This is not seen in B.
Page 14, line 5 from bottom: What does “semiquantitative” mean?
Page 15, line 3: The red traces do not depict the changes.
Page 15, line 6: What is electrophysical.
Page 15, para 1, last line: “…‘on top of’ a steady inward current of about 0.15 pA/pF.” What does ‘on top’ mean? It is not clear to me.
Fig. 10F: Do not cut the y-axis. Start from 0.
Page 15, line 3 from bottom: “dose-dependent modulation by ISO”. How is this done? I cannot find any information in the main text. Maybe it is hidden in any supplementary information. Unfortunately, I did not find any supplementary information.
Page 16 (roughly): A critical question is how the new model differs from the previous model. I could not find any information in the main text. Maybe it is in any supplementary information that I could not find. But I do think this must be spelled out in the main text, including how the calculations were carried out.
Page 16, para 2, line 5: it is said that the firing rate was increase by ISO by a factor of 2-2.5. In Fig. 11A I can only get it to 1.3, which is much less (+30% vs +100-150%).
Fig. 11F: Do not cut the y axis. Start at 0.
Discussion: It is far too long: I suggest at least 50% cut.
Page 21, para 2, lines 6-7: “The Kharche et al. model [38] was chosen as a starting point based on its successful peer review and publication.” I do not understand this. You must use better arguments.
Author Response
Response to Reviewers’ Comments
We would like to thank both Reviewers for their constructive and helpful comments, based on which we have revised our manuscript. We hope you would find our revision satisfactory. Point-by-point response to your comments are listed below.
Reviewer 1
Comment 1: The paper by Hu et al. aims at exploring If (f-current) of mouse SAN cells at 35 degrees in detail, and then implement the gained data in a mathematical model to explore the role of If. While I find the topic very interesting, I have had serious problems to read the paper. There were many inconsistencies between text, figures and legends. I will list them below (mixed up with some minor points) in the order they appear in the paper. A general, and a quite critical point, is that the new data are not very strong and convincing, and therefore the conclusions drawn in the paper are not at all clear. I hope my comments below will substantiate this. If I have missed the point, I am happy to be informed.
Response: Thank you for your comprehensive and very helpful review of the original version of this paper. Many of the points that you have raised are relevant and the corrections and additional information that we have added to the R-1 version significantly clarify and improve our presentation. Below, we provide point-by-point responses to your questions and constructive criticisms and we have also made changes and corrections in the text, the Figure Legends and the Figures themselves. In doing so we have addressed and dealt with the majority of your concerns.
However, it is not possible for us to respond to some of your requests for new experimental data. The major reason for this is that most of the experimental data presented in this paper, although not previously published; in fact, was generated between 12 and 15 years ago. The fact that the functional role of If in the myogenic pacemaker response in adult mouse SAN is not fully understood, was the main reason that Dr. Zhang and I responded to a note from Dr. Delisle regarding the possibility of submitting a paper for this Ion Channel series or compendium.
Appropriately, you raise the question: What is new? The answer, that is now more clearly spelled out in our Discussion is that If definitely does have a role in adult SAN pacemaker activity but that the functionality is conferred by the residual activation of this current and the corresponding ion fluxes driven by the waveforms of the pacemaker depolarization and the action potential. This insight has not been delivered by previous papers or reviews in this field.
Comment 2: Page 2, first paragraph, line 5: I do not think that five classical reviews (1-5) need to be cited.
Response: We believe that I (WRG) understand why you may take this position. However, I do not agree, and in fact, increasingly find in this field and others that manuscripts are not properly or fully introduced or put in a proper historical context. One example of this is described in the editorial cited below.
Petersen, OH. When a discovery is a rediscovery: Do we know the history of our own subject? Function, 2021, 2:zqab030.
Comment 3: Page 3, last para, lines 8-9: “reduced data” is, in my mind, an unorthodox expression. I understand what you mean bur I suggest to spell out it out.
Response: Thank you for this comment. We have clarified this point by simply removing the descriptor ‘reduced’. In addition, in response to your questions regarding the accuracy of our data, we have removed the misleading statistical information that appeared below each panel in part C of Figure 1 and included this information in the Figure Legend using only single decimal place indications of each of these parameters.
Comment 4: Figures in general: It took me some time to figure out what was figure legends and what was text. I do not know if this I because the form of the submission system or not. A large part of the figure legends were incorporated in the main text with similar font size as the main text, while the first part of the figure legend was in another format. But I solved this. A comment to the authors is that the legends were quite fluffy, with much repetition of the main text. I suggest to condense these legends substantially. It I also difficult to find information about the panel. I suggest to write clearly “A)…” and so on.
Response: Thank you for this comment. We agree with one of your concerns and in the case of each Figure have ensured that the information in the Figure Legend and in the text for Results correspond and agree. In the R-1 version some redundancy remains but we have a strong preference for providing important details in Figure Legends and corresponding Results sections. Your comment concerning Figure Legend formatting is well taken, and that was due to the auto-formatting of the submission system. What apparently has been done is that in each case the title of the Figure has been properly included, but the Figure Legend has been formatted in such a way that it appears to be disconnected because there is a one line space between the title of the Figure and the text of the Figure Legend. Presumably we can ask the press to address this if our R-1 paper is accepted for publication. We have also restructured figure legends into (A), (B), (C), (D) etc.
Comment 5: Fig. 1: There are too many digits in the mean, SD and SEM data. You do not have that precision in your recordings. I think the relation between SD and SEM is incorrect if N = 33. The expression “overshoot potential” is not needed. Why not just “potential”? Overshoot was a surprise in 1939, but not now.
Response: Thank you for these two comments. Your ‘too many digits’ concern is valid and has been dealt with by using single decimal for the mean, SD and SEM data, which are now moved to the figure legend. Your second point regarding the overshoot potential may be valid as it relates to contemporary information that was available to squid axon investigators in the 1940s. However, it is certainly not valid regarding the details of the action potential in mammalian SAN primary pacemaker activity. In this field, an early and important finding was that the action potential in pacemaker myocytes, in fact, exhibited very little if any ‘overshoot’ in the syncytium. In contrast, and interestingly, it appears that in the adult mouse SAN the action potential waveform does exhibit overshoot even in the very small area corresponding to the primary pacemaker. Accordingly, we want to retain this terminology and make this distinction.
Comment 6: Page 4, line 6 from bottom: msec is ued throughout this paper. Shouldn’t the abbreviation in the SI system be used? (ms).
Response: Thank you very much for the comment. You are correct. Throught the manuscript, we have changed “msec” to “ms”, “sec” to “s”.
Comment 7: Page 4, lines 3-5 from the bottom: “This prepulse was applied to inactivate the Na+ current and also to activate L-type Ca2+ channels thus ensuring that each preparation that was included in this study generated robust ICaL, IKr and If records.” I do not fully understand this. In the recordings it looks as if the Ca current inactivate during the 200 ms prepulse. This is mentioned in the legend. How does this match the cited sentence?
Response: Thank you for this comment. You are correct in pointing out a discrepancy in the descriptions that appear in the text vs. the Figure Legend. We have altered the text on page 4 and Figure 2 legend to clarify our meaning and to make the description consistent with the Figure Legend.
Comment 8: Fig. 2: Which voltage do you use for the tail, at 1.2 s? I cannot find the information
Response: All of the tail currents developed at a membrane potential of -60 mV; that is, at the holding potential for the preparation. This information has now been added to the Figure Legend and the corresponding text.
Comment 9: Page 5, last two paragraphs: The language is sometimes unnecessary cumbersome. These are two sections. I suggest that you are more to the point. For instance “These results can be used for inspection of the regions of interest” can be shortened. There are many similar sentences in the text.
Response: We note and appreciate your concern or at least your stylistic preference. In preparing the R-1 manuscript this concern has been dealt with on a sentence-by-sentence basis and a number of changes have been introduced as shown in the modified text provided in red. In addition, a number of full sentences have been removed, although these are not shown in the R-1 manuscript. We appreciate the diligence of your review and expect that our responses are appropriate.
Comment 10: Fig. 3:
-You claim that ISO increases the current at -20 to -50 and that this is even more pronounced at more negative voltages. This is one of the major findings of the paper. This is definitely not easy to see, and my question is if this increase is statistically significant? There is no discussion about statistical tests at all in the paper. This is a very important point. If it is not statistically significant increase, there is not increase.
Response: In this case you make a very important point. We concede that the data generated in ISO produces only small changes in the -20 to -50 mV range and that we not only have no evidence for the statical significance but in fact also have no data that would constitute a bracketed control. Accordingly, we have altered text relevant to Figure 3 to indicate that this is an interesting preliminary finding.
Comment 11: -What does “Col 15 vs Col 16” mean?
Response: Now this uncessary labelling in Figure 3 has been removed. However, the way in which this data was logged and the procedure for extracting and displaying both raw data and isochronal I-V curves often did involve obtaining difference currents by subtracting two data sets that were denoted in this way. This procedure and terminology does apply to Panels B and D of Figure 4.
Comment 12: -It is said that A is from a pulse protocol with 200 ms pulses and that B is from one with 1 s pulses. Is this true? Personally I would have measured 200 ms after 200 ms in the 1 s pulse protocols. It would be good if this can be clarified. And if you had different pulse protocols, why?
Response: This comment appears to apply to Figure 3 and if so it requests an important clarification. These two data sets were indeed obtained with two completely separate sets of voltage clamp command pulses applied to the same cell. Accuracy and reproducibility in semiquantitative studies of If of the kind that form the basis of this paper requires extreme diligence in applying or challenging these small myocytes with unphysiological voltage clamp commands. In particular, there is the real possibility of producing an artefactual change in intracellular sodium concentration by applying large and long hyperpolarizing voltage clamp commands. We wanted to avoid this while at the same time seeking to obtain data sets under both physiological conditions (200 ms) and conditions that approximated maximal or steady-state responses (1 s).
Comment 13: -In the legend you write that you have used pulses down to -110 mV, but no such data are presented. What is correct?
Response: Thank you for this comment. We believe that the Figure Legend is incorrect; -110 should have been stated as -100 and this change has been made in Figure 3 legend.
Comment 14: Page 6, second para, line 4: You use the expression “quasi instantaneous current change” (and sometimes “quasi steady-state”) in many place in the text. What is meant? Why quasi? Isn´t is a real instantaneous current?
Response: Thank you for the comment. In choosing this terminology, we are attempting to be both accurate and conservative – an approach that you will fully appreciate given your detailed knowledge of squid axon electrophysiology and biophysics done in the late 1930s and early 1940s and published in the classical Hodgkin and Huxley papers. In this context and for these reasons, amphotericin-mediated patch clamp current records cannot be considered to be instantaneous because the capacitative transient can and does obscure the current changes occurring within the first 10 to 20 ms. We have addressed your point and clarified this by describing what we mean by quasi instantaneous in the revised text. Perhaps more importantly, generating convincing fully activated or steady-state current voltage relationships or activation curves for If is extremely difficult because doing so requires the experimentalist to apply protocols and work under experimental conditions that almost certainly alter or disrupt intracellular sodium levels and therefore moderate and could significantly change localized intracellular calcium levels.
Comment 15: Page 6, 5-6 lines from the bottom: What is ‘paired pulse” protocol? I cannot find any information about this in the legend to fig 4, where it is said to be described.
Response: We apologize for this omission. The information that you have requested is now included in the Results section near Figure 4 and in the Legend of Figure 4. Along with this, I have clarified how an estimate of the reversal potential for If was obtained from the raw data shown in this Figure.
Comment 16: Page 7, lines 1-2: “It is apparent from the results in the right hand panel that these types of recordings of If do indeed, exhibit a quasi-instantaneous current change…” I am not sure I understand. I cannot see any instantaneous current in the right panel. (Maybe, maybe in the -135 mV trace). But the following sentence points at no instantaneous current in the right panel.
Response: Your concern here is legitimate and we appreciate the fact that you have pointed out this deficiency. We have useful (and we hope accurate and convincing) new text in the relevant text for the Results of Figure 4.
Comment 17: Page 7, second para, lines 2-5: “Inspection of the current changes that take place during the 500 msec. depolarizing clamp step to membrane potentials in the range -35 to +45 mV reveal that under baseline conditions the reversal potential is near -15 mV +/- 10 mV.” I have no idea how I can figure out that the reversal potential is -15 mV.
Response: The original text was incomplete and certainly unclear with respect to the points that you raise here. In the Results section and in the legend to this Figure the double pulse protocol has been clarified and additional description has been added regarding this qualitative estimate of the reversal potential for If.
Comment 18: Fig. 4: Are panel A and C from the same cell? If so, why is Cm so different? Which voltage I used for the tail (2.1-2.6 s). Why is the outward current so different in shape in the two panels? I cannot detect any difference in current size between B and D. Didn’t Ba and Ni remove the instantaneous current, and should we not expect different currents in panels B and D. Have I missed something?
Response: Once again these are interesting points. The data sets in Panels A and C are not from the same cell. The revised text now specifies the voltage levels that were used to estimate the reversal potentials. It is not known why the outward currents in response to the P2 pulse are quite different but it is likely to do with the complex ways in which barium and/or nickel can influence the permeability and/or the gaiting of If channels. When you compare the early onset components of the current records in Panels A and C you can quite clearly see that the quasi-instantaneous current components that are prominent in Panel A are much reduced in Panel C. Your question refers to Panel D but it seems to me that you were asking about the differences between Panels A and C.
Comment 19: Legend to Fig 4: If there is 10 mV between each step how is then possible that you start at -50 and end at -135 mV? The last sentence states that “The resulting conductance-voltage relation was fitted to a Boltzmann function (defined as: G = Gmax/(1 + exp[(Vm – Vh)/s])) and plotted as shown in Panel C.” This does not fit anything in Fig 4. I am confused.
Response: In Figure 4 the holding potential was -45 mV and not -40 mV as stated in the Legend. In addition, the 10 mV steps were applied from -55 to -135 mV. I apologize for including incorrect information in the original manuscript. In addition, in the Legend of this Figure the last sentence was misplaced. It should have been the last sentence in the Legend of Figure 7. Now this has been corrected.
Comment 20: Page 8, last line: “not shown” is not OK for me. If you have the data, they must be shown.
Response: Thank you for this comment. In response in the last sentence of page 8 we have removed all of the wording following the expression ‘much better fit’.
Comment 21: Fig. 5: Why is the trace for -100 mV missing?
Response: Although the experimental notes associated with this original protocol and the resulting curve fitting are quite complete, I have no basis for being able to answer this question other than to say that the protocol involved 20 mV hyperpolarizing steps when the membrane potential during the step was larger than -90 mV. It turns out that this choice was a good one because if the additional data would have been included in this diagram you may well have commented that the Figure, although well-constructed, was quite cluttered and difficult for you to interpret.
Comment 22: Legend to Fig 5: It is said that the pulse length is 10 s, but as far as I can see it is 20 s in the figure. It is said that you use pulses down to -135 mV, but I can just find down to -120 mV. It is stated that “For deactivation, a 10 sec step to -120 mV was utilized to fully activate If; and then its deactivation kinetics were studied by applying 10 sec. steps to membrane potentials in the range -50 to -110 mV.” This is not seen at all.
Response: You are correct on both accounts. Each hyperpolarizing step lasted 20 seconds and the largest step shown is to -120 mV. The Figure Legend has been corrected. Once again you are correct with regard to the statements in your last two sentences. The information provided relates to Figure 6 and new text has now been added to point this out.
Comment 23: Page 9, Eq 1: This cannot be correct. At the voltage Vm=V0 the equation is undetermined.
Response: Thank you for your comment. In this revised version, we refer details about the fit functions to Equation 5-6 in the Supplementary section.
Comment 24: Page 9, lines 5-9 from bottom: “Moreover, when the SAN beating rate increases approximately 2.5-fold in the presence of ISO the timedependent changes in If during the shortened diastolic intervals will be even smaller. In fact, the activation of If that occurs during each action potential does not fully deactivate within the following diastolic interval.” How do you know this? You do not present any data for If at voltage more positive to -50 mV. In addition, do you have any data on time constants for If under ISO exposure?
Response: Thank you for the comment. We have revised the relevant text in this part to make the text more clarified.
Comment 25: Fig. 6: Time constants from a double exponential fit without amplitudes are quite meaningless. You should also present the fraction of amplitudes at different voltages. Furthermore, you have pooled data from deactivation and activation. I definitely think that data should be presented for the separately. In the analysis, they can be pooled.
Response: Thank you. The fit function details and amplitude information is presented in the Supplemnetary materials Section I, equation 5 & 6, which are in the format that are integrated into the model of If. The fraction of If fast and slow component is also presented there in Equation 9. In addition We have attached a plot of the original data in which the kinetic information based on analyses for the time courses of activation and deactivation are clearly indicated. In terms of the formalism for the analysis that we have adopted and utilized, I see no reason why these data sets cannot be combined as has been done in Figure 6.
Comment 26: Legend to Fig. 6: This is, in my ind, one example of a too wordy legend. It must be stated which equation is used to get the parameter values.
Response: Thank you. This Figure Legend has been reduced in length substantially – by about 30%.
Comment 27: Page 10, lines 2-5 (after the legend): “A decision was made to attempt to record the raw data that is required for deriving this information by applying voltage clamp protocols that utilized ramp, as opposed to rectangular, command waveforms.” Unnecessary wordy.
Response: Again, we agree and have reduced the text significantly.
Comment 28: Page 10, para 2, line 4-6: “A typical estimated leakage current, assuming a seal resistance in the 10 Gigaohm range is shown in red in Figure 7A (assuming that this current is linear over the voltage range of interest).” I do not understand why you have to assume a seal resistance. Can’t you measure it?
Response: Thank you for this comment. In the R-1 text we now indicate that a 10 Gigaohm seal resistance was the minimal requirement and for that reason we have used it in this calculation as an exemplar.
Comment 29: Fig. 7B: It is a little bit confusing that you have switched colors. In Fig. 5 the fit was red and data in black. Now it is the opposite. I thought the black curve was a Boltzmann fit, but there is a slight shakiness of the curve. Why?
Response: Thank you for the comment. Now we have corrected the use of colour: black for raw and red for the fit. We have no information that would be helpful to me or you in attempting to provide reasons for the ‘slight shakiness’ of this curve.
Comment 30: Legend to Fig 7: Again too wordy for my taste. I think the way voltage ramps are a little bit confusing. Sometimes they are called ramps, sometimes they are linear (but even square-like voltage clamp steps are linear), sometimes they are triangular. I suggest to use a consistent language. The used equation should have a number.
Response: Thank you for this comment. We agree with both. The equations are now numbered sequentially and an attempt has been made to describe the ramp voltage clamp commands with consistent terminology.
Comment 31: Page 11, lines 6-8 from bottom: “A typical steady-state current trajectory in this range of potentials is shown in Figure 8A and the conductance voltage relationship derived from this data is shown in Panel B.” In the figure it is the opposite.
Response: You are correct and we appreciate the careful review that you have provided. We have corrected these panel descriptors for Figure 8.
Comment 32: Fig. 8: Why is the conductance in A given in nS/pF while the current in B is given only in pA? Why I there no noise in all the panels of this figure. We would expect massive noise in C. I think this is a very critical point in my review. These are the data that are important.
Response: Thank you for this important question. Once again my (WRG) response is perhaps not complete because of the data and data files being quite old and my use and interpretation of them relying entirely on comprehensive but not perfect experimental records and annotation. As far as I can tell, the absence of the noise that you are expecting to be present is due to the fact that these records were both heavily filtered and have been signal averaged using a total of either 4 or 6 identical voltage ramps. The information provided in Panels B and C could certainly have been expressed as a current density but this was not done in the records that I have access to. Recognizing that you and most readers will want this information, I have attempted to address this by stating the average capacitance of the SAN pacemaker myocytes that were studied in this series of experiments. I hope that this response suffices.
Comment 33: Legend to Fig. 8: The gain is not 300-fold. It is 20-fold.
Response: Thank you for this comment and correction. I believe that the increase in gain comparing Panels B and C is 30-fold.
Comment 34: Page 13, line 2: 600 ms should be 60 ms and 1 s should be 100 ms if I interpret the figure correctly.
Response: Thank you for this comment. The information provided in the original manuscript was correct. That is, the two numbers stated at the bottom of page 12 and the top of page 13 should in fact be 600 ms and 100 ms as originally written.
Comment 35: Page 13 and Fig. 9: I have problems with this figure and associated text: The colors (blue and black) are extremely difficult to differentiate in my printed version. If I correctly dissect the colors I have problem to get the text in line with the figures. For instance, from 0.04 to 0.06 ISO makes the current more negative in panel A(i). This time interval corresponds to a voltage of about -40 to -60 mV. But in Panel B, the ISO induced current is positive. I am confused. From 0.06 and onwards, the ISO induced current in A(i) switches from a negative to a positive. This is not seen in B.
Response: Thank you for this comment. This Figure was not constructed in an optimal way. The data obtained in ISO is now shown in red and the control data is shown in black and this is consistent with the other control vs. ISO data in the R-1 manuscript. The description of the protocol and the results have both been re-written in an attempt to clarify the results obtained. In addition, and importantly, with respect to this comment that you have made (and your comment associated with Figure 8), the new text now makes it clear that this is an interesting preliminary finding. This is a particular case where the absence of a bracketed control results in firm conclusions from this interesting finding being impossible to put forward.
Comment 36: Page 14, line 5 from bottom: What does “semiquantitative” mean?
Response: When I choose the word ‘semiquantitative’ with respect to our modelling, I want to convey that as presented in this paper the modelling provides a useful illustration of the consequences of our experimental data but that neither this model nor those published by others provide a basis for firm conclusions regarding unique mechanisms for ionic currents of interest such as If.
Comment 37: Page 15, line 3: The red traces do not depict the changes.
Response: I am uncertain what this comment indicates or requests. Accordingly, and consistent with other efforts to make it clear to the reader that our work is preliminary, I have changed the word ‘depict’ to the phrase ‘provide an indication of the consequence of changes in these dynamics parameters’.
Comment 38: Page 15, line 6: What is electrophysical.
Response: I have changed the word ‘electrophysical’ to ‘electrophysiological’. When I chose the word electrophysical, I may have had a premonition regarding your strong interest in some of the 1939 squid axon data and the fact that the three principal investigators all had undergraduate degrees in Physics.
Comment 39: Page 15, para 1, last line: “…‘on top of’ a steady inward current of about 0.15 pA/pF.” What does ‘on top’ mean? It is not clear to me.
Response: Thank you for pointing this out. The phrase ‘on top’ has been replaced with ‘simultaneously with’.
Comment 40: Fig. 10F: Do not cut the y-axis. Start from 0.
Response: Thank you for the suggestion. Now we have rescaled the y-axis to start from 0.
Comment 41: Page 15, line 3 from bottom: “dose-dependent modulation by ISO”. How is this done? I cannot find any information in the main text. Maybe it is hidden in any supplementary information. Unfortunately, I did not find any supplementary information.
Response: You raise an important point here and we certainly appreciate the fact that you persisted in completing this review while not being able to access the information in the Supplement section. The supplementary material was provided as a separate file to the main text, with details about how ISO were simulated. In the revised submission, we do hope this material is accessible to you.
In a number of Figures in that section (Figures S5 to S10) 2.b of the Supplemetary Materials we offer what we believe to be an interesting extension of our theoretical work by illustrating possible consequences of dose-dependent changes in selected ionic currents. We acknowledge that this is illustrative but not conclusive.
Comment 41: Page 16 (roughly): A critical question is how the new model differs from the previous model. I could not find any information in the main text. Maybe it is in any supplementary information that I could not find. But I do think this must be spelled out in the main text, including how the calculations were carried out.
Response: Details of the modifications to the original model were provided in the Supplementary materials. We apologise for it not being accessible to you in the previous round of the submission. In brief, the new If formulation is different to the original ones by incorporating a fast and a slow component of If with their time constants being refitted to experimental data presented in this study.
Comment 42: Page 16, para 2, line 5: it is said that the firing rate was increase by ISO by a factor of 2-2.5. In Fig. 11A I can only get it to 1.3, which is much less (+30% vs +100-150%).
Response: Thank you. This statement was based more closely on Figure 1 (experimental data) than on the computational output that is shown in Figure 11. The text has been changed in accordance with your correction.
Comment 42: Fig. 11F: Do not cut the y axis. Start at 0.
Response: Thank you once again for the suggestion. In the revised version the y-axis of Figure 11 Panel F starts at 0.00 values.
Comment 43: Discussion: It is far too long: I suggest at least 50% cut.
Response: On re-reading this section, I can appreciate your point and have done some considerable editing. However, in the end, I have only achieved about a 20% reduction in part because Reviewer #2 put forward what I consider to be an opposite request. In response to your concern, I seriously considered removing the material regarding the consequences of mutations in If for human SAN pacemaker activity. However, Reviewer #2 had a strong preference for including additional translational information and the previous extensive and meaningful work on mutations provided the best example of this that I am aware of.
Comment 44: Page 21, para 2, lines 6-7: “The Kharche et al. model [38] was chosen as a starting point based on its successful peer review and publication.” I do not understand this. You must use better arguments.
Response: Thank you for the suggestion. In the revised version, we have changed the expression as:
“The Kharche et al., model [38] was chosen as a starting point based on its quite extensive validation against key features of experimental data describing most of the major transmembrane currents in mouse SAN myocytes.”
Reviewer 2 Report
Title: “Frequency-dependent Properties of the Hyperpolarization-activated Cation Current, If, in Adult Mouse Heart Primary Pacemaker Myocytes”
I read this paper and I think that:
- The authors should provide a table gathering the main data from literature: type of study, results, outcomes, etc. Please provide.
- The authors should dedicate a paragraph dealing with the clinical implication and therapeutic targets derived from their researches.
- The authors can discuss the role of heart rate in cardiovascular diseases. Specifically, they can discuss the paper from Scicchitano P et al. Drug Des Devel Ther. 2014 Jun 3;8:689-700.
Author Response
Response to Reviewers’ Comments
We would like to thank both Reviewers for their constructive and helpful comments, based on which we have revised our manuscript. We hope you would find our revision satisfactory. Point-by-point response to your comments are listed below.
Reviewer 2
Comment 1: I read this paper and I think that:
Response: Thank you for evaluating our paper and providing thoughtful but brief suggestions for clarifications and changes. Our detailed response to your suggestions are provided below and additional information and clarification can also be found in our responses to Reviewer #1.
Comment 2: The authors should provide a table gathering the main data from literature: type of study, results, outcomes, etc. Please provide.
Response: This is a useful suggestion and we have carefully considered adding Tables of the kind that you request. However, a previous review provides a very comprehensive set of Tables that provide the information that you request. In the publication by Verkerk et al., (2009), Tables 1 through 4 provide this information. This review article is cited as Reference 16 in our R-1 manuscript and we have now added text guiding the reader to this detailed information in Tables 1 to 4.
Comment 3: The authors should dedicate a paragraph dealing with the clinical implication and therapeutic targets derived from their researches.
Response: Thank you for this suggestion. In the original manuscript detailed reference was made to some aspects of the clinical implications of our findings through our citation and treatment of information concerning the functional effects of mutations in HCN gene products and related alterations in the pharmacological and biophysical properties of If. We are uncertain and reluctant to attempt to bring out the ways in which adrenergic tone and its effects on If may be relevant since our data has all been obtained from a mouse heart preparation in which both heart rate and adrenergic tone are considered outliers with respect to human physiology or pathophysiology. In both the original manuscript and the R-1 version this reservation is stated in the Discussion section.
Comment 4: The authors can discuss the role of heart rate in cardiovascular diseases. Specifically, they can discuss the paper from Scicchitano P et al. Drug Des Devel Ther. 2014 Jun 3;8:689-700.
Response: Thank you for this suggestion. We were not aware of this review. However, after reading it, we are concerned that a direct citation regarding the clinical contexts that it deals with may be somewhat misleading given the known and marked differences, particularly in heart rate between the adult mouse and the adult human heart under either physiological or pathophysiological conditions. As a compromise, which we hope you will find successful, we have cited this review as Reference [85] in the last sentence of the Discussion section where it provides a useful, general context.
