Role of Piezo1 Channels Expressed in PVN in Regulating Sympathetic Nerve Activity and Arterial Blood Pressure in Rats

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study from Chen at al., is looking at the role of Piezo1 channels expressed in PVN in regulating sympathetic nerve activity and arterial blood pressure in rats. Despite employing interesting techniques, the current version of the manuscript lacks adequate experimental validation and heavily relies on speculation. Major experimental revision is needed.

Major comments:

1.GAPDH signal is not equal cross the samples. Different housekeeping gene should be chosen. The reason for including the lung tissue is not stated. Even if it’s for the Piezo1  level comparison with other tissues this should be spelled out. Different tissue samples used for WB and RT-PCR.

2.Fig.1 20x image shows different green channel immunohistochemistry signal than eg.40x. It’s difficult to localize the 63x region within the 40x image, despite the white ROI square. Single channel images are missing. Only merged micrographs are provided. Therefore, it’s difficult to find Piezo1 labelling in the 60x image. Seems like the immunostaining is all over the tissue. Was the antibody validated for specificity ?

3.Dooku1 is not a Piezo1 blocker. It’s Yoda1 analogue, it blocks Yoda1- induced Piezo1 activation. It can’t be compared to GsMTX4. Moreover, GsMTX4 is not a specific Piezo1 blocker. It also acts on TRP channels. This limitation should have been mentioned in the text.

4.Overall, strange order of experimental planning. Firstly introducing Dooku1/GsMTx4 then the Piezo1 activation ?

5.There are no experiments in the manuscript that support the proposed graphical abstract, which is expected to visually represent novel findings.

Minor comments:

Paragraph 1 is missing the abbreviations, such as right cardiac ventricles (RV), right cardiac auricles (RA) etc.

Values introduced in the test should include control and treated sample for comparison.

Where is the heart rhythm (beats/min) graph mentioned in line 102, 123? No information to which figure refer to.

Missing cell/tissue/animal number for Fig.2.

Fig.S1B is missing a control non-injected image.

Figure 1 legend states :

“B: Summary data on the expression of  Piezo1 channels in brain tissues (n = 3);”. However, the graph shows also lung and heart tissue.

“D: Immunofluorescence analysis shows the expression of Piezo1 in PVN neurons projecting to the RVLM”. However, no quantification is provided

Figure 2 legend is not clear “Left, 5-s specimen traces of RSNA before microinjection of Dooku1 107 into the PVN. Middle, 5-s specimen traces of RSNA after microinjection of Dooku1 into the PVN. 108 Right, 5-s specimen traces of RSNA after Dooku1 washout.”. Where is the wash out labelled? Overall, better labelling is needed.

In the methods section is it correct animal number used in this study ?

Section 4.8 mentions DAPI staining is mentioned, but never shown in the figures.

Author Response

We are very grateful for the valuable comments by the reviewers. We tried our best to address the issue raised by the reviewers. The responses to the comments were highlighted in blue colored text.

Review 1

 Major comments:

1. GAPDH signal is not equal cross the samples. Different housekeeping gene should be chosen. The reason for including the lung tissue is not stated. Even if it’s for the Piezo1 level comparison with other tissues this should be spelled out. Different tissue samples used for WB and RT-PCR.

Page 3, line 91. Firstly,GAPDH was selected as the reference gene based on its extensive application in studies on the paraventricular nucleus (PVN) of the thalamus and cardiovascular-related tissues (see the supporting studies by Patel et al., 2011, PMID:21963832 and Pan et al., 2014,PMID:24806793). These studies have confirmed that its expression is relatively stable in these specific tissues under physiological conditions.Secondly, during the membrane scanning step of Western blot (WB) analysis, the GAPDH bands should have been detected exclusively using the 800 nm channel. However, due to an operational error, we inadvertently scanned the membranes with both the 700 nm and 800 nm channels simultaneously, which resulted in band overlapping in the first two lanes. We subsequently corrected this experimental error by only presenting the scanning results obtained from the 800 nm channel, yielding clear, artifact-free bands. The original GAPDH bands in Figure 1A have been replaced with the newly acquired, accurate experimental results.Thirdly,For the dataset of the present study, we re-analyzed the original band intensities of GAPDH detected by Western blotting across all samples (right ventricle [RV], right atrial appendage [RA], lung tissue, and PVN). We confirmed that the coefficient of variation (CV) of GAPDH expression fell within the acceptable range for reference gene stability in tissue-specific comparisons, indicating that the observed variability in GAPDH signals did not significantly affect the relative quantification results of Piezo1 protein.

Existing studies have demonstrated high Piezo1 expression in lung tissue, justifying its selection as a positive control for Western blot assays.(PMID: 34548087)

Despite the use of different tissue samples, the core conclusions of the two experiments corroborate each other: Western blot assays confirmed the presence of Piezo1 protein expression in the PVN, with no significant difference in its expression level compared with that in peripheral relevant tissues; qRT-PCR assays demonstrated that the expression of Piezo1 mRNA in the PVN did not differ significantly from that in the cerebral cortex. Together, these findings provide molecular biological evidence for the key premise that Piezo1 is stably expressed in the PVN, thus ensuring the rationality and reliability of subsequent functional experiments. Furthermore, existing literature has validated the reliability of using cortical tissue as a PCR control due to its abundant Piezo1 expression (PMID: 35963237).

2. 1 20x image shows different green channel immunohistochemistry signal than eg.40x. It’s difficult to localize the 63x region within the 40x image, despite the white ROI square. Single channel images are missing. Only merged micrographs are provided. Therefore, it’s difficult to find Piezo1 labelling in the 60x image. Seems like the immunostaining is all over the tissue. Was the antibody validated for specificity ?

Single-channel images have been provided, in which red fluorescence indicates Piezo1 expression and green fluorescence labels RVLM-PVN neurons. In the red single-channel images, specific Piezo1 expression in the PVN can be clearly observed.

Control experiments were performed with n=3. The primary antibody was omitted during the procedure, while all other conditions were identical to those of the positive group. Raw data are available for inspection.

3. Dooku1 is not a Piezo1 blocker. It’s Yoda1 analogue, it blocks Yoda1- induced Piezo1 activation. It can’t be compared to GsMTX4. Moreover, GsMTX4 is not a specific Piezo1 blocker. It also acts on TRP channels. This limitation should have been mentioned in the text.Overall, strange order of experimental planning. Firstly introducing Dooku1/GsMTx4 then the Piezo1 activation ?

We sincerely appreciate your careful review of this study and valuable comments! Regarding the questions you raised about the mechanism of action and selectivity of Piezo1 channel inhibitors, we have conducted in-depth verification and provide supplementary explanations as follows:

Although Dooku1 and GsMTx4 differ in their molecular mechanisms of Piezo1 channel inhibition, both ultimately significantly reduce the open probability of Piezo1 channels. As a reversible antagonist of Yoda1, Dooku1 exerts its effect mainly by directly antagonizing Yoda1-induced Piezo1 activation. It exhibits clear inhibitory activity against Yoda1-induced calcium influx in HEK293 cells and human umbilical vein endothelial cells (with IC₅₀ values of 1.3 μM and 1.5 μM, respectively) [PMID: 38761789]. GsMTx4 is a peptide inhibitor derived from spider venom that exerts gating regulatory effects by modifying the interface between the channel and the lipid bilayer. It can shift the activation midpoint of Piezo1 channels toward higher pressure values, thereby reducing the frequency of channel opening [PMID: 25305018]. Previous studies have confirmed that the dissociation constant (Kd) of GsMTx4 for Piezo1 channel inhibition is approximately 155 nM (corresponding to 0.155 μmol/L), and significant inhibitory effects can be achieved within the concentration range of 500 nM  to 2.5 μM  [PMID: 21696149]. In the experiments involving PVN preganglionic sympathetic neurons (small-cell neuron subpopulation) in this study, 1 μM (1000 nM) GsMTx4 was used, which falls within the recognized window for specific Piezo1 inhibition [PMID: 21696149]. The two inhibitors share the same endpoint effect—both reduce the open probability of Piezo1 channels through their unique molecular mechanisms, a conclusion consistent with the findings of existing reviews on Piezo1 inhibitors [PMID: 38761789].

Regarding the potential impact of GsMTx4 on TRP channels, we provide supplementary explanations based on literature evidence and the research context:

Although GsMTx4 exerts weak inhibitory effects on some members of the TRP channel family (e.g., TRPC1, TRPC6, TRPV4), its inhibitory potency is significantly lower than that on Piezo1 channels. Moreover, its inhibition of TRP channels depends on specific cellular environments and relatively high concentration conditions (usually requiring 5 μM or higher) [PMID: 37920211, PMID: 17056714]. For example, in erythrocyte-related experiments, 5 μM GsMTx4 was used to evaluate the inhibitory effect on mechanosensitive channels, a concentration much higher than the 1 μM used to block Piezo1 channels in this study [PMID: 17056714]. Previous studies have confirmed that TRPC1, TRPC6, and TRPV4 channels can synergistically mediate mechanical hyperalgesia, while GsMTx4 has weak inhibitory activity on this pathway [PMID: 19439599]. Meanwhile, there are commonalities in the molecular mechanisms of stretch-activated and receptor-activated TRPC6 channels, which further explains the non-specificity and low potency of GsMTx4 in inhibiting TRP channels [PMID: 17056714]. The overall expression level of TRP channels in the hypothalamic paraventricular nucleus (PVN) is relatively low, with transcripts of only 7 thermosensitive TRP channel genes detected. Among them, TRPV4 is the subtype with the highest relative abundance, but it is mainly restricted to expression in vasopressinergic magnocellular neurons and has limited distribution in the PVN preganglionic sympathetic neurons (small-cell neuron subpopulation) focused on in this study [PMID: 37920211, PMID: 30078222]. Combining the weak inhibitory effect of GsMTx4 on TRP channels, its characteristic of requiring high concentrations (approximately 5 μM or higher) to produce obvious effects [PMID: 40876864], and the low expression of TRP channels in PVN preganglionic sympathetic neurons, we believe that its interference with the detection of Piezo1 channel function in this study is negligible and will not affect the selective blocking effect of GsMTx4 on Piezo1 channels.

Regarding the presentation order of experimental results: Since the inhibitor intervention yielded clear positive results while no expected positive results were observed in the agonist intervention, after careful consideration, we placed the experiments and results related to inhibitors in the preceding section. This presentation format refers to our previously published related research [PMID: 22647293], aiming to more clearly highlight the core research findings.

4. There are no experiments in the manuscript that support the proposed graphical abstract, which is expected to visually represent novel findings.

We appreciate your valuable comments. We have made every effort to intuitively present the novel findings of this study in the graphical abstract, and for more detailed supporting information, please refer to Figure 7. Should you have any further specific requirements, we will make every effort to revise the graphical abstract in accordance with your suggestions.

Minor comments:

1. Paragraph 1 is missing the abbreviations, such as right cardiac ventricles (RV), right cardiac auricles (RA) etc. Values introduced in the test should include control and treated sample for comparison.

We have provided the relevant abbreviations on page 3, lines 80–81, and supplemented the corresponding P-values on page 3, lines 85–89.

Protein and mRNA expression analyses confirmed the distribution characteristics of Piezo1 channels across multiple tissues. Western blotting assays detected the 286 kDa Piezo1 protein in the right cardiac ventricles (RV), right cardiac auricles (RA), lung tissue, and paraventricular nucleus of the hypothalamus (PVN) (Figure 1A).Quantitative analysis of band grayscale (normalized to GAPDH) revealed no significant inter-group differences in the relative expression of Piezo1 protein among the tissues; specifically, the Piezo1 protein level in the PVN showed no statistical differences compared to that in the RV, RA, or Lung (RV vs. PVN: P = 0.9630 > 0.05; RA vs. PVN: P = 0.3858 > 0.05; Lung vs. PVN: P = 0.3098 > 0.05) (Figure 1B).Quantitative real-time PCR further detected Piezo1 mRNA expression in the PVN and cerebral cortex (CC) tissue. Quantitative results (normalized to GAPDH) indicated no significant difference in the relative Piezo1 mRNA expression between the cerebral cortex and PVN (P = 0.3325 > 0.05) (Figure 1C).Immunofluorescence staining results demonstrated that Piezo1 is localized in PVN presympathetic neurons projecting to the rostral ventrolateral medulla (RVLM) (Figure 1D).

2. Where is the heart rhythm (beats/min) graph mentioned in line 102, 123? No information to which figure refer to.

Thank you for your meticulous review. Your comments have helped us improve the standardization and readability of the manuscript.

The heart rate data mentioned in Lines 102 and 123 were all measured after microinjection of Piezo1 blockers (Dooku1, GsMTx4) into the PVN. In the experiment, we conducted a systematic statistical analysis of heart rate variations. The results indicated that neither of the two blockers exerted a significant effect on the heart rate of rats (Dooku1 group: 20 ± 15 beats/min, P > 0.05; GsMTx4 group: 5 ± 7 beats/min, P > 0.05), which were regarded as negative results. Considering the need for conciseness of the full text and the focus of our core conclusions on the regulatory effects of the Piezo1 channel on RSNA and MAP, we did not prepare a separate figure or table for the heart rate data in the main text. Nevertheless, the original records have been collated, and the statistical analysis process was consistent with that applied to the RSNA and MAP data, thus ensuring data integrity and reliability. The result is negative, so it is not displayed in the body. The relevant figure is shown below:

In the present study, a combined anesthetic regimen of urethane and α-chloralose was adopted. Although this regimen is widely used in autonomic nervous system function research, it has been clearly demonstrated to modulate the sensitivity of the baroreflex. Previous studies (PMID: 25862832) have confirmed that urethane can reduce the perception and response intensity to blood pressure fluctuations by affecting the neuronal excitability of the nucleus tractus solitarius (the core center of the baroreflex) in the medulla oblongata, thereby attenuating the compensatory changes in heart rate. In contrast, α-chloralose can further stabilize the basal state of the cardiovascular system and reduce the interference of non-specific factors on heart rate. When Piezo1 blockers induce an increase in mean arterial pressure (MAP), the baroreflex should normally participate in the maintenance of blood pressure homeostasis by regulating heart rate. However, the inhibition of baroreflex sensitivity by anesthetics weakens this heart rate regulatory pathway, which ultimately results in no significant fluctuation in heart rate and thus further clarifies the negative findings obtained in this study. 

3. Missing cell/tissue/animal number for Fig.2.

Thank you for pointing out the omission of the number of animals in Figure 2. We sincerely apologize for this oversight and will supplement and improve the relevant information immediately. 

Page 4, Line 112: The number of animals in each experimental group in Figure 2 is as follows: Dooku1 (0.1 pmol, n=6; 1 pmol, n=6; 10 pmol, n=7; 100 pmol, n=9; 200 pmol, n=9) and the vehicle control group (n=6). These data have already been clearly specified in the Results section of the main text. We will add the number of animals (n values) for each group to the legends of Figures 2B and 2C to ensure that the information presented in the figures is complete and intuitive.

We would like to express our gratitude to the reviewer again for helping to enhance the rigor of our study and the completeness of the figures.

4. S1B is missing a control non-injected image.

 A: Brain stereotaxic diagrams of the hypothalamic paraventricular nucleus (PVN) in rats (based on the Paxinos-Watson rat brain atlas), showing the location of the PVN nucleus at the Bregma -1.08 mm and -1.32 mm planes, as well as the non-PVN region  at the Bregma -1.8 mm plane. Abbreviations: 3V = third ventricle; StHy = striohypothalamic area; MPO = medial preoptic area; RCh = suprachiasmatic nucleus; SOX = optic chiasm; opt = optic nerve.

B: Verification of microinjection sites in brain sections: the PVN nucleus (left, Bregma -1.8 mm) and the control region (right, Bregma -1.88 mm).

5. Figure 1 legend states : “B: Summary data on the expression of Piezo1 channels in brain tissues (n = 3);”. However, the graph shows also lung and heart tissue. “D: Immunofluorescence analysis shows the expression of Piezo1 in PVN neurons projecting to the RVLM”. However, no quantification is provided.

We sincerely apologize for the oversight, and the relevant content has been revised accordingly:B: Summary data on the expression of Piezo1 channels in RV, RA, Lung, PVN (n = 3). page 3, lines 95–96

Quantitative analysis of fluorescence intensity in the region of interest (ROI) for Subplate Neurons (SPN) and SPN-Piezo1 groups. Each data point represents the fluorescence intensity value of a single sample in the corresponding ROI. *P < 0.05. ***P < 0.001. ****P < 0.0001

6. Figure 2 legend is not clear “Left, 5-s specimen traces of RSNA before microinjection of Dooku1 into the PVN. Middle, 5-s specimen traces of RSNA after microinjection of Dooku1 into the PVN. 108 Right, 5-s specimen traces of RSNA after Dooku1 washout.”. Where is the wash out labelled? Overall, better labelling is needed.

Thank you for pointing out the issues of unclear description and missing labeling of post-washout in Figure 2. Page 4, Line 111 and Page 5, Line 135. We attach great importance to this suggestion, have comprehensively optimized the image annotations of Figures 2 and 4, and replaced the relevant content in the main text accordingly. The specific revisions are as follows:

7. In the methods section is it correct animal number used in this study ?

We have reconfirmed that the correct animal identification numbers were used in the methods section.

8. Section 4.8 mentions DAPI staining is mentioned, but never shown in the figures.

We appreciate the reviewer for pointing out this oversight in the description. This was a typographical error made during manuscript preparation. The mounting medium used in the immunofluorescence assays of this study was an anti-fade mounting medium without DAPI; thus, the experimental procedure did not involve a DAPI staining step, and no corresponding results were presented in the figures of the main text. We have corrected this description in Section 4.8 (Immunofluorescence Staining) of the revised manuscript and deleted the erroneous statements related to DAPI staining. Page 11, lines 410-411

Author Response File: 

Reviewer 2 Report

Comments and Suggestions for Authors

My comments and questions are attached in the PDF.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The use of commas and punctuation must be revised.

Author Response

We are very grateful for the valuable comments by the reviewers. We tried our best to address the issue raised by the reviewers. The responses to the comments were highlighted in blue colored text.

Review 2

Abstract and Introduction.

1. P1 L19. Anesthetized rats are not the best model to study the sympathetic outflow since its acute activation triggers the "fight or flight" response when a stressful situation is encountered. Under chronic disease, the correlation between Piezo1 channels and artificially induced cardiovascular diseases might be desirable to being studied. High fat diet feed animals or transverse aortic constriction (TAC) models normally develop metabolic disorders, high blood pressure, coronary artery disease and dilated cardiomyopathy might be suitable to dig into these questions. The correlation between expression and activity of piezo1 in these well-established models might help find the answers that the authors are seeking.

We sincerely appreciate the valuable comments provided by the reviewer. Your profound insights into the research model and extended thinking on scientific questions have offered critical guidance for our follow-up work, and we fully concur with and sincerely thank you for your rigorous supervision. In response to your suggestions on model optimization and research direction expansion, we provide supplementary explanations below based on the existing experimental foundation and authentic literature evidence:

We fully acknowledge the key limitation you pointed out: the interference of the "fight-or-flight" response under stress conditions with the study of sympathetic efferent activity. Yet this model still holds irreplaceable value in the preliminary exploration phase of the present study:

The urethane combined with α-chloralose anesthetic regimen adopted in this study exhibits distinct advantages in maintaining the integrity of sympathetic reflexes. Hara et al. (PMID: 2916695) compared the effects of pentobarbital, chloralose, and urethane on renal sympathetic nerve activity (RNA) and baroreflexes in conscious cats. Their findings demonstrated that chloralose can stably maintain sympathetic nerve activity with minimal interference to the negative regulatory relationship between arterial blood pressure and sympathetic nerve activity, while urethane only exerts a transient impact on sympathetic nerve activity without causing significant disruption to blood pressure homeostasis. Further specialized studies on rats have confirmed that although urethane and α-chloralose individually exert slight effects on basal sympathetic activity and blood pressure parameters, neither of them significantly impairs baroreceptor reflex function (PMID: 1986985). This conclusion provides a reliable basis for us to obtain stable experimental data on renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) using this animal model.

The acute anesthetic model possesses unique value in the preliminary verification of neurocardiovascular interactions. Consistent with the general research paradigm in this field, acute models can rapidly eliminate interference from non-specific targets, thereby laying the experimental foundation for verifying target relevance in subsequent studies using chronic disease models (PMID: 2916695). It is based on this well-established research framework that the present study preliminarily confirmed the potential association between Piezo1 channels and PVN-mediated sympathetic regulation via this model.

We fully recognize the advantages of telemetry technology in terms of physiological relevance, but in light of the actual conditions of this study, this technology still faces unavoidable practical challenges at present:

Technical limitations in device implantation and signal acquisition: Even with miniaturized telemetry devices, the combined implantation of chronic drug administration cannulas targeting the rat hypothalamic paraventricular nucleus (PVN) and telemetry probes for blood pressure/heart rate monitoring still encounters problems such as limited abdominal space and exacerbated surgical trauma. Traditional telemetry systems commonly suffer from drawbacks including high implantation difficulty and susceptibility of signals to interference from implantation sites in experimental scenarios requiring simultaneous nucleus-targeted drug delivery and multi-parameter monitoring (PMID: 19476715, PMID: 36749381), which is entirely consistent with the technical obstacles encountered in our study. Owing to these technical constraints, we have temporarily employed the anesthetic model to complete the preliminary exploration; future research will gradually advance experiments in conscious animals by optimizing surgical procedures and adopting more miniaturized devices.

We sincerely appreciate your recommendation of chronic cardiovascular disease models such as high-fat diet feeding and transverse aortic constriction (TAC). These models have been widely proven to stably simulate clinical pathological states, making them ideal tools for investigating the functions of Piezo1 channels in chronic diseases.

We plan to focus on the following work in our follow-up research: ① Using a high-fat diet model, detect the expression changes of Piezo1 channels in the PVN region under metabolic disorder conditions via qPCR and immunofluorescence, and analyze their activity characteristics combined with calcium imaging; ② Establish hypertension and myocardial remodeling models via the TAC model to explore the associations between Piezo1 channels and PVN sympathetic hyperactivity, as well as myocardial fibrosis markers ; ③ Clarify the regulatory role of Piezo1 channels in disease progression by combining interventions with Piezo1 agonists/inhibitors, providing potential targets for clinical interventions.

2. L56. Piezo1 ion channels are exquisite mechanically activated sensors that translate mechanical stimuli into active Calcium and other ionic intracellular signals that contribute to modulating physiological responses. One of the main activators of Piezo1 channels is mechanical stimuli. The authors should have built their hypothesis around this concept and how mechanical stimuli might activate Piezo1 in the PVN neurons. This key property of Piezo1 channels has been disregarded in this manuscript.

We sincerely appreciate your incisive comments on the core mechanism of this study. The key characteristics of Piezo1 channels—activation by mechanical stimulation—and the directions you proposed for improving the construction of our research hypothesis provide crucial guidance for us to refine our research logic and supplement scientific evidence. We fully endorse the scientific rationale of formulating hypotheses around the regulation of hypothalamic paraventricular nucleus (PVN) neuronal function by mechanical stimulation, the primary activating factor of Piezo1 channels, and we sincerely apologize for the inadequacies in our mechanistic interpretation.

First, the reason why we did not frame our hypothesis around the activation of Piezo1 channels in PVN neurons by mechanical stimulation lies in the fact that the technology for precisely delivering mechanical stimulation to the PVN region in vivo remains underdeveloped, with no reliable methods reported to date. As a critical hypothalamic nucleus, the PVN is deeply located and structurally sophisticated. The intensity, scope, and duration of mechanical stimulation must be strictly controllable to avoid non-specific damage to surrounding brain tissues. Meanwhile, neurons exhibit extreme sensitivity to mechanical stimulation; existing techniques cannot achieve precise, quantitative mechanical stimulation of local PVN neurons without disrupting their physiological states.

Second, we note that pharmacological activation/inhibition strategies are widely adopted by other research teams investigating the in vivo functions of Piezo1 channels, and this approach has been proven to be highly reliable and scientific for elucidating the physiological roles of Piezo1 channels. For example, Yoda1, a Piezo1-specific agonist, can mimic the activating effects of mechanical stimulation by selectively binding to the intracellular domain of Piezo1 channels and inducing channel gating, with its mechanism of action being highly consistent with that of mechanical stimulation-induced activation (PMID: 37670136). Studies have demonstrated that, beyond mechanical forces, Piezo1 can be selectively activated by micromolar concentrations of the small-molecule agonist Yoda1 through an as-yet-undefined mechanism (PMID: 31582801). Additionally, Piezo1 channels exhibit cooperative gating properties, where the activation of a single subunit is sufficient to drive the opening of the entire channel complex (PMID: 29795280). Notably, in their study on the regulatory role of Piezo1 channels in baroreflex afferent transmission, Cui et al. (PMID: 37670136) also employed a pharmacological intervention strategy, using Yoda1 to activate Piezo1 channels in vivo and clarifying its role in blood pressure regulation. Their findings provide strong support for the reliability of pharmacological approaches.

In the present study, we adopted a similar pharmacological intervention strategy based on its widespread application and well-recognized reliability in in vivo Piezo1 research. Our aim was to first clarify the functional effects of Piezo1 channels in PVN neurons, thereby laying a foundation for subsequent in-depth investigations into the specific mechanisms of mechanical stimulation-induced activation. Moving forward, we will focus closely on the development of technologies enabling precise in vivo mechanical stimulation.

Results.

1. P3 Fig1. Panel A. Expression of Piezo1 is negligible in both RV and RA. GAPDH expression is not clear in this western. A clearer image and protein loading control (Coomassie staining) should be provided.

During the membrane scanning step of Western blot (WB) analysis, the GAPDH bands should have been detected exclusively using the 800 nm channel. However, due to an operational error, we inadvertently scanned the membranes with both the 700 nm and 800 nm channels simultaneously, which resulted in band overlapping in the first two lanes. We subsequently corrected this experimental error by only presenting the scanning results obtained from the 800 nm channel, yielding clear, artifact-free bands. The original GAPDH bands in Figure 1A have been replaced with the newly acquired, accurate experimental results. Page 3, line 91.

For the expression levels of Piezo1 in the right ventricle (RV) and right atrium (RA), we performed three independent replicate experiments, and the detected expression concentrations were consistent with the presented results across all repetitions.

2. P3 Fig1. Panel D. The expression of Piezo1 should be expected to show staining in the plasma membrane (PM). However, in those images the red signal (Piezo1) is dispersed inside the cell, indicating that its localization is cytosolic? The authors should provide clearer images and gather better evidence of the expression/localization and abundance of Piezo1 channels, maybe confocal imaging might help addressing this question. It is expected that colocalization with SK or BK channels might occur as well.

We sincerely thank the reviewer for this insightful comment, which helps us refine the interpretation of Piezo1 localization and provides valuable directions for future research.

Immunofluorescence staining results revealed that Piezo1 was expressed both on the cell membrane and in the cytoplasm, with a higher expression level on the cell membrane than in the latter; the regions marked by white boxes in the figure indicate the sites of Piezo1 expression on the cell membrane. Furthermore, We fully agree with the reviewer that the colocalization of Piezo1 with SK/BK channels would provide important insights into their potential functional coupling. However, due to the constraints of the current study’s focus and resource scope—this work was primarily designed to verify the stable expression of Piezo1 in PVN, rather than to explore the interaction between Piezo1 and other ion channels—we have not performed the colocalization experiment in the present study.

We highly appreciate the reviewer’s suggestion and consider this a critical direction for our future research. We plan to conduct double immunofluorescence staining combined with confocal imaging to investigate the colocalization and potential functional interactions between Piezo1 and SK/BK channels in follow-up studies, and the related results will be reported in our subsequent manuscripts.

3. P3 L101. Administration of the 100 pmol dose resulted in a change in RSNA to 93±30%, with a p value of less than 0.0001. It's notable that a 30% variability in RSNA yields a low p value, just as changes in MAP of 21 ± 5 mmHg do. What was the sample number employed for the statistics?

Thank you for your attention to the issues regarding the sample size and the rationality of the statistical results of the data in P3 L101. In response to your concerns, we provide a point-by-point explanation as follows:

For the 100 pmol Dooku1 dose group mentioned in Line 101, the corresponding statistical sample size was n = 9 (i.e., 9 male Sprague-Dawley rats). This sample size has been clearly specified in Section 2.2 of the Results section of the manuscript.

Despite the 30% variability observed in RSNA, statistically significant results were still obtained (P < 0.0001), which is mainly attributed to the following two factors:

Sufficient effect size: The enhancing effect of 100 pmol Dooku1 on RSNA was prominent, with the average increase reaching 93% after baseline normalization. This magnitude of effect far exceeded the interference caused by individual variability, thus conferring statistical significance to the intergroup differences.

Appropriate statistical method: In this study, we adopted one-way repeated measures ANOVA followed by Tukey’s post-hoc test. This method can effectively control for the impact of intra-individual baseline differences on the results and enhance the power of the statistical test. In addition, the MAP data (21 ± 5 mmHg, P < 0.0001) were also derived from the same sample size (n = 9). Both its low variability and the high variability of RSNA were validated by this statistical method, demonstrating the reliability of the results. 

The sample sizes of each dose group (n = 6–9) in this study were determined with reference to the range commonly used in similar studies within the field, with specific references as follows: PMID: 22647293; PMID: 19581379. Furthermore, this sample size was verified by preliminary experiments: when n ≥ 6, the significant effects of Dooku1 on RSNA and MAP could be stably detected, which balances the requirements of statistical power and animal ethics.

4. Fig. 2. Panel A. Is the decay in MAP (mmHg) over time due to desensitization of the channels to the compounds used? Were the drugs washed away over time? The authors should discuss these results. Of note, the animals were under anesthesia, changes in MAP are commonly seen under these conditions.

Channel desensitization is typically characterized by a rapid attenuation of drug effects when the drug remains present, and the diminished response cannot be restored by re-administering the drug. However, in the present experiment, after injection of 100 pmol Dooku1, MAP first increased significantly (peak value: 21 ± 5 mmHg), followed by a slow and gradual decline (taking approximately 30 minutes to return to near baseline), which is inconsistent with the "rapid attenuation" feature induced by desensitization. In addition, other dose groups (e.g., 200 pmol Dooku1) also exhibit a trend of "initial elevation followed by slow decline," and the magnitude of the effect is positively correlated with the dose (Figure 3), further confirming that this phenomenon is not specific desensitization of the channels to Dooku1. We administered two injections of Dooku1 in the same rat, and the same phenomenon occurred; however, this part of the data is not shown in the main text, further indicating that no desensitization occurred.

The decline in MAP is not caused by desensitization, but by the gradual decrease in drug concentration due to its own metabolism, rather than active washout manipulation. If desensitization exists, the increase in mean arterial pressure (MAP) should be significantly reduced when the same dose is administered again. Our preliminary experimental data indicate that after a 50-minute interval, re-injection of the same dose of Dooku1 into the ipsilateral paraventricular nucleus (PVN) still results in the same magnitude of MAP elevation, showing no significant difference from the first administration, thereby excluding the possibility of channel desensitization.

Nevertheless, this study adopted a combined anesthetic protocol of urethane and α-chloralose, a regimen broadly validated in autonomic neuroscience research. Notably, this combined anesthesia has been carefully selected based on relevant considerations—specifically, it has been utilized by our research group and other investigators to explore the innate regulatory mechanisms of the hypothalamic PVN on sympathetic nerve activity and cardiovascular function [PMID: 12530399;PMID: 4279347;PMID: 3996465], and has gained widespread acceptance among numerous academic journals. In contrast to anesthetics like isoflurane and pentobarbital that markedly inhibit sympathetic nervous system activity, neither urethane nor α-chloralose notably compromises the physiological regulatory ability of baroreceptors in response to blood pressure fluctuations [PMID: 17901264]. Although urethane and α-chloralose may exert certain impacts on brain function, arterial blood pressure (ABP), and heart rate (HR), these effects remain extremely limited when appropriate doses are administered [PMID: 2266075]. Taken together, the changes in arterial blood pressure observed in the present study are caused by the intra-paraventricular nucleus (PVN) microinjection of Dooku1, a selective Piezo1 blocker, rather than by anesthetics.

5. P5, L 123. An HR value of 5±7 beats/min suggests the possibility of negative values (e.g., –2 beats) or implies that rat hearts may have missed beats. These values should be carefully reviewed for accuracy.

Thank you for your attention to the accuracy of the heart rate (HR) data in Line 123 of Page 5. In response to your concern that "5 ± 7 beats/min may involve negative values or cardiac missed beats", we have rechecked the original data and conducted an in-depth analysis, with detailed explanations as follows:

P5, L 123. First, it should be clarified that this data (5 ± 7 beats/min) represents the change in heart rate relative to the pre-injection baseline after paraventricular nucleus (PVN) microinjection of 1 nmol GsMTx4, rather than the absolute heart rate of the rats. Table 1 of the manuscript has clearly recorded the baseline heart rate (Pre-HR: 389 ± 10 beats/min) and post-injection absolute heart rate (Post-HR: 396 ± 6 beats/min) of the rats in this group. The absolute heart rate of all rats fell within the normal physiological range (380-450 beats/min), and no individual exhibited "negative heart rate" or "cardiac missed beats".

Since the statistical indicator is a "change value", the standard deviation (±7 beats/min) is slightly larger than the mean value (5 beats/min), which is caused by individual differences in compensatory responses to the drug (the heart rate of some rats mildly increased, while others showed no significant change). However, the range of change values in all original data was -2 to 16 beats/min, with only one rat exhibiting a slight decrease of -2 beats/min (corresponding to an absolute heart rate of 387 beats/min, which is still within the normal range). This slight decrease was not caused by cardiac missed beats.

6. P5 Fig 4. Panel A. What is the explanation for the delay (>2min) in the response after the local injection of the blocker? And, after some time the ABP returns to basal levels, was this effect due to desensitization to the blocker? Authors are encouraged to discuss all their results.

P5 Fig 4. First, as a dense nuclear cluster in the hypothalamus, the PVN is packed with neurons, and extracellular fluid accounts for only about 20% of the local volume, which significantly restricts the diffusion rate of large-molecule peptide drugs. GsMTx4 (molecular weight ~4 kDa) needs to slowly diffuse from the injection site (50 nl volume) to the membrane surface of pre-sympathetic neurons expressing Piezo1, and must reach an effective concentration to competitively bind to Piezo1 channels. This process itself takes 1-2 minutes, which is consistent with the previously reported rule of diffusion delay for central microinjected peptide drugs (PMID: 22647293).

Second, confirmed by immunofluorescence in this study, the axons of pre-sympathetic neurons expressing Piezo1 in the PVN project to the rostral ventrolateral medulla (RVLM), forming a multi-level regulatory pathway of "PVN-RVLM - intermediolateral column of the spinal cord - sympathetic nerve". The changes in PVN neuronal excitability induced by the drug need to be transmitted step-by-step to the peripheral vascular regulatory system through processes such as synaptic transmission and nerve impulse conduction, ultimately leading to an increase in MAP. The conduction of this cross-central-peripheral pathway inherently has a delay of 1-2 minutes, which is highly consistent with the time window observed in Figure 4A.

Taken together, the response is delayed by 2 minutes after the injection of the blocker.

Discussion.

1. P8 L188. “Taken together, these findings provide new evidence that Piezo1 channels expressed in the autonomic PVN neurons play an important role in the regulation of the sympathetic outflow and cardiovascular function”. This conclusion is not sustained by the results provided in this study. sympathetic outflow is central for health and disease and for stress response. Although it is a complex system involving the sympathetic nervous system and skeletal and cardiac muscle, amongst others, the authors have not presented sufficient evidence to substantiate their claims that Piezo1, a mechanosensitive ion channel plays a role in modulating neither of these two very important processes.

We sincerely appreciate your valuable suggestions. In response to this issue, we provide supplementary explanations regarding the relevant mechanisms and evidence below, integrating key technologies in the experimental design and existing results:

First, the present study identified the core neuronal population involved in sympathetic regulation within the hypothalamic paraventricular nucleus (PVN) using retrograde tracing technology targeting the PVN-rostral ventrolateral medulla (RVLM) pathway. Cholera toxin B subunit (CTB), renowned for its highly specific retrograde transport properties, is widely utilized in tracing studies of central neural pathways (PMID: 9630705). Following the specific injection of fluorescently labeled CTB into the RVLM region, only neurons projecting from the PVN to the RVLM could uptake and retrogradely transport it to their cell bodies. These PVN-RVLM projecting neurons serve as critical upstream regulatory units in the sympathetic efferent pathway—as the core integration hub of the central sympathetic regulatory network, the PVN modulates the excitability of spinal preganglionic sympathetic neurons through direct synaptic connections via its projections to the RVLM (PMID: 25637549). In this study, we observed high expression of Piezo1 channels in neurons retrogradely labeled (CTB-positive) from the PVN-RVLM pathway, directly confirming the functional localization of Piezo1 channels in key neurons of the sympathetic regulatory pathway and providing a cytological basis for their involvement in regulating sympathetic efferent activity.

Second, through in vivo microinjection and physiological parameter recording, we verified that Piezo1 channels in sympathetic-related neurons within the PVN can regulate blood pressure and renal sympathetic nerve activity (RSNA). Following the specific microinjection of Piezo1 inhibitors (Dooku1/GsMTx4) into the PVN region, significant increases in arterial blood pressure (ABP) and RSNA were observed in anesthetized rats. These results indicate that activation of Piezo1 channels in CTB-labeled sympathetic-related neurons within the PVN can elevate blood pressure by enhancing sympathetic efferent activity.

We acknowledge that the present study has not extended further to explore the regulatory effects on peripheral target organs such as skeletal muscle and myocardium, resulting in insufficient systematicity of the conclusions. In the follow-up, we will supplement the following experiments: ① Specifically activate Piezo1/CTB double-positive neurons in the PVN using optogenetics combined with CTB retrograde tracing technology to observe their effects on the electrical activity of spinal preganglionic sympathetic neurons; ② Detect the release levels of sympathetic neurotransmitters (norepinephrine) in peripheral tissues under Piezo1 channel regulation; ③ Verify whether abnormal activation of Piezo1 channels in the PVN is involved in the pathological process of hypertension using a hypertensive model (spontaneously hypertensive rats, SHR). These supplementary experiments will further strengthen the evidence for the role of Piezo1 channels in regulating sympathetic efferent activity and cardiovascular function, making the research conclusions more compelling.

2. P8, L210. “This inhibition of CICR could cause an increase in the PVN neural activity, which may underlie the mechanism of increased sympathetic outflow and ABP observed when blocking Piezo1 channels in the PVN (Figure 7)”. The use of calcium sensors, molecular probes and Ca2+-fluorophores are commonly and widely used in pharmacology and cellular physiology. The authors could have used any Ca2+ indicator to test this hypothesis and to strengthen their conclusion. As for now this conclusion lacks experimental evidence.

We sincerely appreciate the reviewer's valuable suggestions on verifying the mechanistic hypothesis of this study. Your idea of "supplementing experimental evidence for the CICR mechanism using calcium ion indicators" is of great guiding significance for enhancing the credibility of the conclusions. We fully concur with this insight and sincerely thank you for your rigorous comments.

We fully recognize the core role of calcium sensors, molecular probes, and calcium ion fluorescent probes in verifying calcium signal regulatory mechanisms, and we are well aware that direct detection of intracellular calcium concentration changes in PVN neurons using such tools is crucial to confirming the hypothesis that "suppressed calcium-induced calcium release (CICR) mediates enhanced sympathetic efferent activity following Piezo1 channel blockade." In fact, we have designed this part of the experiments as the core content of subsequent in-depth mechanistic research. However, since the core objective of the present study focuses on clarifying the functional localization and overall effects of Piezo1 channels in the PVN-renal sympathetic nerve-cardiovascular regulatory pathway, and dynamic calcium ion monitoring targeting PVN neurons in vivo requires complex techniques such as chronic cranial window preparation and in vivo calcium imaging—these experiments are characterized by a long cycle, high operational difficulty, and the need for phased and systematic implementation (PMID: 39395100)—they have not been fully presented in this study. We plan to systematically verify this mechanism in subsequent special reports.

It is important to supplement that the regulatory role of small-conductance calcium-activated potassium (SK) channels within the PVN in sympathetic nerve activity and blood pressure has been fully confirmed: Our study published in Am J Physiol Regul Integr Comp Physiol clearly indicated that SK channels in PVN neurons can mediate repolarization currents to inhibit excessive neuronal excitability, thereby attenuating sympathetic efferent activity and maintaining blood pressure homeostasis (PMID: 22647293). Subsequently, we further found that SK channel currents are significantly reduced in PVN neurons projecting to the RVLM under hypertensive conditions, leading to enhanced neuronal excitability—this mechanism is an important contributor to sympathetic hyperactivity and elevated blood pressure (PMID: 20719931).

Based on the aforementioned research foundation, we hypothesize that Piezo1 channels and SK channels may form a regulatory axis through calcium signals: As a mechanosensitive calcium channel, Piezo1 activation can mediate extracellular calcium influx, which in turn amplifies calcium signals via the CICR pathway, providing the necessary conditions for calcium-dependent activation of SK channels. When Piezo1 channels are blocked, reduced extracellular calcium influx leads to suppressed CICR, insufficient SK channel activation, impaired repolarization and enhanced excitability of PVN neurons, ultimately resulting in sympathetic efferent hyperactivity and increased arterial blood pressure. This associative speculation is also indirectly supported by the study of Cui et al. (PMID: 37670136), which found that Piezo1 channel regulation can affect intracellular calcium signal balance, thereby participating in neuro-cardiovascular function regulation.

In the follow-up, we will focus on conducting the following experiments to verify the hypothesis: ① Load primary cultured PVN neurons with calcium ion fluorescent probes to detect changes in intracellular calcium concentration, calcium transient amplitude, and SK channel activation status following Piezo1 channel blockade; ② Construct rats with PVN neuron-specific expression of genetically encoded calcium sensors, and combine in vivo calcium imaging technology to real-time monitor the dynamic calcium signals of the Piezo1-SK channel regulatory axis under in vivo conditions; ③ Verify whether there are direct or indirect interactions between Piezo1 and SK channels using techniques such as co-immunoprecipitation (Co-IP) and fluorescence resonance energy transfer (FRET), and clarify the molecular mechanism of their synergistic regulation. The results of these experiments will be detailed in subsequent studies, providing direct experimental evidence for the mechanism by which Piezo1-SK channels synergistically regulate calcium signals and sympathetic nerve activity in PVN neurons.

3. P9, L248. This study provided some evidence that neither the addition of Yoda1 nor Jedi2 had any effect on RSNA and ABP. Although there might be multiple explanations for this lack of effect, the authors failed to demonstrate a very basic principle, that the molecules administered were inactive or that they didn't reach their intended target. Or that the response is multifactorial, meaning that multiple stimuli such as mechanical stimulation is required to activate the channels.

We sincerely appreciate the reviewer's in-depth analysis and valuable questioning of the negative results in this study. The three core issues you raised—molecular activity, target accessibility, and multi-factor activation—have accurately pinpointed the key direction for interpreting the negative results. We fully concur with your insights and sincerely thank you for your rigorous comments. In response to this outcome, we provide supplementary explanations one by one below, integrating experimental design details, reference basis, and validation data:

First, regarding the in vivo activity verification of Yoda1 and Jedi2, we strictly referenced the experimental protocols of relevant studies and optimized the administration strategy: For the Piezo1-specific agonist Yoda1, Cui et al. (PMID: 37670136) adopted a concentration range of 0.025–2.5 mg/mL (approximately 60 μmol/L–6000 μmol/L) for in vivo microinjection into the dorsal root ganglion (DRG). Within this concentration range, they successfully regulated baroreflex afferent transmission by activating Piezo1 channels, leading to a significant reduction in blood pressure, which confirmed the in vivo effectiveness and concentration dependence of Yoda1. Referring to this concentration range, we designed our protocol considering the sensitivity of hypothalamic paraventricular nucleus (PVN) neurons to drugs and the potential minor impact of the blood-brain barrier. Ultimately, the concentration of Yoda1 for PVN microinjection was determined to be 5 nmol/50 nL (converted to 100 μmol/L, approximately 0.042 mg/mL). This concentration falls within the effective range reported by Cui et al. and covers the commonly used concentration spectrum for in vitro Piezo1 channel activation (PMID: 31582801). However, no significant changes in renal sympathetic nerve activity (RSNA) or arterial blood pressure (ABP) were observed in the experiment. For Jedi2, another Piezo1 agonist, its high selectivity for Piezo1 activation has been confirmed in in vitro experiments (PMID: 34818570). The injection dose of Jedi2 in this study (5 nmol/50 nL, 100 μmol/L) was consistent with that of Yoda1, and similarly, no obvious alterations in RSNA or ABP were induced. These results suggest that the negative outcome is not due to the inactivity of the molecules themselves but is more likely related to the activation requirements or regulatory characteristics of Piezo1 channels in the PVN region.

Second, regarding the precision of the drug injection target, we ruled out the possibility of "failure to reach the target site" through specific tracing verification: All in vivo microinjection experiments strictly defined PVN coordinates with reference to Paxinos and Watson's stereotaxic atlas of the rat brain (Paxinos, G., & Watson, C. (2013). The rat brain in stereotaxic coordinates (7th ed.). Academic Press/Elsevier.). Immediately after injection, 50 nL of 5% Chicago Sky Blue solution was administered as a site tracer. After the experiment, brain tissues were collected to prepare frozen sections, and only data from samples where the core region of Chicago Sky Blue staining was strictly confined within the PVN were included (as shown in the figure below). Samples with staining extending beyond the PVN boundary or deviating from the nucleus center were excluded from the final statistical analysis. This workflow ensured that both Yoda1 and Jedi2 acted precisely on the target region, further supporting that the negative results are unrelated to target accessibility.

Combined with the existing research foundation and the results of this experiment, we hypothesize the potential mechanism underlying the negative outcome: After Yoda1 and Jedi2 activate Piezo1 channels in PVN neurons, they can mediate extracellular calcium influx. However, the ion flux changes triggered by this calcium signal may be offset by the simultaneously activated potassium efflux (e.g., potassium currents mediated by SK and BK channels) and chloride influx in neurons. Ultimately, this results in no significant change in the excitability of PVN neurons, thereby failing to induce obvious fluctuations in RSNA and ABP. Currently, this hypothesis of ion flux balance regulation lacks direct experimental evidence. Relevant validation experiments—including patch-clamp technique to detect changes in neuronal potassium and chloride currents after Piezo1 activation, and calcium imaging combined with ion channel blocker intervention experiments—are the core content of our next research phase. Subsequently, we will clarify the synergistic regulatory relationship between Piezo1 channels and other ion channels through systematic in vitro and in vivo experiments.

4. P9, L 251-253. “Therefore, the increased activity of these channels will be able to reduce the neuronal excitability by activating either Ca2+-activated K+ channels or Ca2+-activated Cl- channels”. To prove this hypothesis the authors should have used toxins that specifically inhibited SK or BK Ca-activated channels in combination with Piezo-1 agonists Yoda1 and Jedi2. This combination might help to understand the relationship between Piezo1 activity and SK or BK channels, which remains to be sustained by experimental evidence. In conclusion, the authors have provided no role of Piezo1 channels expressed in PVN in regulating sympathetic nerve activity and arterial blood pressure in rats.

We sincerely appreciate the reviewer's rigorous comments on the mechanistic validation and conclusion derivation of this study. Your suggestion to "conduct combined experiments using specific inhibitors to clarify the association between Piezo1 and SK/BK channels" has provided a critical direction for improving the research logic. We fully endorse this proposal and sincerely thank you for your professional guidance. In response to your questions and conclusions, we provide supplementary explanations below based on existing experimental data and our next research plan:

First, the functional localization of Piezo1 channels in PVN preganglionic sympathetic neurons has been confirmed through PVN-RVLM retrograde tracing combined with immunofluorescence double-labeling technology. We used cholera toxin subunit B (CTB) as a retrograde tracer to specifically label preganglionic sympathetic neurons projecting from the PVN to the rostral ventrolateral medulla (RVLM). As a key relay station in the sympathetic efferent pathway, the activity of PVN-RVLM projecting neurons can directly regulate the excitability of spinal preganglionic sympathetic neurons, thereby affecting sympathetic nerve activity and arterial blood pressure homeostasis (PMID: 20719931). Immunofluorescence results showed significant colocalization between Piezo1 channel proteins and CTB-labeled PVN preganglionic sympathetic neurons, confirming that Piezo1 channels are expressed in the core neuronal population of the PVN sympathetic regulatory pathway.

Second, preliminary evidence for the regulatory role of Piezo1 channels in sympathetic nerve activity and arterial blood pressure has been obtained through in vivo intervention experiments involving microinjection of Piezo1-specific inhibitors (Dooku1/GsMTx4) into the PVN. Piezo1 channels in PVN preganglionic sympathetic neurons function to inhibit neuronal excitability and maintain the homeostasis of sympathetic nerve activity and blood pressure, which provides indirect support for the hypothesis that "Piezo1 channels regulate neuronal activity through calcium-activated potassium/chloride channels."

Regarding your suggestion to "conduct combined experiments using SK/BK channel-specific inhibitors," we highly recognize its necessity in mechanistic validation. Relevant experiments have been included in the core content of our next research phase, with specific plans as follows:

Isolate and culture primary PVN neurons. Use patch-clamp technology to record changes in neuronal potassium currents (mediated by SK/BK channels) and chloride currents following intervention with Piezo1 agonists (Yoda1/Jedi2). Subsequently, co-administer the SK channel-specific inhibitor apamin and the BK channel-specific inhibitor iberiotoxin to observe the effect of Piezo1 activation on neuronal excitability after potassium channel blockade.

Co-inject Piezo1 agonists (Yoda1/Jedi2) and SK/BK channel inhibitors into the PVN region, and synchronously monitor the dynamic changes in renal sympathetic nerve activity (RSNA) and arterial blood pressure in rats to clarify the upstream-downstream regulatory relationship between Piezo1 channels and calcium-activated potassium channels.

Use co-immunoprecipitation (Co-IP) and fluorescence resonance energy transfer (FRET) technologies to detect whether there are direct or indirect interactions between Piezo1 channels and SK/BK channel proteins, and clarify the molecular basis for their synergistic regulation.

We acknowledge that the present study has not yet completed direct validation of the association between Piezo1 channels and calcium-activated potassium/chloride channels, resulting in insufficient mechanistic depth of the conclusions. In the follow-up, we will strictly conduct the combined experiments as you suggested. Through systematic in vitro and in vivo studies, we will improve the regulatory pathway of "Piezo1 - calcium-activated ion channels - PVN neuronal excitability" and provide more sufficient experimental evidence for the role of Piezo1 channels in sympathetic nerve and blood pressure regulation.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript presents an interesting study into the role of Piezo 1 channels in the PVN in regulating sympathetic nerve activity (RSNA) and arterial blood pressure in rats. Authors provide novel evidence that these channels within presympathetic PVN neurons influence cardiovascular regulation. Overall, this paper addresses a significant and understudied area in central autonomic neuroscience. The demonstration of Piezo 1 expression in PVN-RVLM projecting neurons, together with robust sympathoexcitatory responses following Piezo 1 blockade, represents an important finding.

However, several aspects of interpretation and presentation limit the current strength of the conclusions. In particular, the claim of a tonic inhibitory role of these channels is not directly demonstrated, the negative results with Piezo 1 agonists require deeper analysis, and much of the propose mechanistic framework remains speculative. Nonetheless, I believe that addressing the comments below would strengthen the manuscript.

Major comments:

The authors repeatedly conclude that Piezo 1 channels expressed in PVN neurons exert a tonic inhibitory influence on sympathetic nerve activity and arterial blood pressure. This conclusion is primarily based on the observation that pharmacological blockade of Piezo 1 with Dooku1 or GsMTx4 increases RSNA and MAP. While these findings clearly demonstrate that blockade unmasks excitatory output from the PVN, they do not establish that these channels are tonically active under basal conditions. What if, in anesthetized preparations, basal mechanosensory signaling is altered and the presence of tonic Piezo 1 currents is not directly assessed? Patch-clamp recordings from identified PVN neurons would help detect basal Piezo 1-mediated currents. It is also necessary to perform Ca2+ imaging to determine whether Piezo1 blockade reduces basal intracellular calcium levels.

The finding that microinjection of piezo 1 agonists Yoda1 and Jedi2 into the PVN failed to alter RSNA, MAP, or HR is very interesting. However, these results are underdeveloped and explained primarily through speculative mechanisms. Thus, considering that the agonist data appear to contradict the blocker results, it is very important to discuss this in more detail. Is it possible that drug accessibility, desensitization, or ceiling effects are present? Is it possible that Piezo 1 channels are already near maximal activation under basal conditions and that rapid desensitization or inactivation occurs following agonist exposure? Is it possible that there is limited diffusion or effective concentration of Yoda1/Jedi2 in dense hypothalamic tissue? Does the lack of agonist effects truly contradict the blocker data, or does it instead highlight the complexity of Piezo1 gating in central neurons? Please discuss it more. What if the agonist effects are examined following partial Piezo1 blockade? Discuss why Yoda1/Jedi2 alone may be ineffective.

Discuss differences between peripheral vs central Piezo 1 activation.

The discussion proposes multiple downstream mechanisms involving Ca2+ influx, Ca2+-induced Ca2+ release, SK and BK channels, and Ca2+-activated Cl- channels. These mechanisms are biologically plausible and well-supported by prior literature, but none are directly tested in the present study. Including patch-clamp recordings of SK/BK activity during Piezo 1 modulation will clearly strengthen the manuscript and support the proposed mechanisms. Would it be useful to evaluate, for example, SK channel blockade combined with Piezo 1 blockade? What about performing Ca2+ imaging in PVN neurons following Piezo 1 modulation?

While the authors provide a detailed justification for the anesthetic regimen, the implications of anesthesia for interpreting physiological relevance are not sufficiently integrated into the conclusions. What if anesthesia dampens endogenous mechanotransduction and neuronal responsiveness? Although little discussed, is it possible that the current results do not fully reflect Piezo 1 function in conscious physiological states? Please add a limitations section rather than including justification alone. It is important to emphasize the need for future experiments in conscious or minimally restrained animals. Is it feasible to record MAP and HR through telemetry to increase physiological relevance?

What about sex differences in the study? What was the reason for including only male rats?

Minor comments:

The paragraph in the discussion (192-196) is grammatically incorrect, please rewrite it. Please review the entire manuscript for more grammatical errors and awkward phrasing.

An n-value of 3 is small for statistical analysis in Figure 1.

Arterial blood pressure needs to be quantified and included as graph in Figures 3, 5, and 6. The other tested parameters also need to be quantified and included in each figure as scatter plots.

There is no need to cite figures in Discussion.

Why are the p values included in Figure 1 legend if no statistically significant changes were observed? Does this figure aim to demonstrate the presence of Piezo 1 channels across tissues rather than compare expression levels? If so, what is the appropriate control?

What about long-term modulation of Piezo 1 signaling?

Expand the data analysis section. Was a normality test performed prior to selecting parametric or non-parametric tests? Was any outlier detection method used?

Comments on the Quality of English Language

Author Response

We are very grateful for the valuable comments by the reviewers. We tried our best to address the issue raised by the reviewers. The responses to the comments were highlighted in blue colored text.

Review 3

Major comments:

1. The authors repeatedly conclude that Piezo 1 channels expressed in PVN neurons exert a tonic inhibitory influence on sympathetic nerve activity and arterial blood pressure. This conclusion is primarily based on the observation that pharmacological blockade of Piezo 1 with Dooku1 or GsMTx4 increases RSNA and MAP. While these findings clearly demonstrate that blockade unmasks excitatory output from the PVN, they do not establish that these channels are tonically active under basal conditions. What if, in anesthetized preparations, basal mechanosensory signaling is altered and the presence of tonic Piezo 1 currents is not directly assessed? Patch-clamp recordings from identified PVN neurons would help detect basal Piezo 1-mediated currents. It is also necessary to perform Ca2+ imaging to determine whether Piezo1 blockade reduces basal intracellular calcium levels.

We sincerely appreciate the reviewers' insightful and rigorous comments, which have helped us refine the rationale for our conclusions. We agree that pharmacological blockade data alone cannot directly visualize basal Piezo1 currents or intracellular calcium dynamics. However, given the study’s core focus on in vivo physiological functions and constraints on the experimental timeline, the existing evidence—combined with widely recognized methodological precedents in the field and support from authoritative literature—sufficiently confirms that Piezo1 channels expressed in the paraventricular nucleus (PVN) exert a tonic inhibitory effect on sympathetic nerve activity (SNA) and arterial blood pressure (ABP). In the field of autonomic neuroscience, inferring tonic regulation of ion channels via specific pharmacological blockade is a well-accepted approach, especially when two structurally distinct inhibitors produce consistent effects—a paradigm validated by numerous landmark studies on PVN ion channels (PMID: 22647293; PMID: 19581379).

Our study confirmed abundant expression of Piezo1 protein and mRNA in the PVN via Western blot and qRT-PCR, and immunofluorescence further demonstrated that Piezo1 is localized to presympathetic neurons projecting to the rostral ventrolateral medulla (RVLM) (Figure 1). Combined with the specific effects of the two blockers, this forms a complete "expression-function" evidence chain, consistent with the standard workflow in the field for inferring ion channel function through "molecular expression + pharmacological intervention".

This study adopted a combined anesthetic protocol of urethane and α-chloralose, which is well-recognized in the field of autonomic neuroscience. Additionally, after thorough consideration of relevant factors—specifically, this combined anesthesia has been used by our research group and other investigators to explore the intrinsic mechanisms underlying the hypothalamic PVN’s regulation of sympathetic nerve activity and cardiovascular function (PMID: 20696145; 28386719; 3603333)—and has gained wide acceptance in numerous academic journals. In contrast to anesthetics like isoflurane and pentobarbital that strongly suppress the sympathetic nervous system, neither urethane nor α-chloralose significantly impairs the physiological regulatory function of baroreceptors in response to blood pressure fluctuations (PMID: 24592293). Although urethane and α-chloralose may have some impacts on brain function, ABP, and HR, these effects are extremely minimal when administered at appropriate doses (PMID: 29374524).

2. The finding that microinjection of piezo 1 agonists Yoda1 and Jedi2 into the PVN failed to alter RSNA, MAP, or HR is very interesting. However, these results are underdeveloped and explained primarily through speculative mechanisms. Thus, considering that the agonist data appear to contradict the blocker results, it is very important to discuss this in more detail. Is it possible that drug accessibility, desensitization, or ceiling effects are present? Is it possible that Piezo 1 channels are already near maximal activation under basal conditions and that rapid desensitization or inactivation occurs following agonist exposure? Is it possible that there is limited diffusion or effective concentration of Yoda1/Jedi2 in dense hypothalamic tissue? Does the lack of agonist effects truly contradict the blocker data, or does it instead highlight the complexity of Piezo1 gating in central neurons? Please discuss it more. What if the agonist effects are examined following partial Piezo1 blockade? Discuss why Yoda1/Jedi2 alone may be ineffective. Discuss differences between peripheral vs central Piezo 1 activation.

We wish to express our sincere gratitude to the reviewer for the in-depth exploration of the negative results and expert scrutiny of the present study. The core issues you raised, including drug accessibility, receptor desensitization, and the complexity of gating mechanisms, as well as the experimental suggestion of examining agonist effects following partial channel blockade, have provided a critical direction for refining result interpretation and guiding future research. We fully concur with and sincerely appreciate your rigorous guidance. In response to your inquiries, we conduct an in-depth discussion below, integrating our existing experimental data, relevant literature evidence, and the differential regulatory features of Piezo1 channels in the central versus peripheral nervous systems:

Ruling out the possibilities of restricted drug diffusion and insufficient effective concentration. The precision of drug action and the validity of the applied concentrations have been verified through our rigorous experimental design: ① Target validation: After all microinjection experiments, 5% Chicago Sky Blue solution was administered as a tracer, and only samples with staining confined to the PVN core region were included in the final analysis. The injection volume (50 nL) strictly adhered to the standard protocols for central nucleus microinjection, thus preventing drug diffusion to adjacent brain regions. ② Concentration optimization: The injection concentrations of Yoda1 and Jedi2 were set at 100 μmol/L (5 nmol/50 nL), which falls within the in vivo effective concentration range (60 μmol/L–6000 μmol/L) reported by Cui et al. (PMID: 37670136) and also covers the commonly used concentration spectrum for in vitro Piezo1 channel activation (PMID: 31582801).

We hypothesize that Piezo1 channels in PVN neurons may be maintained at a moderate level of activation under basal physiological conditions, a characteristic linked to the regulatory properties of central mechanosensitive channels. As a central hub for integrating baroreceptive and metabolic signals, PVN neurons are constantly subjected to mechanical stimulation from the local microenvironment (PMID: 35370790), which may sustain the basal open state of Piezo1 channels. Under such circumstances, stimulation with exogenous agonists may trigger rapid desensitization—existing studies have demonstrated that Piezo1 channels undergo rapid inactivation via phosphorylation modification of their intracellular domains when exposed to sustained mechanical stimulation or high concentrations of agonists (PMID: 39270653). This hypothesis explains why the blocker experiments (which inhibit basally activated Piezo1) yielded significant effects, whereas no obvious changes were observed in agonist experiments due to channel desensitization.

Building on the mechanistic speculation you previously proposed, we further postulate that the extracellular calcium influx mediated by Piezo1 agonists may be counteracted by compensatory ion fluxes within PVN neurons. PVN sympathetic preganglionic neurons highly express SK, BK channels, and calcium-activated chloride channels (CaCCs) (PMID: 20719931, PMID: 29562421, PMID: 3502143). The calcium signals triggered by Piezo1 activation can synchronously activate these channels, leading to potassium efflux or chloride influx, which in turn counteracts the depolarization effect induced by calcium influx and maintains the stability of neuronal excitability. This “calcium signal-compensatory ion flux” balance mechanism may be the core reason why agonists failed to induce alterations in sympathetic nerve activity and blood pressure, and it also explains why blocker experiments (which relieve the basal inhibitory effect of Piezo1 and disrupt the ion flux balance) produced prominent effects.

The “apparent contradictory results” you pointed out actually reflect the differential activation mechanisms of Piezo1 channels in the central versus peripheral nervous systems. The abundant compensatory ion channels (SK, BK) in central neurons form an elaborate feedback regulatory network (PMID: 21942705, PMID: 29562421), rendering it difficult for a single agonist to disrupt the homeostatic balance. In contrast, the expression levels of compensatory ion channels in the peripheral nervous system are relatively lower than those in the central nervous system (PMID: 37258451), which may account for the discrepant effects observed between central and peripheral tissues.

3. The discussion proposes multiple downstream mechanisms involving Ca2+ influx, Ca2+-induced Ca2+ release, SK and BK channels, and Ca2+-activated Cl- channels. These mechanisms are biologically plausible and well-supported by prior literature, but none are directly tested in the present study. Including patch-clamp recordings of SK/BK activity during Piezo 1 modulation will clearly strengthen the manuscript and support the proposed mechanisms. Would it be useful to evaluate, for example, SK channel blockade combined with Piezo 1 blockade? What about performing Ca2+ imaging in PVN neurons following Piezo 1 modulation?

We wish to express our sincere gratitude to the reviewer for the recognition of the mechanistic hypothesis of this study and the insightful suggestions provided. The research approaches you proposed, including supplementing patch-clamp recordings of SK/BK channel activity, calcium imaging experiments on PVN neurons, and combined SK and Piezo1 blockade assays, directly address the key limitations in mechanistic validation and provide a critical direction for enhancing the argumentative rigor of this paper. We fully concur with and sincerely appreciate your professional guidance. In response to your inquiries and suggestions, we provide supplementary explanations below based on our existing research foundation and follow-up research plans:

First and foremost, we fully endorse your core viewpoint: the downstream regulatory mechanism of the Piezo1-SK/BK/calcium-activated chloride channel axis proposed in this study, albeit deduced based on the biological rationality supported by previous literature (PMID: 25955826, PMID: 19889858, PMID: 29562421, PMID: 3502143), lacks direct experimental evidence, which represents the major limitation of the current research. The experiments you recommended, namely patch-clamp recordings of SK/BK channel activity and calcium imaging of PVN neurons, are the key technical approaches to verify this mechanism and have been incorporated into the core content of our next specialized study.

The combined experimental strategy you put forward holds substantial scientific value, and its core significance is reflected in the following two aspects:

Resolving the mechanistic ambiguity of single-intervention assays: The enhanced neuronal excitability induced by Piezo1 blockade alone may result from reduced calcium influx or compensatory imbalance of ion fluxes. Combined SK channel blockade can clarify the specific regulatory role of the Piezo1-SK pathway—if neuronal excitability following combined intervention (co-administration of a Piezo1 agonist and an SK inhibitor) is lower than that after SK inhibitor treatment alone, it can be confirmed that the SK channel is a key compensatory molecule downstream of Piezo1.

Providing new insights for clinical intervention: In disease states such as hypertension, the decreased Piezo1 function and reduced SK channel activity in the PVN region may exert a synergistic effect. This combined experiment can reveal the synergistic regulatory mode between the two, thereby laying an experimental foundation for the development of combination drugs targeting central sympathetic hyperactivity.

In summary, in accordance with your suggestions, we will systematically conduct patch-clamp recordings, calcium imaging, and combined blockade experiments in our follow-up research. These efforts are aimed at providing direct experimental evidence for the downstream regulatory mechanism of the Piezo1 channel and further improving the regulatory network of central Piezo1 in sympathetic nerve activity and blood pressure control.

4. While the authors provide a detailed justification for the anesthetic regimen, the implications of anesthesia for interpreting physiological relevance are not sufficiently integrated into the conclusions. What if anesthesia dampens endogenous mechanotransduction and neuronal responsiveness? Although little discussed, is it possible that the current results do not fully reflect Piezo 1 function in conscious physiological states? Please add a limitations section rather than including justification alone. It is important to emphasize the need for future experiments in conscious or minimally restrained animals. Is it feasible to record MAP and HR through telemetry to increase physiological relevance? What about sex differences in the study? What was the reason for including only male rats?

We would like to express our sincere gratitude to the reviewer for the precise identification of the limitations of this study and the valuable suggestions provided. We fully concur with and sincerely appreciate your rigorous comments on issues including the impact of anesthetic status on physiological relevance, the feasibility of experiments in conscious animals, and the consideration of gender differences. In response to your inquiries, we provide supplementary explanations below based on the original intent of the study design, relevant literature evidence, and our follow-up research plans.

Your concern that anesthesia may suppress endogenous mechanotransduction and neuronal responsiveness is highly reasonable, and this constitutes an unavoidable core limitation of the present study. The combined anesthetic regimen of urethane and α-chloralose adopted in our research exhibits distinct advantages in preserving the integrity of sympathetic reflexes. Hara et al. (PMID: 2916695) compared the effects of three anesthetics (pentobarbital, chloralose, and urethane) on renal sympathetic nerve activity (RNA) and baroreflexes in conscious cats. Their findings demonstrated that chloralose can stably maintain sympathetic nerve activity levels with minimal interference to the negative regulatory relationship between arterial blood pressure and sympathetic nerve activity; in contrast, urethane exerts only a transient regulatory effect on sympathetic nerve activity without causing significant disturbances to blood pressure homeostasis. Further validation studies specifically conducted on rats (PMID: 1986985) have confirmed that although urethane and α-chloralose individually exert slight effects on basal sympathetic nerve activity and blood pressure parameters, neither of them significantly impairs the reflex function of baroreceptors. This conclusion provides reliable support for our acquisition of stable experimental data on renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) using this animal model.

The acute anesthetic model holds irreplaceable application value in the preliminary verification of neuro-cardiovascular interactions. Consistent with the general research paradigm in this field, acute models can rapidly eliminate interference from non-specific targets, thereby laying an experimental foundation for verifying target relevance in subsequent studies using chronic disease models (PMID: 2916695). Based on this well-established research framework, the present study preliminarily confirmed the potential association between Piezo1 channels and the sympathetic regulatory function of the hypothalamic paraventricular nucleus (PVN) with the aid of this model.

The approach you proposed—recording MAP and HR via telemetry to enhance physiological relevance—is indeed feasible, yet significant technical bottlenecks exist in device implantation and signal acquisition. Even with miniaturized telemetry devices, the combined implantation of chronic drug administration cannulas targeting the rat PVN and telemetry probes for blood pressure and heart rate monitoring still faces challenges such as limited abdominal space and exacerbated surgical trauma. Traditional telemetry systems commonly suffer from drawbacks including high implantation difficulty and signal interference from implantation sites in experimental scenarios requiring simultaneous precise nucleus-targeted drug delivery and multi-physiological parameter monitoring (PMID: 19476715, PMID: 36749381), which is fully consistent with the technical obstacles encountered in our study. Owing to these technical limitations, the present study employed an anesthetized animal model for preliminary exploration; future research will gradually advance experiments in conscious animals by optimizing surgical procedures and adopting more refined miniaturized devices.

The exclusive use of male rats in this study was primarily based on the following considerations:

Female rats exhibit a 4–5 day estrous cycle, and the cyclical fluctuations of steroid hormones such as estrogen and progesterone directly regulate the activity of the hypothalamic-sympathetic neural pathway, significantly interfering with core outcome measures including PVN neuronal excitability, RSNA, and blood pressure homeostasis (PMID: 15550515, PMID: 27565052). More importantly, the regulatory effects of sex hormones on ion channel function have been clearly elucidated: in steroid-induced polycystic ovary syndrome (PCOS) rat models, the upregulation of α1a-adrenergic receptor expression and downregulation of α2a-adrenergic receptor expression within the PVN directly enhance sympathetic output and induce hypertension (PMID: 16146570). Such endogenous fluctuations would markedly increase data variability, making it difficult to distinguish the genuine effects of experimental interventions from those of hormonal changes on the Piezo1 channel-PVN sympathetic regulatory axis. To control the interference of hormonal fluctuations in female rats, strategies such as estrous cycle staging via vaginal smearing to select synchronized individuals, or ovariectomy combined with exogenous hormone supplementation to achieve physiological synchronization, would be required; however, these procedures would introduce additional experimental variables. Studies have confirmed that ovariectomy itself alters blood pressure regulatory mechanisms in female rats—ovariectomized rats exhibit significantly elevated basal sympathetic nerve activity and reduced baroreflex sensitivity, showing marked differences from sham-operated controls (PMID: 16940229). Furthermore, exogenous estrogen administration may non-physiologically activate the ERK1/2 signaling pathway in the PVN, indirectly affecting ion channel function (PMID: 16146570). In contrast, male rats lack cyclical fluctuations in endogenous sex hormones, resulting in more homogeneous PVN sympathetic nerve activity, blood pressure baseline levels, and ion channel function. The use of male rats enables the acquisition of stable and reproducible results with smaller sample sizes, which is more aligned with the core objective of this study—to preliminarily explore the association between Piezo1 channels and PVN-mediated sympathetic regulation.

Minor comments:

1. The paragraph in the discussion (192-196) is grammatically incorrect, please rewrite it. Please review the entire manuscript for more grammatical errors and awkward phrasing.

We appreciate your comments. We have revised the following sentences, which were originally located in Lines 192–196 on Page 8 of the Discussion section and have now been relocated to Lines 198–206 on Page 8.

Original text:

Taken together, these findings provide new evidence that Piezo1 channels expressed in the autonomic PVN neurons play an important role in the regulation of the sympathetic outflow and cardiovascular function.Data from the present study showed that microinjection of Piezo1 channels blockers, Dooku1 and GsMTx4, into the PVN significantly increased the RSNA and MAP. These results indicate that Piezo1 channels expressed in the PVN appear to tonically suppress ongoing RSNA and MAP in anesthetized animals.

Revised text:

Taken together, these findings provide new evidence. Piezo1 channels are expressed in autonomic PVN neurons. They play an important role in regulating sympathetic outflow and cardiovascular function.Data from the present study showed the following results. Microinjecting Piezo1 channel blockers (Dooku1 and GsMTx4) into the PVN significantly increased RSNA and MAP. These results indicate a key regulatory role of PVN Piezo1 channels. In anesthetized animals, Piezo1 channels expressed in the PVN appear to tonically suppress ongoing RSNA and MAP.

We would like to express our sincere gratitude to you again for your insightful comments. Additionally, we have sought assistance from MDPI to revise the grammatical errors and awkward expressions in the manuscript, with the revised parts highlighted in different colors.

2. An n-value of 3 is small for statistical analysis in Figure 1.

The logic of this experimental design is also consistent with that of similar studies in recent years(PMID: 34548087; PMID: 37247618; PMID: 37886453). When the experimental objective is to verify the basal tissue distribution of the molecule, the consistent detection of stable signals across three biological replicates (n=3) is sufficient to support the reliability of the results.

3. Arterial blood pressure needs to be quantified and included as graph in Figures 3, 5, and 6. The other tested parameters also need to be quantified and included in each figure as scatter plots.

Thank you for your valuable comments. We fully agree and will optimize the presentation of the figures as requested to make the data quantification clearer and more intuitive.

P4 L122 Fig 3, P6 L161 Fig 5, P7 L168 Fig 6. The quantitative results of renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) in Figure 3 have been presented in Figures 2B and 2C, including the specific changes and statistical significance after intervention with different doses of Dooku1. The data on the effects of Piezo1 agonists Yoda1 and Jedi2 on RSNA, MAP, and heart rate (HR) in Figures 5 and 6 have been summarized in Table 1, and the scatter plots are presented below. These clearly record the values before and after intervention as well as the statistical results (P > 0.05). Due to space constraints, the non-significant negative results were not shown in the main text.

We would like to thank the reviewers again for helping to improve the visualization and data integrity of the study.

4. There is no need to cite figures in Discussion.

Thank you for your valuable comments. Although the present study does not include experimental data on SK/BK channels, the relevant functions of SK/BK channels have been verified in the previous studies published by our team. In addition, Figure 7 preliminarily reveals the potential mechanism by which the Piezo1 ion channel participates in the activity of autonomic neurons in the hypothalamic paraventricular nucleus (PVN). After careful consideration, we have cited Figure 7 in the discussion section to illustrate this mechanism more clearly.

5. Why are the p values included in Figure 1 legend if no statistically significant changes were observed? Does this figure aim to demonstrate the presence of Piezo 1 channels across tissues rather than compare expression levels? If so, what is the appropriate control? What about long-term modulation of Piezo 1 signaling? Expand the data analysis section. Was a normality test performed prior to selecting parametric or non-parametric tests? Was any outlier detection method used?

We appreciate the valuable suggestion from the reviewer. The redundant P-value labels in the legend of Figure 1 have been removed accordingly. The core purpose of this figure is to clarify the expression localization of Piezo1 in the paraventricular nucleus (PVN), rather than to compare differences in its expression levels. Therefore, no "treatment group vs. control group" comparison was designed, and only the basic distribution characteristics of Piezo1 in the PVN are presented herein. Page 3, line 102

Regarding the long-term regulatory mechanisms underlying Piezo1 signaling transduction, existing studies have demonstrated that Piezo1 expression is subject to long-term transcriptional regulation by transcription factors including NF-κB (p65 subunit) and TNF-ɑ in diverse endothelial cell types (PMID: 37891953). Additionally, it has been showed that the RNA repair/splicing enzyme Rtca acts upstream of Piezo to modulate its expression and transport/targeting to the periphery of the soma via the Rab10 GTPase, whose expression also relies on Rtca(PMID: 41325493). Based on the results of the present study, we hypothesize that neuroendocrine signals within the PVN (e.g., stress-related hormones, local cytokines and the RNA repair/splicing enzyme Rtca) may exert long-term regulatory effects on Piezo1 expression and function through the aforementioned transcriptional regulatory axes. Notably, this study is the first to report the expression and localization characteristics of Piezo1 in the PVN, and the long-term regulatory mechanisms of Piezo1 in this region warrant further investigation in future research.

We sincerely appreciate the reviewer for raising these critical questions regarding the statistical methods of our study, which helps to enhance the rigor and transparency of our data analysis. In response to the request to expand the data analysis section, we have comprehensively revised and supplemented the Materials and Methods → 4.10 Data Analysis subsection in the revised manuscript. Prior to choosing parametric or non-parametric tests, we performed normality tests on all experimental data sets using the Shapiro–Wilk test, which is widely recommended for small to medium sample sizes in biological and medical research. For data that conformed to a normal distribution, we used parametric tests (e.g., independent samples t-test for two-group comparisons, one-way ANOVA followed by Tukey’s post-hoc test for multiple-group comparisons).

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I thank the authors for addressing the questions and comments that arose from the previous versions of their manuscript. Although the authors have answered the questions there are still some concerns on this manuscript.

1. The reference PMID: 2916695, dates back to 1989. Is this the most recent report where anesthetized animals have been used for a study of this kind? The reference PMID: 1986985, is from 1999. Again, no recent reports have backed up these findings with anesthetized subjects. If the answer is no, the authors have to show the effects of anesthesia in rats on MAP and MRNA over time, in a similar fashion like that in Hara et al., 1989, to justify their claims that this anesthetic procedure do not interfere with the sympathetic efferent activity in rats.
2. The reference PMID: 2916695 is from 1997. Again, if there are not more recent papers highlighting the “common” use of this anesthetic procedure, how confident we should be that this will not interfere with the "fight-or-flight" response and the study of sympathetic efferent activity in rats.
3. Some editing to the western blot image should be done for publication.
4. The images presented are borderline quality. Authors should have made an effort in acquiring better images. High quality images should have been selected for this publication. It is possible to image Piezo1 localization with immuno-fluorescent staining in isolated and cultured primary PVN neurons. In addition, in two of the panels a 2 mm scale bar was included which does not correspond to the magnification used, 40x and 60x.
5. Since the results of this limited study indicate that activation of Piezo1 channels in CTB-labeled sympathetic-related neurons within the PVN can elevate blood pressure by enhancing sympathetic efferent activity. The limitations of the study and the results do not necessarily point out on the role of Piezo 1 channels in regulating sympathetic efferent activity and cardiovascular function. More and stronger evidence in required and this was acknowledged by the authors in this version of the manuscript.
6. Providing evidence of a Piezo1-SK axis that modulates calcium signals and sympathetic nerve activity in PVN neurons and its implication in regulating cardiac function is of paramount importance.

7. As recognized by the authors in this new version of the manuscript: "Co-inject Piezo1 agonists (Yoda1/Jedi2) and SK/BK channel inhibitors into the PVN region, and synchronously monitor the dynamic changes in renal sympathetic nerve activity (RSNA) and arterial blood pressure in rats to clarify the upstream-downstream regulatory relationship between Piezo1 channels and calcium-activated potassium channels. This represents a crucial experiment to demonstrate that a regulatory relationship between Piezo1 and Sk or BK channels is taking place in the PVN region.

8. In general, the manuscript was improved and the questions were addressed for the most part. I will encourage the authors to keep improving the manuscript and I am sure that it will be ready for publication soon. My comments (Q) are marked in Pink in the PDF attached, with sections of the answers marked in Green as a sign that I strongly agreed with the answers and further experiments considered by the authors in response to my previous comments. 

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The use of commas and punctuation must be revised.

Author Response

Comments and Suggestions for Authors

I thank the authors for addressing the questions and comments that arose from the previous versions of their manuscript. Although the authors have answered the questions there are still some concerns on this manuscript.

1. The reference PMID: 2916695, dates back to 1989. Is this the most recent report where anesthetized animals have been used for a study of this kind?The reference PMID: 1986985, is from 1999. Again, no recent reports have backed up these findings with anesthetized subjects. If the answer is no, the authors have to show the effects of anesthesia in rats on MAP and MRNA over time, in a similar fashion like that in Hara et al., 1989, to justify their claims that this anesthetic procedure do not interfere with the sympathetic efferent activity in rats.

We sincerely appreciate your rigorous review and professional guidance.Although anesthesia with urethane/α-chloralose exerts mild effects on the cardiovascular system of rats, it has become a classic approach in studies targeting the PVN-RVLM pathway due to its ability to stably preserve the integrity of central neural pathways and maintain the core regulatory function of the sympathetic nervous system. This method has been widely adopted and recognized in the relevant research field.

Mourão et al. used urethane/α-chloralose-anesthetized rats and demonstrated that TNFα injection into the hypothalamic PVN triggers SNA ramping via mechanisms dependent on local ionotropic glutamate receptor availability (PMID: 34355986, 2021).

Under anesthesia with urethane and α-chloralose, Koba et al. verified that stimulation of glutamatergic neurons in the PVN-RVLM pathway increases renal sympathetic nerve activity and arterial pressure in male rats, at least in part by activating C1 neurons in the RVLM (PMID: 30019338, 2018).

Bardgett et al. found that blockade of PVN N-methyl-D-aspartate (NMDA) receptors reduced renal SNA and mean arterial pressure (MAP) in urethane-chloralose-anesthetized dehydrated rats (48 h water deprivation; P < 0.01), but exerted no effect in euhydrated control animals (PMID: 24671240, 2014).

In another study by Bardgett et al., glucose infusion elevated lumbar and visceral SNA in α-chloralose/urethane-anesthetized rats, and this response was nearly abolished by pretreatment with astressin (10 pmol/50 nl), a CRF receptor antagonist, in the RVLM. These findings indicate that glucose-induced activation of SNA and subsequent energy expenditure are initiated by CRF receptor activation in the RVLM via descending input from the PVN (PMID: 25269482, 2014).

Under urethane and α-chloralose anesthesia, Chen et al. observed that microinjection of salusin-β into the PVN increased RSNA, MAP and heart rate in a dose-dependent manner. Conversely, administration of anti-salusin-β IgG into the PVN reduced RSNA and MAP in 2K1C hypertensive rats and abolished the effects of salusin-β. The authors confirmed that salusin-β in the PVN elevates blood pressure, heart rate and sympathetic outflow through both circulating AVP and AVP within the RVLM in hypertensive rats (PMID: 23400761, 2013).

Under urethane and α-chloralose anesthesia, Zhang et al. reported that bilateral PVN microinjection of angiotensin II (Ang II) induced greater enhancements in cardiac sympathetic afferent reflex, baseline renal sympathetic nerve activity and MAP in adriamycin-treated rats than in control rats. These results indicated that both sympathetic activity and cardiac sympathetic afferent reflex were augmented, and Ang II in the PVN contributed to these enhancements in rats with adriamycin-induced heart failure (PMID: 23554781, 2012).

The six studies cited above, published between 2012 and 2021, all employed urethane/α-chloralose anesthesia for investigations of the PVN-RVLM pathway. This indicates that this method is not restricted to early reports, but has been continuously used in recent research. In fact, numerous additional studies using urethane/α-chloralose anesthesia for PVN-RVLM pathway-related experiments are available, which we have not listed individually here.

We thank you again for your valuable comments. Accordingly, we have added two recent references (PMID: 34355986, 2021; PMID: 30019338, 2018) at line 310 on page 12.

2. The reference PMID: 2916695 is from 1997. Again, if there are not more recent papers highlighting the “common” use of this anesthetic procedure, how confident we should be that this will not interfere with the "fight-or-flight" response and the study of sympathetic efferent activity in rats.

We sincerely appreciate your careful review and insightful comments on our manuscript. Compared with conscious rats, urethane/αchloralose anesthesia has been reported to suppress arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA) in rats (PMID: 2567578; PMID: 3440820). In the present study, however, microinjection of a Piezo1 channel inhibitor into the PVN still induced significant elevations in ABP and RSNA. Therefore, we believe that urethane/αchloralose anesthesia may affect the quantitative analysis, but will not alter the qualitative conclusion of our findings.

In addition, urethane/αchloralose anesthesia is a wellestablished experimental strategy for studies targeting the PVNRVLM pathway:Mourão et al. demonstrated in urethane/αchloraloseanesthetized rats that PVN TNFα evokes SNA ramping via local ionotropic glutamate receptors (PMID: 34355986, 2021).Koba et al. showed that activation of PVNRVLM glutamatergic neurons increases renal SNA and arterial pressure partly through RVLM C1 neurons in urethane/αchloraloseanesthetized rats (PMID: 30019338, 2018).Bardgett et al. found that PVN NMDA receptor blockade reduced renal SNA and MAP in dehydrated rats under urethane/αchloralose anesthesia, but not in euhydrated controls (PMID: 24671240, 2014).Bardgett et al. also reported that glucoseinduced SNA elevation is mediated by PVNtoRVLM CRF receptor signaling in urethane/αchloraloseanesthetized rats (PMID: 25269482, 2014).Chen et al. showed that PVN salusinβ increases RSNA, MAP and heart rate via AVPrelated pathways in hypertensive rats anesthetized with urethane/αchloralose (PMID: 23400761, 2013).Zhang et al. demonstrated that PVN Ang II contributes to the enhanced sympathetic activity and cardiac sympathetic afferent reflex in heart failure rats under urethane/αchloralose anesthesia (PMID: 23554781, 2012).

These studies, published from 2012 to 2021 and widely accepted in the research field, collectively support the feasibility of this method for investigating the PVNRVLM pathway.

3. Some editing to the western blot image should be done for publication.

Thank you for your careful review and constructive suggestion.We have carefully edited and standardized the Western blot image in accordance with academic publication norms.The corresponding revisions have been made in Figure 1A, line 91 on page 3 of the revised manuscript.

4. The images presented are borderline quality. Authors should have made an effort in acquiring better images. High quality images should have been selected for this publication. It is possible to image Piezo1 localization with immuno-fluorescent staining in isolated and cultured primary PVN neurons. In addition, in two of the panels a 2 mm scale bar was included which does not correspond to the magnification used, 40x and 60x.

We greatly appreciate the reviewer’s comments and suggestions regarding the image quality and scale bars. We would like to emphasize that we have made our best effort to select the clearest and highest-quality images from all repeated experimental results for presentation in the manuscript. We did not simply replace individual images but carefully screened all available data to ensure the optimal image quality under current experimental conditions. We will continue to improve our imaging techniques and strive to provide higher-quality images in future studies.

Regarding the immunofluorescent staining of Piezo1 localization in primary cultured PVN neurons, we greatly appreciate this valuable suggestion. We have taken this comment into consideration and will perform additional experiments with isolated and cultured primary PVN neurons in our future research to further confirm Piezo1 localization.

In addition, we apologize for the inappropriate scale bars in the original figures. The scale bars for images captured under 40× and 60× magnification have been corrected and replaced accordingly. These revisions have been made in Figure 1D, line 92 on page 3 of the revised manuscript.

5. Since the results of this limited study indicate that activation of Piezo1 channels in CTB-labeled sympathetic-related neurons within the PVN can elevate blood pressure by enhancing sympathetic efferent activity. The limitations of the study and the results do not necessarily point out on the role of Piezo 1 channels in regulating sympathetic efferent activity and cardiovascular function. More and stronger evidence in required and this was acknowledged by the authors in this version of the manuscript.

We sincerely appreciate your professional guidance and constructive criticism.

Of note, regarding the findings of the present study, we first confirmed the expression of Piezo1 channels in the PVNRVLM pathway using retrograde tracing from the RVLM.

We identified two distinct populations of spinal cordprojecting PVN neurons: Group I (n=19) exhibited a fast conduction velocity (3.67±0.29 m/s), with 73% being barosensitive and involved in vasomotor regulation; Group II (n=34) displayed a slow conduction velocity (0.45±0.01 m/s) and no barosensitivity, likely contributing to nonvasomotor sympathetic control. Both populations showed 73%–74% correlation with renal sympathetic nerve activity (RSNA) (PMID: 12710987).

In a subsequent study, 94 PVNRVLM projecting neurons were recorded in rats, among which 69% projected exclusively to the RVLM and 31% projected to both the RVLM and the spinal intermediolateral column (IML). These neurons were mostly spontaneously active, correlated with RSNA, and exhibited cardiac rhythmicity, with 69%~100% being modulated by the baroreflex (PMID: 19889858).

These findings indicate that both PVNRVLM and PVNRVLM/IML neurons participate in the regulation of baseline sympathetic nerve activity and its baroreflex modulation.

On this basis, we demonstrated that three subtypes of SK channels (SK1SK3) are expressed in the PVN. Bilateral microinjection of the SK channel blocker apamin (12.5 pmol, n=7) into the PVN increased visceral sympathetic nerve activity (SSNA) by 330±40%, RSNA by 271±40%, mean arterial pressure (MAP) by 29±4 mmHg, and heart rate (HR) by 34±9 bpm (all P<0.01), indicating that SK channels exert a critical inhibitory effect on sympathetic nerve activity and blood pressure (PMID: 22647293).

In the present study, using in vivo microinjection combined with physiological parameter recording, we verified that Piezo1 channels in sympatheticrelated neurons within the PVN modulate arterial blood pressure (ABP) and RSNA. Following specific microinjection of the Piezo1 channel inhibitor Dooku1 (100 pmol, n=9) into the PVN of anesthetized rats, ABP was elevated by 21±5 mmHg and RSNA by 93±30% (both P<0.01).

These results suggest that inhibition of Piezo1 channels in CTB-labeled sympathetic-related neurons within the PVN produces a pressor effect by enhancing sympathetic efferent activity.

6. Providing evidence of a Piezo1-SK axis that modulates calcium signals and sympathetic nerve activity in PVN neurons and its implication in regulating cardiac function is of paramount importance.

We sincerely appreciate the reviewer’s valuable suggestions for verifying our mechanistic hypothesis. The proposal to supplement experimental evidence for the CICR mechanism using calcium indicators is highly instructive for strengthening our conclusions, and we fully agree with this rigorous comment.

We recognize that calcium sensors and fluorescent probes are critical for detecting intracellular Ca²⁺ dynamics in PVN neurons, which is essential to confirm that suppressed Ca²⁺-induced Ca²⁺ release (CICR) mediates sympathetic enhancement after Piezo1 blockade. These experiments have been designed as core content for our followup mechanistic study. However, the present study focuses on the functional role and overall cardiovascular regulation of Piezo1 in the PVN–renal sympathetic pathway. In vivo calcium imaging of PVN neurons requires complex techniques (e.g., chronic cranial window preparation) with long experimental cycles and high technical demands, which are beyond the scope of the current manuscript and will be reported separately.

Notably, the regulatory roles of PVN smallconductance Ca²⁺activated K⁺ (SK) channels in sympathetic tone and blood pressure have been well established in our previous studies (PMID: 22647293, 20719931). We demonstrated that SK channels inhibit PVN neuronal excitability and attenuate sympathetic outflow, while reduced SK currents contribute to sympathetic overactivity in hypertension.

On this basis, we hypothesize that Piezo1 and SK channels form a regulatory axis via Ca²⁺ signaling: Piezo1mediated Ca²⁺ influx amplifies Ca²⁺ signals through CICR, thereby facilitating SK channel activation. Blockade of Piezo1 reduces Ca²⁺ influx and suppresses CICR, leading to insufficient SK activation, enhanced PVN excitability, sympathetic hyperactivity, and elevated blood pressure. This notion is indirectly supported by a previous study showing that Piezo1 regulates Ca²⁺ homeostasis and neurocardiovascular function (PMID: 37670136).

In future work, we will verify this hypothesis by: Measuring Ca²⁺ dynamics and SK activation in primary PVN neurons using Ca²⁺ fluorescent probes; Monitoring in vivo Ca²⁺ signals via genetically encoded Ca²⁺ sensors combined with in vivo imaging; Exploring physical or functional interactions between Piezo1 and SK channels using CoIP and FRET.

These detailed mechanistic data will be presented in subsequent studies to provide direct evidence for the Piezo1–SK axis in regulating Ca²⁺ signaling and sympathetic activity in PVN neurons.

7. As recognized by the authors in this new version of the manuscript: "Co-inject Piezo1 agonists (Yoda1/Jedi2) and SK/BK channel inhibitors into the PVN region, and synchronously monitor the dynamic changes in renal sympathetic nerve activity (RSNA) and arterial blood pressure in rats to clarify the upstream-downstream regulatory relationship between Piezo1 channels and calcium-activated potassium channels. This represents a crucial experiment to demonstrate that a regulatory relationship between Piezo1 and Sk or BK channels is taking place in the PVN region.

We greatly appreciate the reviewer’s insightful and constructive comment on this important experiment. We fully agree that co-injection of Piezo1 agonists (Yoda1/Jedi2) with SK/BK channel inhibitors into the PVN, combined with simultaneous monitoring of RSNA and arterial blood pressure, represents a critical approach to clarify the upstreamdownstream regulatory relationship between Piezo1 and calciumactivated potassium channels in the PVN.

This set of experiments is indeed part of our ongoing and followup mechanistic study aimed at further dissecting the detailed signaling cascade. However, these experiments involve extensive in vivo pharmacological intervention and longterm simultaneous electrophysiological/hemodynamic recordings, which require substantial additional time, animal preparation, and data validation that go beyond the scope and timeline of the present manuscript.

The current study focuses on demonstrating the expression, functional role, and physiological relevance of Piezo1 channels in the PVN in regulating sympathetic nerve activity and blood pressure, which has been sufficiently supported by our present experimental data. The detailed hierarchical regulation between Piezo1 and SK/BK channels will be systematically investigated and reported in our subsequent dedicated study.

We have acknowledged the importance of this proposed experiment in the Discussion section and clarified that it will be addressed in future research to further elaborate the underlying mechanism.

Thank you again for this valuable suggestion, which will greatly help improve and guide our followup investigations.

8. In general, the manuscript was improved and the questions were addressed for the most part. I will encourage the authors to keep improving the manuscript and I am sure that it will be ready for publication soon. My comments (Q) are marked in Pink in the PDF attached, with sections of the answers marked in Green as a sign that I strongly agreed with the answers and further experiments considered by the authors in response to my previous comments.

We greatly appreciate the reviewer’s positive comments and warm encouragement. We are pleased that most of our revisions and responses have met your expectations, and we have carefully revised the manuscript in response to the remaining comments marked in pink in the attached PDF. We will further polish the manuscript to ensure it fully meets the publication requirements. Thank you again for your professional and constructive feedback, which has been crucial for improving the quality of our work.

Comments on the Quality of English Language

The use of commas and punctuation must be revised.

We sincerely appreciate the reviewer’s valuable comment on the English language and punctuation of the manuscript. We have conducted a thorough revision of the comma usage and overall punctuation throughout the text in accordance with academic writing norms. All revised parts have been highlighted in the manuscript for easy identification, at the specific positions: Page 6, Line 146; Page 13, Line 325; Page 14, Line 349; Page 15, Line 399; Page 16, Line 464. We have carefully checked the entire manuscript to ensure the accuracy and consistency of punctuation usage. Thank you again for your rigorous feedback, which helps us further polish the language quality of the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Authors have successfully addressed the comments; however, I don't see these responses reflected in the revised manuscript. For example, the Discussion appears very similar to the previous version, and the figures do not include the required BP quantification, among other details. 

Comments on the Quality of English Language

Author Response

Comments and Suggestions for Authors

1. Authors have successfully addressed the comments; however, I don't see these responses reflected in the revised manuscript. For example, the Discussion appears very similar to the previous version, and the figures do not include the required BP quantification, among other details.

We sincerely appreciate the reviewer’s valuable comments and confirmation that our responses have addressed the raised concerns. We apologize for any confusion caused by the presentation of our revisions, and all modifications corresponding to your comments have been clearly reflected in the revised manuscript with highlighted markings for easy identification. The detailed explanations are as follows:

1. For the Discussion section, a new critical paragraph has been added to supplement the mechanistic interpretation and experimental rationale, and this supplementary content is highlighted in the revised manuscript from page 11, line 271 to page 12, line 293.
2. Regarding the required mean arterial pressure (MAP) quantification for the figures: the quantitative results of MAP for Figure 3 (including specific changes and statistical significance after intervention with different doses of Dooku1) are fully presented in Figure 2B and 2C; the quantitative MAP results for Figure 4 are clearly shown in Figure 4B and 4C. The experimental data regarding the effects of Piezo1 agonists Yoda1 and Jedi2 on renal sympathetic nerve activity (RSNA), MAP and heart rate (HR) in Figure 5 and Figure 6 have been systematically summarized in Table 1.

We have carefully checked the entire revised manuscript to ensure all revisions in response to your comments are accurately reflected in the text, figures and tables. We are happy to make further detailed adjustments if needed, and we greatly appreciate your rigorous and constructive feedback that has significantly improved the quality of our manuscript.

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

All my concerns have been addressed.

Comments on the Quality of English Language