Flow-Dependent Modulation of Endothelial Ca2+ Dynamics by Small Conductance Ca2+-Activated K+ Channels in Mouse Carotid Arteries
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis study investigates the role of Small conductance Ca²⁺-activated K⁺ channels (KCa2.3) in endothelial cell Ca²⁺ signaling under flow conditions using an endothelial-specific Ca²⁺ fluorophore (cdh5-GCaMP) mouse model. The findings suggest that KCa2.3 amplifies flow-induced Ca²⁺ signaling, with potential implications for vasodilation in disease models such as hypertension. While the study addresses an important topic, several critical issues need to be clarified before publication:
Major Concerns
1. Mechanosensitive Ca²⁺ Channel Identification
The key finding that KCa2.3 amplifies flow-induced Ca²⁺ influx is significant. However, as KCa2.3 is not mechanosensitive and requires Ca²⁺ influx to activate, the study lacks clarity on the identity of the upstream mechanosensitive channel that initiates the Ca²⁺ signal.
2. Discrepancy Between Static and Flow Conditions
The results in Figure 2 show no difference in Ca²⁺ signaling under static conditions when KCa2.3 is inhibited by apamin, whereas Figure 4 demonstrates a significant increase in Ca²⁺ signal when a KCa2.3 agonist is used. This inconsistency raises concerns about the mechanism.
Clarification Needed: If a Ca²⁺-permeable channel spontaneously opens under static conditions, inhibiting KCa2.3 with apamin should theoretically reduce Ca²⁺ levels. The absence of this effect suggests that KCa2.3 activation may involve additional factors or pathways under flow conditions. The authors should explain why activation of KCa2.3 potentiates Ca²⁺ signals in Figure 4 if no change is observed under static conditions with apamin.
3. Discrepancy in Event Counts (Figures 1D and 3B)
The number of events in Figures 1D and 3B (red curves) is noticeably different. This discrepancy is critical, as it could influence the interpretation of apamin’s effect on Ca²⁺ influx.
Additional Suggestions
Mechanistic Insights into KCa2.3 Activation: The study would benefit from additional mechanistic insights into how KCa2.3 contributes to amplified Ca²⁺ signaling under flow conditions. Exploring whether KCa2.3 activation involves feedback from Ca²⁺-dependent hyperpolarization or changes in membrane potential could strengthen the findings.
Conclusion
The study presents novel findings on the role of KCa2.3 in endothelial Ca²⁺ signaling under flow conditions, with implications for vascular physiology and pathology. However, several critical issues, including the identification of the upstream mechanosensitive channel, discrepancies in experimental data, and inconsistencies in the interpretation of KCa2.3's role, must be addressed to ensure the validity and impact of the conclusions.
This manuscript has the potential to make a valuable contribution to the field once these issues are clarified.
Author Response
Reviewer comment: The key finding that KCa2.3 amplifies flow-induced Ca²⁺ influx is significant. However, as KCa2.3 is not mechanosensitive and requires Ca²⁺ influx to activate, the study lacks clarity on the identity of the upstream mechanosensitive channel that initiates the Ca²⁺ signal.
Response: We thank the reviewer for this query and agree that the source of shear stress detection and transduction is important. However, this appears to be a complex mechanism, with multiple channels, molecular components and signaling entities potentially contributing to flow-induced Ca2+ entry. In fact, there is little consensus on a primary shear stress sensor and mechanism, and past work suggests the endothelium may have multiple levels of response depending on the nature (laminar, oscillatory, pulsatile) and degree of the prevailing flow profile and expression profile. This includes differential roles of cation channels such as peizo-1, purinergic ATP-sensing receptors (P2X4), and transient receptor potential channels, that may themselves be coupled to other signaling components and to each other at the membrane. These collective influences can elicit both rapid and extended effects on vascular function and structure (e.g. vasodilation, channel/receptor trafficking, gene expression). We did not set out to address this question directly. Rather, we focused the current study on determining whether KCa2.3 contributes to modifying the broader endothelial Ca2+ profile (regardless of the specific Ca2+ source(s)) with the idea that, if true, future studies will directly address interactions with mechanism(s) of Ca2+ influx and/or release. Based on our current findings, subsequent studies are warranted and now being pursued to elucidate the mechanism of the Ca2+ amplification observed. We have clarified our rationale and findings in the manuscript (line 264)
Reviewer comment: Mechanistic Insights into KCa2.3 Activation: The study would benefit from additional mechanistic insights into how KCa2.3 contributes to amplified Ca²⁺ signaling under flow conditions. Exploring whether KCa2.3 activation involves feedback from Ca²⁺-dependent hyperpolarization or changes in membrane potential could strengthen the findings.
Response: We thank the reviewer for this suggestion and agree. We have expanded this concept in the discussion. While this study was focused on the potential impact of KCa2.3, future studies are aimed at identifying key flow-linked elements coupled with KCa2.3 and extending the mechanistic insights.
Reviewer comment: The results in Figure 2 show no difference in Ca²⁺ signaling under static conditions when KCa2.3 is inhibited by apamin, whereas Figure 4 demonstrates a significant increase in Ca²⁺ signal when a KCa2.3 agonist is used. This inconsistency raises concerns about the mechanism.
Clarification Needed: If a Ca²⁺-permeable channel spontaneously opens under static conditions, inhibiting KCa2.3 with apamin should theoretically reduce Ca²⁺ levels. The absence of this effect suggests that KCa2.3 activation may involve additional factors or pathways under flow conditions. The authors should explain why activation of KCa2.3 potentiates Ca²⁺ signals in Figure 4 if no change is observed under static conditions with apamin.
Response: We thank the reviewer for this insightful comment. Although basal dynamic Ca2+ activity is always present in the endothelium, our data suggest that under static conditions this activity is not adequate to drive sufficient KCa2.3 channel opening to influence the overall Ca2+ signal profile. This is supported by the non-effect of apamin under basal conditions. However, CyPPA is a positive modulator of KCa2.3 channels; it increases the Ca2+ sensitivity of the channels, thereby increasing their relative Ca2+-dependent activation. Our interpretation is that addition of CyPPA under static conditions increases KCa2.3 Ca2+ sensitivity sufficiently to allow even basal Ca2+ signals to elicit KCa2.3 activation capable of influencing the Ca2+ profile. Overall, we believe our data indicates that KCa channels are capable of augmenting Ca2+ signals...under basal conditions this influence is essentially undetectable unless facilitated by sensitization of the channels to Ca2+ i.e. to allow adequate activation to amplify Ca2+ signals. Under flow conditions, however, the shear stress-induced Ca2+ signals alone are able to amplify the KCa2.3 influence...moreover, Ca2+ sensitization via CyPPA in the presence of flow further expands this amplification. We have more clearly articulated this in the manuscript (line 301).
Reviewer comment: The number of events in Figures 1D and 3B (red curves) is noticeably different. This discrepancy is critical, as it could influence the interpretation of apamin’s effect on Ca²⁺ influx.
Response: We thank the reviewer for this observation and comment. The histograms shown represent full distributions of all recorded events in each group. The number of events per bin for each parameter can be different depending on whether most events are very similar (i.e. in size or duration) or are spread over a larger range. Indeed, this tendency for skewed distributions on a linear scale (where most data points pile up on the left side) is one reason we also plot data on a log scale; this allows evaluation and comparison of data when redistributed over the full range from smallest to largest. While the basal frequency of events is characteristic to a field, there is some variability among different vascular preparations (see Fig 1B). Primarily for this reason, we performed all experiments within the same fields before and after treatment. This allowed us to account for variation and perform paired analysis before and after perturbation as well as inspection of changes at individual sites.
Reviewer comment: Mechanistic Insights into KCa2.3 Activation: The study would benefit from additional mechanistic insights into how KCa2.3 contributes to amplified Ca²⁺ signaling under flow conditions. Exploring whether KCa2.3 activation involves feedback from Ca²⁺-dependent hyperpolarization or changes in membrane potential could strengthen the findings.
Response: We thank the reviewer for this suggestion and we agree. We have expanded this concept in the discussion. While this study was focused on characterizing the overall impact of KCa2.3 channels on the broad Ca2+ profile along the arterial intima, our extended studies are aimed at identifying key flow-linked elements coupled with KCa2.3 and extending these mechanistic insights. These mechanistic targets have been emphasized in the discussion (line 338).
Reviewer 2 Report
Comments and Suggestions for AuthorsIn presented manuscript authors used carotid arteries from Cdh5-GCaMP8 mice to investigated the impact of Kca2.3 channels on applied shear stress compared to no-flow condition. Paper is well structured and results are clearly presented, however I have few comments:
-Is there any correspondent author, and why sign for affiliation is not added? -Why is in footnote of paper year 2022? -How old were animals? And how many them were used? Why you used both sexes? -Ln108 please introduced abbreviation CyPPA -Add in M&M how apamin and CyPPA were dissolved, in which solvent -Ln160 on what referred n=4, experiments, animals, arteries? Do 4 experiments were enough for stastical analysis? The same is applicable for Ln216. -Ln166 is apamin specif or non-specific blocker of KCa2.3 channels? -Ln294 introduce abbreviation for IP3R -Ln296 introduce abbreviation for TRPA1 -Ln295-298 arteries were from which animal/human, please specified in manuscript. -Ln303 what is Rab GTPase Rab11A, please provide more information and why that is important here.Author Response
Reviewer comment: Is there any correspondent author, and why sign for affiliation is not added? -Why is in footnote of paper year 2022?
Response: We thank the reviewer for these helpful comments. The title page has been updated.
Reviewer comment: How old were animals? And how many them were used? Why you used both sexes?
Response: All mice were 3-6 months of age. For all experiments N values refer to number of animals (33 mice were used in total). It is our common practice to include both sexes for inclusive evaluation in cardiovascular studies. Where findings may lead to follow-up evaluations, no specific sex based differences were targeted in the current study and no extended sex-based trends were indicated by the data.
Reviewer comment: Ln108 please introduced abbreviation CyPPA -Add in M&M how apamin and CyPPA were dissolved, in which solvent
Response: This information has been added. Thank you.
Reviewer comment: Do 4 experiments were enough for stasitical analysis? The same is applicable for Ln216.
Response: Thank you for this query. As indicated in response to Reviewer 1, variability among animals and preparations is an important consideration. To reduce variance from independent observations and focus on discrete measurable changes, we performed all observations (longitudinally) in the same continuous fields before and after perturbation. This provided the requisite statistical power through paired comparisons and allowed for assessment of cellular recruitment/de-recruitment within the field.
Reviewer comment: Ln166 is apamin specif or non-specific blocker of KCa2.3 channels?
Response: Apamin is a selective inhibitor of small conductance Ca2+-activated K+ channels (KCa2.1, 2.2 and 2.3). In the vasculature, KCa2.3 is the predominant subtype, being highly expressed within the endothelium. Endothelial KCa2.3 (along with the intermediate conductance channel KCa3.1) have been characterized as key contributors to endothelial signaling and vasoregulation of the arterial vasculature.
Reviewer comment: Ln294 introduce abbreviation for IP3R -Ln296 introduce abbreviation for TRPA1
Response: This information has been added. Thank you.
Reviewer comment: Ln295-298 arteries were from which animal/human, please specified in manuscript.
Response: For the data/findings described, rat cerebral arteries and mouse mesenteric arteries were employed. This has been clarified in the text.
Reviewer comment: Ln303 what is Rab GTPase Rab11A, please provide more information and why that is important here.
Response: We thank the reviewer for this important query. Rab GTPases regulate anterograde and retrograde trafficking in cells and Rab11A is a Ca2+-activated GTPase. Previous findings suggest endothelial stimulation (flow, agonist) elicits Rab11A activation through TRPV4-dependent Ca2+ influx, and this causes endosomes containing SK3 channels to be trafficked to the membrane. We have now elaborated on this in the manuscript (line 325).
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsN/A
