Within our bodies, cells and tissues are constantly being pushed and pulled by their surrounding environment. These mechanical forces are then transformed into electrical or chemical signals by cells. This process is crucial for many biological structures, such as blood vessels, to develop correctly, and is also a key part of our senses of touch and hearing.

In 2010, researchers discovered a group of ion channels—proteins embedded in the membrane that surrounds a cell—that open up when a force is applied and allow calcium and other ions to enter the cell. This movement of ions generates the electrical response of the cell to the applied force. However, not much is known about the roles of these ‘Piezo’ ion channels.

Red blood cells experience significant forces when they pass through narrow blood vessels. In a disease called xerocytosis, the red blood cells become severely dehydrated and shrink. In 2013, researchers discovered that patients with this disease have mutations in the gene that codes for the Piezo1 protein: a Piezo protein that has also been linked to a role in blood vessel development in embryos. This suggested that Piezo1 may regulate the volume of red blood cells.

Cahalan, Lukacs et al.—including some of the researchers who worked on the 2010 and 2013 studies—have now investigated the role of Piezo1 in red blood cells in more detail. Applying strong forces to red blood cells from mice caused calcium to rapidly enter cells through Piezo1 channels.

Cahalan, Lukacs et al. then deleted the Piezo1 gene from red blood cells. This made the cells larger and more fragile than normal cells because they contained too much water. To investigate how Piezo1 regulates water content, the cells were treated with a chemical compound called Yoda1. This compound was shown in a separate study by Syeda et al. to activate Piezo1 channels. Activating Piezo1 caused a second type of ion channel to open up as well, which allowed potassium ions and water molecules to leave the cell. This resulted in the cell becoming dehydrated.

This work raises the possibility that Piezo proteins are involved in other diseases where red blood cell volume is altered. In particular, many believe that Piezo1 may be involved in sickle cell disease, a possibility that can now be tested using the tools described in this study.

Mammalian red blood cells (RBCs) are rather unique in that they lack a nucleus and many organelles and that they traverse the circulatory system several hundred thousand times in their life cycle. RBCs experience significant mechanical forces while recirculating that influence their physiology in many ways, including changes in deformability (Chien, 1987), ATP release (Sprague et al., 2001), NO release (Yalcin et al., 2008), and Ca²⁺ influx (Larsen et al., 1981; Dyrda and et al., 2010), the latter of which can influence RBC volume. Changes in RBC volume can affect their membrane integrity and ability to travel through capillaries with diameters smaller than the RBCs themselves. The critical importance of RBC volume regulation is demonstrated by several pathologies resulting from either overhydration or dehydration of RBCs (Gallagher, 2013). However, the molecular mechanisms by which RBCs sense mechanical forces and the effects of these forces on volume homeostasis have remained unclear.

Recent studies have identified a conserved family of mechanosensitive non-selective cation channels, Piezo1 and Piezo2 (Coste et al., 2010, 2012). Piezo1 responds to a wide array of mechanical forces, including poking, stretching, and shear stress, and is essential for proper vascular development in mice (Nilius and Honore, 2012; Li et al., 2014; Ranade et al., 2014). The potential role of Piezo1 in RBC physiology is most clearly demonstrated by many gain-of-function mutations in Piezo1 that have been identified in patients with the RBC disease xerocytosis, also called dehydrated hereditary stomatocytosis (DHS) (Zarychanski et al., 2012; Albuisson et al., 2013; Andolfo et al., 2013; Bae et al., 2013). In addition, whole body treatment of zebrafish with Piezo1 morpholino affects RBC volume homeostasis (Faucherre et al., 2014). Finally, the Piezo1 locus has also been implicated in a genome-wide association screen for affecting the RBC mean corpuscular hemoglobin concentration (MCHC) in humans (van der Harst et al., 2012). However, whether any of these effects are due to Piezo1 expression on the RBCs themselves is not understood nor is the normal role of Piezo1 in mammalian RBC physiology.

We first investigated whether Piezo1 is expressed on mouse RBCs using Piezo1^P1-tdTomato mice that express a Piezo1-tdTomato fusion protein from the Piezo1 locus (Ranade et al., 2014). Both peripheral blood RBCs (Figure 1A) and developing bone marrow pro-RBCs (Figure 1B) from Piezo1^P1-tdTomato mice exhibited increased tdTomato fluorescence by flow cytometry compared to those from Piezo1^+/+ mice. Peripheral RBCs from Piezo1^P1-tdTomato mice had clear expression of a ∼320 kDa Piezo1-tdTomato fusion protein by Western blot (Figure 1A). To further investigate the role of Piezo1 in RBC physiology, we set out to genetically ablate it. Mice deficient in Piezo1 die in utero, so we deleted Piezo1 specifically in the hematopoietic system. We bred Vav1-iCre mice, which express Cre recombinase early in hematopoiesis (Shimshek et al., 2002), to mice where exons 20–23 of Piezo1 are flanked by loxP sites (P1^f), thus generating viable, fertile Vav1-iCre P1^f/f (Vav1-P1cKO) mice (Figure 1—figure supplement 1A). Vav1-P1cKO lymphocytes exhibited >95% deletion of piezo1 transcript, demonstrating efficient Cre-mediated excision (Figure 1—figure supplement 1C). Hematological analysis of blood from Vav1-P1cKO mice revealed significant changes in RBC physiology without significant anemia (Table 1). Notably, compared to WT mice, Vav1-P1cKO mice had elevated (% of WT ± SEM) mean corpuscular volume (MCV, 109.51 ± 1.51) and mean corpuscular hemoglobin (MCH, 103.14 ± 0.48) and reduced mean corpuscular hemoglobin concentration (MCHC, 94.37 ± 1.08), suggesting that Piezo1-deficient RBCs were overhydrated. Since increased MCV can also be observed in the dehydrated RBCs in xerocytosis, we further tested whether Piezo1-deficient RBCs were actually overhydrated. Overhydrated RBCs exhibit increased osmotic fragility and increased size as measured by forward scatter using flow cytometry. Blood from Vav1-P1cKO mice exhibited both of these characteristics (Figure 1C and Figure 1—figure supplement 2A), demonstrating that Piezo1-deficient RBCs are overhydrated. While Vav1-P1cKO RBCs were overhydrated, scanning electron microscopy of WT and Vav1-P1cKO RBCs revealed that Vav1-P1cKO RBCs had relatively normal discoid morphology, unlike more severe overhydration pathologies such as spherocytosis (Figure 1—figure supplement 2B). Regardless, these results suggest that Piezo1 expression on RBCs is a negative regulator of RBC volume.

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Because changes in RBC volume commonly result in pathology in the spleen, we compared Vav1-P1cKO spleens with those of WT littermates. Although they appeared visibly darker and redder following H&E staining, spleens from Vav1-P1cKO mice exhibited normal formation of both red and white pulp without an evident expansion of red pulp or increased iron deposition (Figure 1—figure supplement 2C,D). However, flow cytometric analysis of splenic RBC subpopulations revealed an increased number of fully mature Ter119⁺ CD71⁻ RBCs, but not immature Ter119⁺ CD71⁺ RBCs (Figure 1D), suggesting that the darker splenic color is due in part to retention of overhydrated circulating mature RBCs. Consistent with this, immature splenic RBCs had similar forward scatter in WT and Vav1-P1cKO mice, indicating that they were of similar size, while fully mature RBCs in Vav1-P1cKO exhibited increased forward scatter indicative of increased size (Figure 1—figure supplement 2A). We also found that Vav1-P1cKO mice exhibited significantly lower plasma haptoglobin concentrations, indicative of intravascular hemolysis in vivo (Figure 1E). Thus, Piezo1-deficient RBCs have increased fragility and are aberrantly retained within the spleen, suggesting that Piezo1 helps maintain RBC integrity and normal recirculation.

Piezo1 is a mechanically activated, calcium-permeable non-selective cation channel. RBCs experience significant mechanical forces during circulation; we, therefore, sought to determine whether acute application of mechanical force could cause Ca²⁺ influx and whether any Ca²⁺ influx observed was dependent on Piezo1. However, many of the existing methods of mechanical stimulation available to us proved unsuitable for this purpose. Patch-clamp experiments proved impractical as conditions that allowed the formation of gigaohm seals in a cell-attached setting resulted in cell rupture upon application of negative pressure. Additionally, RBCs were also too small and fragile for indentation using a glass probe with or without simultaneous electrophysiological recording. Calcium imaging studies using shear stress in laminar flow chambers did not yield detectable increases in intracellular Ca²⁺.

We, therefore, developed a mechanical stimulation assay combining the advantage of calcium imaging (no gigaohm seal necessary) and patch clamp (a pipette to capture the cell). We used micropipettes with a long, tapered tip and optimized their size and shape (tip diameter ∼1–1.5 μm) so that RBCs could only partially enter into the pipette (Figure 2A and Figure 2—figure supplement 1). In the presence of 0.05% BSA, mechanical stimulation of these cells was possible without causing cell rupture by applying negative pipette pressure using a high-speed pressure clamp device (Figure 2A). This method allowed for quantitative and reproducible application of force while detecting changes in intracellular Ca²⁺. Application of negative pressure induced a rapid rise in intracellular Ca²⁺ levels in Fluo-4-loaded WT RBCs with a threshold of ∼ −5 mmHg and a P₅₀ of −9.72 ± 0.51 mmHg (Figure 2B,C and Video 1). As seen in Figure 2D, repeated applications of negative pressure induced a mild rundown in the calcium response. To account for this, we normalized all responses in Figure 2C to the average of two maximal (−25 mmHg) stimuli flanking each test pulse (Figure 2C). Following removal of a half-maximal −10 mmHg stimulus, Ca²⁺ levels declined to baseline with an average t_1/2 of 21 ± 1.8 s. This increase in intracellular Ca²⁺ concentration must be a result of influx rather than receptor-mediated store release as RBCs do not possess intracellular stores. To conclusively determine whether this Ca²⁺ influx was Piezo1-mediated, we subjected Vav1-P1cKO RBCs to similar negative pressure stimulation. We found that Ca²⁺ influx was not detectable in any Vav1-P1cKO RBCs, even at pressures up to −35 mmHg (Figure 2D). Experiments using Vav1-P1cKO RBCs were conducted using the same pipettes that previously elicited Ca²⁺ influx in WT RBCs, allowing for equivalent mechanical stimulation between genotypes. These results demonstrate that RBCs can respond to mechanical force by allowing Ca²⁺ to enter the cell and that this Ca²⁺ influx is dependent on Piezo1.

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The ability of mechanical force to cause Ca²⁺ entry through Piezo1, coupled with the observed overhydration of Piezo1-deficient RBCs, suggests an important role for Piezo1-dependent Ca²⁺ influx in regulating RBC volume following mechanical stress. To directly test the effect that Piezo1 activation could have on RBC volume, we utilized the recently identified Piezo1-selective activator Yoda1 (Syeda et al., 2015). WT or Vav1-P1cKO RBCs were loaded with the calcium-sensitive dye Fluo-4 and then treated with 15 μM Yoda1. Yoda1 caused robust increases in fluorescence of WT, but not in Vav1-P1cKO RBCs. In contrast, both WT and Vav1-P1cKO RBCs exhibited similar Ca²⁺ influx in response to the Ca²⁺ ionophore A23187 (Figure 3A).

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A rise in intracellular Ca²⁺ in response to Yoda1 would be expected to activate KCa3.1, also known as the Gardos channel, which can mediate K⁺ efflux, H₂O efflux, and RBC dehydration (Maher and Kuchel, 2003). In fact, RBCs from one strain of mice lacking KCa3.1 exhibit increased size and osmotic fragility similar to what is seen in Vav1-P1cKO mice (Grgic et al., 2009). We tested whether Yoda1 could cause RBC dehydration, and whether any possible dehydration was mediated through KCa3.1 activation by Piezo1-dependent Ca²⁺ influx. Treatment of WT RBCs, but not Vav1-P1cKO RBCs, with 15 μM Yoda1 led to a rapid change in their shape, progressing from discocytes to echinocytes to spherocytes, similar to what has been demonstrated for treatment with A23187 (Steffen et al., 2011) (Figure 3B).

We further tested the osmotic fragility of blood following treatment with Yoda1 and/or the KCa3.1 antagonist TRAM-34 (Wulff et al., 2000). Incubation of WT, but not Vav1-P1cKO blood, for 30 min with 5 μM Yoda1 caused a marked reduction in RBC osmotic fragility that was prevented by a 10-min pretreatment with 2 μM TRAM-34 (Figure 3C). Both WT and Vav1-P1cKO RBCs exhibited reduced osmotic fragility when treated for 30 min with 1 μM A23187, demonstrating normal functionality of KCa3.1 in Vav1-P1cKO RBCs (Figure 3D). The lack of any Ca²⁺ influx, shape changes, or changes in osmotic fragility of Vav1-P1cKO RBCs in response to Yoda1 further demonstrates the specificity of Yoda1 on Piezo1. Importantly, TRAM-34 did not block Piezo1 HEK293T cells transfected with Piezo1 (Figure 3—figure supplement 1). These data as a whole suggest a model shown in Figure 3E whereby activation of Piezo1 on RBCs leads to calcium influx, potassium efflux through KCa3.1 that is accompanied by water loss, resulting in RBC dehydration.

Many of the effects exerted by physical forces on cellular physiology remain unclear. Here, we have shown that these forces have a significant effect on RBC physiology by activating the mechanosensitive ion channel Piezo1. Our findings suggest a link between mechanical forces and RBC volume via Ca²⁺ influx through Piezo1. The ability of RBCs to reduce their volume in response to mechanical forces could improve their ability to traverse through small-diameter capillaries and splenic sinusoids. Additionally, it is possible that this reduction in volume could aid in oxygen/CO₂ exchange in the periphery by concentrating hemoglobin within RBCs, which may promote release of oxygen from hemoglobin; in fact, mechanical stimulation of RBCs via optical tweezers has been shown to cause such a release of oxygen (Rao et al., 2009).

It was not previously clear whether the cause of RBC dehydration in DHS patients is due to direct or indirect mechanisms. We have demonstrated that genetic deletion of Piezo1 in blood cells leads to overhydrated, fragile RBCs. We have also shown that mechanical force can cause calcium influx into RBCs that is dependent on Piezo1 expression. We cannot exclude the possibility that deletion of Piezo1 alters RBC membrane properties, resulting in decreased activity of a separate mechanosensitive ion channel rather than calcium entering the cell through Piezo1 itself. We find this unlikely given the relatively mild overhydration of Vav1-P1cKO RBCs and the complete absence of any mechanically induced calcium influx in these cells. Finally, using the first identified selective small molecule activator of Piezo1, we have shown that calcium influx through Piezo1 dehydrates RBCs via the actions of KCa3.1. These findings are consistent with the observation of dehydrated RBCs from DHS patients with gain-of-function Piezo1 mutations (Zarychanski et al., 2012; Albuisson et al., 2013; Andolfo et al., 2013; Bae et al., 2013), further supporting that the dehydration seen in DHS patients is due to increased Piezo1-mediated calcium influx in response to mechanical forces in RBCs themselves.

This work also demonstrates the power of combining both genetic and chemical approaches. Experiments performed on Piezo1-deficient background demonstrate the specificity of Yoda1; meanwhile, the acute effect of Yoda1 on RBC dehydration allows us to conclude that Piezo1 activity can regulate cell volume regulation via KCa3.1 activation and suggests that changes in RBC volume in Vav1-P1cKO mice are not a consequence of developmental compensation or non-cell autonomous mechanisms.

Piezo1 has been proposed by many to mediate a non-selective current observed in sickle cell disease (SCD) called P_Sickle, which acts upstream of KCa3.1 to mediate RBC sickling (Lew et al., 1997; Ranney, 1997; Ma et al., 2012; Demolombe et al., 2013). While KCa3.1 inhibitors can improve many hematological indices of SCD patients, they have thus far been ineffective in preventing painful vasculo-occlusive crises (Ataga et al., 2008). Generation of mice containing conditional deletion of Piezo1 in blood cells combined with full replacement of normal murine hemoglobin genes with sickling human hemoglobin genes could help determine whether Piezo1 mediates both P_Sickle and the pathology of SCD. Inhibiting Piezo1 may modulate other pathways in addition to potentially blocking KCa3.1-dependent dehydration that may have therapeutic benefits in patients with SCD.

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Author details

1. Stuart M Cahalan

   Department of Molecular and Cellular Neuroscience, Howard Hughes Medical Institute, The Scripps Research Institute, La Jolla, United States

   Contribution

   SMC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Viktor Lukacs

   Competing interests

   The authors declare that no competing interests exist.

2. Viktor Lukacs

   Department of Molecular and Cellular Neuroscience, Howard Hughes Medical Institute, The Scripps Research Institute, La Jolla, United States

   Contribution

   VL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Stuart M Cahalan

   Competing interests

   The authors declare that no competing interests exist.

3. Sanjeev S Ranade

   Department of Molecular and Cellular Neuroscience, Howard Hughes Medical Institute, The Scripps Research Institute, La Jolla, United States

   Contribution

   SSR, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Shu Chien

   1. Department of Bioengineering, University of California, San Diego, San Diego, United States
   2. Institute of Engineering in Medicine, University of California, San Diego, San Diego, United States

   Contribution

   SC, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Michael Bandell

   Genomics Institute of the Novartis Research Foundation, San Diego, United States

   Contribution

   MB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

6. Ardem Patapoutian

   Department of Molecular and Cellular Neuroscience, Howard Hughes Medical Institute, The Scripps Research Institute, La Jolla, United States

   Contribution

   AP, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   ardem@scripps.edu

   Competing interests

   The authors declare that no competing interests exist.

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