Review Reports - Biophysical Mechanisms of Vaginal Smooth Muscle Contraction: The Role of the Membrane Potential and Ion Channels

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, the authors reviewed studies on the regulation mechanisms of vaginal smooth muscle (VSM) contraction. Although this review may expand our knowledge in this field, I have some critical concerns and suggestions for improvement.

Major point:

1. Differences in the contraction mechanisms between VSMs and other smooth muscles should be understood to develop therapeutic agents for vaginal contraction disorders with fewer side effects. However, this point is poorly discussed in the manuscript. Muscarinic contractions in the gastrointestinal tract are well known to be mediated by the combined activation of M₂ and M₃ receptors, with a greater contribution from M₃ receptors. TRPC4 and TRPC6 channels have been reportedly responsible for muscarinic cationic current in ileal myocytes (Tsvilovskyy, V.V. et al. Gastroenterology 2009, 137, 1415–1424.). How about VSM? The authors must provide more detailed information about subtypes of receptors and molecular identities of ion channels expressed in VSMs (not only muscarinic receptors and TRPC channels) and compare them to those in other smooth muscles.

2. What kind of VSM changes have been reported in vaginal contraction disorders. Please describe this point in more detail. Readers of Pathophysiology should be primarily interested in this point.

3. Do the authors mean changes in the VSM structure and function induced by pelvic organ prolapse itself or implantation of medical mesh for the prolapse (Line 80)?

4. ATP-sensitive K⁺ channel is not a voltage-gated channel. The description in line 238 must be corrected.

5. In Table 2, the authors described that Cl⁻ channels are involved in the repolarization phase in action potentials/slow waves in smooth muscles. However, Cl⁻ channels expressed in many smooth muscles contribute to depolarization, as the equilibrium potential of Cl⁻ is well above the resting potential (Zholos, A.V. Neurophysiology 31, 173–187 (1999). doi:10.1007/BF02515064). Furthermore, Ca²⁺-activated Cl⁻ channels ANO1/TMEM16A are primarily responsible for generating pacemaker activities in interstitial cells of Cajal (ICC) (Sanders, K.M. et al. Physiol Rev 94: 859–907, (2014). doi:10.1152/physrev.00037.2013.). Thus, Table 2 must be modified.

6. Are there any reports to prove the existence of ICC-like cells in the VSM tissue (Figure 3)?

7. The regulation of ion channels by muscarinic and P2X receptors and its underlying mechanisms in VSM must be discussed.

8. Do the authors mean the release of Ca²⁺ from the intracellular Ca²⁺ store (Line 233)?

9. I suggest adding the description of CRAC channels in VSM in the 6^th section of page 8 and Figure 3.

10. “Vascular smooth muscle” should be changed to “Smooth muscle” in line 36.

11. Figure 1 may not be essential because it provides little information. Please consider deleting it.

Minor point:

1. Replace (a) with (A) and insert (B) after “smooth muscle cells” in lines 141–142 to match with the caption of Figure 2.

2. “,” after Ca²⁺ in line 202 and “-” after Ca²⁺ in lines 204, 218, and 243 were superscripted. Please correct them.

3. Insert “,” before the CRAC channel in line 240.

4. Please make “2” after Ca in line 243, “2+” after Ca in line 342, and “+” after K in line 344 as superscript.

5. Delete “Calcium ions” before “Ca²⁺” in line 309. They are duplicated.

Author Response

Major point:

Differences in the contraction mechanisms between VSMs and other smooth muscles should be understood to develop therapeutic agents for vaginal contraction disorders with fewer side effects. However, this point is poorly discussed in the manuscript. Muscarinic contractions in the gastrointestinal tract are well known to be mediated by the combined activation of M₂ and M₃ receptors, with a greater contribution from M₃ receptors. TRPC4 and TRPC6 channels have been reportedly responsible for muscarinic cationic current in ileal myocytes (Tsvilovskyy, V.V. et al. Gastroenterology 2009, 137, 1415–1424.). How about VSM? The authors must provide more detailed information about subtypes of receptors and molecular identities of ion channels expressed in VSMs (not only muscarinic receptors and TRPC channels) and compare them to those in other smooth muscles.

Response to reviewer:

Thank you for pointing out these essential elements for creating a thorough review. The manuscript has been expanded to include additional content, enhancing its comprehensiveness.

While there's a lack of comprehensive experimental techniques focusing on electrophysiology, calcium dynamics, and force generation in vaginal smooth muscle compared to the GI tract, this review explores potential mechanisms in VSM cells. We draw insights by contrasting them with confirmed data from other smooth muscle types through the study of electrical activities. Ion channels and calcium dynamics are expanded now for more information.

What kind of VSM changes have been reported in vaginal contraction disorders. Please describe this point in more detail. Readers of Pathophysiology should be primarily interested in this point.

Response to reviewer:

Thank you for pointing out these factors. One paragraph is now added to describe the vaginal contraction disorders.

Do the authors mean changes in the VSM structure and function induced by pelvic organ prolapse itself or implantation of medical mesh for the prolapse (Line 80)?

It is pelvic organ prolapse (when the bladder, urethra, rectum, small intestine, uterus, or vagina, slip down from their normal position and bulge into the vagina.), not by any implant.

Response to reviewer:

The line is corrected.

ATP-sensitive K⁺ channel is not a voltage-gated channel. The description in line 238 must be corrected.

Response to reviewer:

The mistake is corrected now and the lines are changed.

In Table 2, the authors described that Cl⁻ channels are involved in the repolarization phase in action potentials/slow waves in smooth muscles. However, Cl⁻ channels expressed in many smooth muscles contribute to depolarization, as the equilibrium potential of Cl⁻ is well above the resting potential (Zholos, A.V. Neurophysiology 31, 173–187 (1999). doi:10.1007/BF02515064). Furthermore, Ca²⁺-activated Cl⁻ channels ANO1/TMEM16A are primarily responsible for generating pacemaker activities in interstitial cells of Cajal (ICC) (Sanders, K.M. et al. Physiol Rev 94: 859–907, (2014). doi:10.1152/physrev.00037.2013.). Thus, Table 2 must be modified.

Response to reviewer:

Thank you for noticing the error and providing the necessary feedback. The table lines are changed now.

Are there any reports to prove the existence of ICC-like cells in the VSM tissue (Figure 3)?

Response to reviewer:

An additional paragraph for ICC is added now and it supports the presence of ICC in VSM cell.

The regulation of ion channels by muscarinic and P2X receptors and its underlying mechanisms in VSM must be discussed.

Response to reviewer:

Additional lines are added now.

Do the authors mean the release of Ca²⁺ from the intracellular Ca²⁺ store (Line 233)?

Response to reviewer:

Yes, it means the sarcoplasmic reticulum store. The line is modified now.

I suggest adding the description of CRAC channels in VSM in the 6^th section of page 8 and Figure 3.

Response to reviewer:

Additional components are added in Figure 3.

“Vascular smooth muscle” should be changed to “Smooth muscle” in line 36.

Response to reviewer:

The error is rectified.

Figure 1 may not be essential because it provides little information. Please consider deleting it.

Response to reviewer:

The figure is modified.

Minor point:

Replace (a) with (A) and insert (B) after “smooth muscle cells” in lines 141–142 to match with the caption of Figure 2.

Response to reviewer:

The errors are rectified.

“,” after Ca²⁺ in line 202 and “-” after Ca²⁺ in lines 204, 218, and 243 were superscripted. Please correct them.

Response to reviewer:

The errors are rectified.

Insert “,” before the CRAC channel in line 240.

Response to reviewer:

The error is rectified.

Please make “2” after Ca in line 243, “2+” after Ca in line 342, and “+” after K in line 344 as superscript.

Response to reviewer:

The errors are rectified.

Delete “Calcium ions” before “Ca²⁺” in line 309. They are duplicated.

Response to reviewer:

The error is rectified.

Reviewer 2 Report

Comments and Suggestions for Authors

Mahapatra and Kumar:

The authors present a review on vaginal smooth muscle (VSM) cells, about their function and disease. They intend to present a biophysical view, combining knowledge from various sources. They describe membrane potential, ion channels calcium regulation in smooth muscles, as well as ways to study VSM contraction.

Major points:

1. Overall, I found that the review lacks depth in any topic that has been taken up by the authors. At a lot of places, as a reader, the review just looks like an information dump, with one whole topic covered in 1-2 lines. For example, various ion channels are described too quickly - giving the reader the feeling that Sodium channels, TRP channels, voltage gated K channels all contribute a little here and there to SMC contraction. While it may be true, specifically what connects particular ion channels to vaginal smooth muscle cell function? What are the known disorders of particular ion channels that have a bearing on vaginal function? The authors just seem to skip the details and move quickly from one topic to the other. This takes away the chance for the review to be comprehensive.

2. The ion channel and calcium dynamics sections seem to be applicable to smooth muscles, but is not in any way specifically tailored for VSM cells. What is the angle here for VSM cells? Do these things work the same way here like all other smooth muscles? If not what is specifically important for VSM cells pertaining to these topics? Overall, specificity to VSM cells is lacking in these sections.

3. Like the previous two points, their model section (6) skips a lot of steps in each point. For example, at one point they just say that calcium binds calmodulin and that leads to myosin activation by phosphorylation. There is no specific mention here of how this happens, where is this phosphorylation happening, what are the implications and how? Things are just mentioned in passing and the relevant depth is just missing. It pertains to all sections of the review in general.

Minor point

4. Which TRP channels are mechanically activated here? Please explain. (Lines 225-227). This underscores my general issue with this review, that details are just lacking.

Comments on the Quality of English Language

Minor english editing is required.

Author Response

Mahapatra and Kumar:

Major points:

Overall, I found that the review lacks depth in any topic that has been taken up by the authors. At a lot of places, as a reader, the review just looks like an information dump, with one whole topic covered in 1-2 lines. For example, various ion channels are described too quickly - giving the reader the feeling that Sodium channels, TRP channels, voltage gated K channels all contribute a little here and there to SMC contraction. While it may be true, specifically what connects particular ion channels to vaginal smooth muscle cell function? What are the known disorders of particular ion channels that have a bearing on vaginal function? The authors just seem to skip the details and move quickly from one topic to the other. This takes away the chance for the review to be comprehensive.

Response to reviewer:

Thank you for pointing out these essential elements for creating a thorough review. The manuscript has been expanded to include additional content, enhancing its comprehensiveness according to reviewers’ queries.

The ion channel and calcium dynamics sections seem to be applicable to smooth muscles, but is not in any way specifically tailored for VSM cells. What is the angle here for VSM cells? Do these things work the same way here like all other smooth muscles? If not what is specifically important for VSM cells pertaining to these topics? Overall, specificity to VSM cells is lacking in these sections.

Response to reviewer:

Like the previous two points, their model section (6) skips a lot of steps in each point. For example, at one point they just say that calcium binds calmodulin and that leads to myosin activation by phosphorylation. There is no specific mention here of how this happens, where is this phosphorylation happening, what are the implications and how? Things are just mentioned in passing and the relevant depth is just missing. It pertains to all sections of the review in general.

Response to reviewer:

Additional paragraphs are added to describe the actin-myosin interaction for the contraction.

Minor point

Which TRP channels are mechanically activated here? Please explain. (Lines 225-227). This underscores my general issue with this review, that details are just lacking.

Response to reviewer:

Additional lines are added now.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for responding and addressing my comments. The manuscript has been improved; however, the authors should revise the manuscript by addressing the following comments:

1 Please remove the word “vaginal” in the caption of Figure 1 because this figure describes the process of excitation–contraction coupling, which is common process in smooth muscle.

2 Do the authors mean “After vaginal delivery” in lines 85-86?

3 In line 113, “mesh” should be removed.

4 Delete “intracellular” from line 129. Furthermore, “electrophysiology” should be changed to “electrophysiological.”

5 Please consider removing “any specific smooth muscle type, including” from line 153.

6 Line 286 should read “Na⁺ channels” instead of “Na⁺ ions”.

7 Include references to L-type and T-type Ca²⁺ channels on line 294 or elsewhere in this paragraph.

8 The sentence in line 301–303 is unclear. Are the authors referring to “urogenital smooth muscle contraction induced by stimulation of cholinergic nerve or by activation of muscarinic receptors due to acetylcholine”?

9 Change “ATP-selective” to “ATP-sensitive” in line 309.

10 Change “action potential” to “AP” in line 322.

11 In line 348, define “KDR.” .

12 In intestinal smooth muscles, TRPC4 channels are responsible for muscarinic cationic current (Tsvilovskyy et al. Gastroenterology 137(4):1415-24. (2009) doi: 10.1053/j.gastro.2009.06.046). Please consider adding a discussion about this point (Line 362–364).

13 Delete “ion” from line 385.

14 I believe ICRAC is an abbreviation for current through CRAC channels (Line 395–396). Thus, the expression of “ICRAC channels” could be incorrect (Line 397). Do the authors mention Orai channels here (Line 395–400)?

15 Please convert “ATP” after “K” on lines 406 and 407 into subscript.

Author Response

Dear reviewer,

Thank you for pointing out these essential elements for creating a thorough review. The manuscript has been expanded to include additional content, enhancing its comprehensiveness.

Revision 2

Please remove the word “vaginal” in the caption of Figure 1 because this figure describes the process of excitation-contraction coupling, which is a common process in smooth muscle.

Response to reviewer:

The word is removed from the manuscript.

Do the authors mean “After vaginal delivery” in lines 85-86?

Response to reviewer:

Yes, it means “After vaginal delivery”. To avoid confusions for readers, we have also replaced this phrase in the manuscript.

In line 113, “mesh” should be removed.

Response to reviewer:

The word is removed from the manuscript.

Delete “intracellular” from line 129. Furthermore, “electrophysiology” should be changed to “electrophysiological.”

Response to reviewer:

The word is changed in the manuscript.

Please consider removing “any specific smooth muscle type, including” from line 153.

Response to reviewer:

The word is removed from the manuscript.

Line 286 should read “Na⁺channels” instead of “Na⁺ ions”.

Response to reviewer:

The word is changed in the manuscript.

Include references to L-type and T-type Ca²⁺channels on line 294 or elsewhere in this paragraph.

Response to reviewer:

Reference added in the manuscript.

The sentence in line 301–303 is unclear. Are the authors referring to “urogenital smooth muscle contraction induced by stimulation of cholinergic nerve or by activation of muscarinic receptors due to acetylcholine”?

Response to reviewer:

The following paragraph is replaced to prevent any confusion.

Specifically, L-type Ca2+ channel antagonists, such as nifedipine, effectively inhibit smooth muscle contraction in the urogenital tract by blocking L-type Ca2+ channels. They also prevent the refilling of intracellular Ca2+ stores by impeding cholinergic nerve stimulation, where acetylcholine activates muscarinic receptors on the membrane.

Change “ATP-selective” to “ATP-sensitive” in line 309.

Response to reviewer:

The word is changed in the manuscript.

Change “action potential” to “AP” in line 322.

Response to reviewer:

The word is changed in the manuscript.

In line 348, define “KDR.” .

Response to reviewer:

Delayed rectifier K is added to this word.

In intestinal smooth muscles, TRPC4 channels are responsible for muscarinic cationic current (Tsvilovskyy et al. Gastroenterology137(4):1415-24. (2009) doi: 10.1053/j.gastro.2009.06.046). Please consider adding a discussion about this point (Line 362–364).

Response to reviewer:

A new reference (Tsvilovskyy et al 2009) is added for the TRPC4 channel.

Delete “ion” from line 385.

Response to reviewer:

The word is changed in the manuscript.

I believe ICRAC is an abbreviation for current through CRAC channels (Line 395–396). Thus, the expression of “ICRAC channels” could be incorrect (Line 397). Do the authors mention Orai channels here (Line 395–400)?

Response to reviewer:

The following paragraph is replaced to prevent any confusion.

ICRAC, a subtype of the CRAC family activated by STIM proteins, exhibits unique properties by selecting Orai1 and Orai2 isoforms.

Please convert “ATP” after “K” on lines 406 and 407 into subscript.

Response to reviewer:

The word is changed in the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I have looked at the revision for the review manuscript. I am satisfied overall with the changes added including the increased focus on contractile mechanisms and ion channels. I am on board with publication.

Comments on the Quality of English Language

Minor edits required.

Author Response

Dear Reviewer,

Thank you for pointing out these essential elements for creating a thorough review. The manuscript is modified with minor edits.

Best regards

Authors

Author Response File: Author Response.pdf