# Reply to Kaestner et al.: Activation of PIEZO1 is not significant for the passage of red blood cells through biomimetic splenic slits

Alexis Moreau, François Yaya, Huijie Lu, Anagha Surendranath, Anne Charrier, Benoit Dehapiot, Emmanuèle Helfer, Annie Viallat, Zhangli Peng

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## To cite this version:

Alexis Moreau, François Yaya, Huijie Lu, Anagha Surendranath, Anne Charrier, et al., Reply to Kaestner et al.: Activation of PIEZO1 is not significant for the passage of red blood cells through biomimetic splenic slits. Proceedings of the National Academy of Sciences of the United States of America, 2025, 122 (2), 10.1073/pnas.2411469121. hal-04869081

HAL Id: hal-04869081

https://hal.science/hal-04869081v1

Submitted on 8 Nov 2025

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# Reply to Kaestner et al.: Activation of PIEZO1 is not significant for the passage of red blood cells through biomimetic splenic slits

Alexis Moreau<sup>a,1</sup> <sup>●</sup>, François Yaya<sup>a</sup>, Huijie Lu<sup>b</sup>, Anagha Surendranath<sup>a</sup> <sup>●</sup>, Anne Charrier<sup>a</sup> <sup>●</sup>, Benoit Dehapiot<sup>c</sup> <sup>●</sup>, Emmanuèle Helfer<sup>a</sup> <sup>●</sup>, Annie Viallat<sup>a,2</sup> <sup>●</sup>, and Zhangli Peng<sup>b,2</sup> <sup>●</sup>

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Intracellular calcium fluorescence intensity upstream (in white) and downstream of the slits (in gray) (A) in the absence or presence of 1 mM calcium in the buffer and (B) in the presence of 1 mM calcium in the buffer and of 5 µM GsMTX4, under ΔP = 500 Pa. Median values are displayed with 25% and 75% percentiles, and min/max values as whiskers. N: number of analyzed cells. Slit dimension: 0.80 × 2.77 × 4.70 µm³. Portions of this figure have been reused from Moreau et al. (1).</figcaption></figure>

We thank the authors for acknowledging the seminal nature of our work (1).

We have not conclusively addressed the question of Piezo1 activation during splenic filtration in vivo. Our aim was to highlight the key mechanisms governing red blood cell (RBC) retention/passage in slits. In the discussion, we were cautious in only suggesting that no PIEZO1 effect is involved in RBC passage.

Kaestner et al. wrote that “Accumulating evidence shows salt solution...contain Ca²⁺” (2). We carefully examined the articles cited supporting this statement.

In ref. 3, the authors use a Gardos channel activator. They observe hyperpolarization and deduce contamination by 4 µmol Ca²⁺. However, they give neither the cause (residual Ca²⁺ from blood plasma?) nor the dosage. Moreover, at low Gardos activator concentrations, no hyperpolarization is observed at 4 µmol Ca²⁺. Finally, hyperpolarization is a slow process (time scale: 500 s).

In ref. 4, the authors use a PIEZO1 activator. They observe hyperpolarization and cite ref. 3 to explain it but provide neither cause nor assay showing contamination. However, they estimate a plausible residual 8 µmol-Ca²⁺ originating from the plasma of the blood sample.

Our experimental conditions differ radically from those described in these articles:

1) We did not use Piezo1 or Gardos activator that promote activation at subphysiological values.

2) We took special precautions to avoid Ca²⁺ contamination. First, we used a Ca²⁺-free commercial buffer prepared from distilled water (See supplier's data sheet available on

the Internet). Second, we diluted the initial 30-µL blood drop in 1 mL (50-fold dilution) prior to centrifugation. The pellet was diluted in 500 µL and centrifuged twice, i.e., two successive 25-fold dilutions before a final 100-fold dilution, so that the residual Ca²⁺ concentration is <1 nmol, far too low to activate Gardos.

3) The slit passage time is a fast process <100 ms.

Finally, we performed a limited experiment using the PIEZO1 inhibitor (GsMTX4) and Fluo4, similarly to our paper's figure 4 (reported here as Fig. 1A).

Although the statistics are limited, the trend is similar: i) an increase in intracellular Ca²⁺ signal after slit exit; ii) no difference in Ca²⁺ signal after exit, regardless of no Ca²⁺, or

Author affiliations: <sup>a</sup>CNRS, Centre Interdisciplinaire de Nanoscience de Marseille, Turing Centre for Living Systems, Aix Marseille Université, Marseille 13009, France; <sup>b</sup>Richard and Loan Hill Department of Biomedical Engineering, University of Illinois, Chicago, IL 60612; and <sup>c</sup>CNRS, Institut de Biologie du Développement de Marseille, Turing Centre for Living Systems, Aix Marseille Université, Marseille 13009, France

Author contributions: E.H., A.V., and Z.P. designed research; A.M., F.Y., H.L., A.S., A.V., and Z.P. performed research; A.C. and B.D. contributed new reagents/analytic tools; A.M., E.H., A.V., and Z.P. analyzed data; and A.M., F.Y., E.H., A.V., and Z.P. wrote the paper.

The authors declare no competing interest.

Copyright © 2025 the Author(s). Published by PNAS. This article is distributed under Creative Commons Attribution License 4.0 (CC BY).

¹Present address: Department of Molecular, Cellular, and Developmental Biology, Quantitative Biology Institute, Yale University, New Haven, CT 06511.

²To whom correspondence may be addressed. Email: annie.viallat@univ-amu.fr or zhpeng@uic.edu.

Published January 2, 2025.
Ca²⁺ and PIEZO1 inhibitor in solution (Fig. 1B). Also, RBC transit times and velocities through the slits are unaffected by Ca²⁺ and/or PIEZO1 and Gardos inhibitors (Paper's figures S9 and 5B). The similar transit times observed irrespectively of the presence of Ca²⁺ or PIEZO1 and Gardos inhibitors show that PIEZO1-Gardos channels do not induce a significant cell volume reduction, in agreement with a previous study (5). Thus, no significant change is expected in hemoglobin concentration and, consequently, in the Fluo4 signal.

Finally, our simulations show that the typical maximum membrane tension is about 421.23 pN/µm (figure 3F), below the critical tension (6) to activate PIEZO1 [1,440 pN/µm (7), 2,468 pN/µm (8), and 7,820 pN/µm (9)].

To conclude, we think that our experiments and conclusions are correct.

An experiment designed jointly by Kaestner et al. and ourselves would be beneficial in reaching a scientific consensus.

**ACKNOWLEDGMENTS.** The project leading to this publication has received funding from France 2030, the French Government program managed by the French National Research Agency (ANR-16-CONV-0001 and ANR-20-CE17-0024). We thank I. Ozerov and F. Bedu from PLANETE micro fabrication facility. We thank Pr. Pierre Buffet and his group for providing us spherocytic and irreversible sickle RBCs. H.L. and Z.P. acknowledge the supports from US NSF grants NSF CBET-1706436/1948347, NSF DMS-1951526, NSF CMMI-2339054, and American Society of Hematology Scholar Award. E.H., A.V., and Z.P. are also supported by a joint NSF-ANR grant (NSF-PHY2210366 and ANR-22-CE95-0004-01). E.H. belongs to the French Consortium Approches Quantitatives du Vivant/Quantitative approaches to living systems.

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8. Y.-C. Lin et al., Force-induced conformational changes in PIEZO1. *Nature* **573**, 230-234 (2019).

9. C. D. Cox et al., Removal of the mechanoprotective influence of the cytoskeleton reveals PIEZO1 is gated by bilayer tension. *Nat. Commun.* **7**, 10366 (2016).