Piezo1 Is a Mechanosensor Channel in Central Nervous System Capillaries

Abstract

Capillaries are equipped to sense neurovascular coupling agents released onto the outer wall of a capillary, translating these external signals into electrical/Ca²⁺ changes that play a crucial role in blood flow regulation and ensuring that neuronal demands are met. However, control mechanisms attributable to forces imposed onto the lumen are less clear. Here, we show that Piezo1 channels act as mechanosensors in central nervous system capillaries. Electrophysiological analyses confirmed expression and function of Piezo1 channels in brain cortical and retinal capillaries. Activation of Piezo1 channels evoked currents that were sensitive to endothelial cell-specific Piezo1 deletion. Using genetically encoded Ca²⁺ indicator mice and an ex vivo pressurized retina preparation, we found that activation of Piezo1 channels by mechanical forces triggered Ca²⁺ signals in capillary endothelial cells. Collectively, these findings indicate that Piezo1 channels are capillary mechanosensors that initiate crucial Ca²⁺ signals and could, therefore, have a profound impact on central nervous system blood flow control.

Keywords: Piezo1; calcium signaling; capillaries; central nervous system; endothelial cells; neurovascular coupling.

Fig. 1:. Changes in pressure/flow evoke endothelial Ca²⁺ transients in the retinal vasculature.

(a) Schematic illustration of the pressurized retina vasculature preparation. Top: Diagram showing the eyes and their circuit connection to the visual cortex of the mouse. Bottom: After first isolating eyes from euthanized mice, the intact retina is obtained without damaging the ophthalmic artery, then the ophthalmic artery is cannulated and the retinal cup is cut open into four retinal petals and pinned to the recording stage. Right: A gravity column is used to control pressure at the ophthalmic artery, and a transducer is used to monitor pressure changes. (b) Representative images of the entire en face ex vivo retinal preparation from a Cdh5-GCaMP8 mouse. Pressure at the ophthalmic artery was elevated from 20 to 80 to 20 mmHg, and Ca²⁺ transients were monitored. Inset: Venules (V), capillaries (C), and arterioles (A) could be characterized based on their angioarchitecture and vessel diameter. Representative images (each from a 30-s recording) depict the median response during a pressure step. (c) Traces of line scans showing Ca²⁺ transients in arterioles, capillaries, and venules in response to elevating intravascular pressure at the cannulated ophthalmic artery from 20 to 80 mmHg (n = 6 retina preparations from 6 mice). (d) Scatter plots of changes in fluorescence in arterioles, capillaries, and venules in response to pressure elevation. Data are presented as means (bold dotted horizontal lines) ± SEM (error bars) (*P < 0.05, ns indicates not significant, Kruskal-Wallis test followed by Dunn’s multiple comparisons test; arterioles, n = 17 ROIs/6 retinas/6 mice; capillaries, n = 20 ROIs/6 retinas/6 mice; venules, n = 19 ROIs/6 retinas/6 mice). (e) Left: Averaged fluorescence from capillary ROIs (yellow; n = 3 ROIs) and non-vascular background (gray; n = 3 ROIs) from the same retina preparation in response to the depicted pressure changes (20→40→60→80→20 mmHg). Solid lines are means and dotted lines are SEM. Right: Fluorescence changes in capillary segments (yellow) and neighboring non-capillary background ROIs (gray). Data were obtained from 3 retina preparations from 3 Cdh5-GCaMP8 mice.

Fig. 2:. Mechanical stimulation evokes a current with an intermediate conductance in CNS capillary ECs.

(a) Experimental protocol for the isolation of retinal cECs. Retinal cups were dissected from isolated eyeballs, homogenized, and then filtered. Retained capillaries were eluted into enzymatic solution for digestion, then washed and triturated (see Methods). Right box: Cell-attached approach for electrophysiology in cECs. A micropipette with slight positive pressure was advanced until it touched a cEC, after which suction was applied to attain a giga-ohm seal. Mechanical stimulation of the attached patch was achieved by delivering calibrated negative pressure. (b) Representative traces measured from a retinal cEC bathed in a 140-mM K⁺ solution using the cell-attached mode and a pipette filled with a 3-mM K⁺ solution. The patch was voltage-clamped at −50 mV, and negative pressure (0 to −40 mmHg) was gradually applied to the patch pipette. (c) Representative traces and amplitude histograms (right) from a retinal cEC without negative pressure (0 mmHg, top), followed by application of negative pressure (−10 mmHg, middle), and then no pressure (bottom). The pipette solution was supplemented with 1 μM RR and 100 μM Ba²⁺. Amplitude histograms and open probabilities (NP_O) reflect pressure-evoked changes. C: closed, O: open. Representative traces (from ≥5-min recordings) depict median responses to pressure.

Fig. 3:. Retinal cECs express functional Piezo1 channels.

(a, b) Representative traces and averaged NP_O values for single-channel openings obtained in the cell-attached mode from retinal cECs in the absence (control) or presence of Yoda1 (5 μM) in the 3 mM K⁺ pipette solution. GSK219 (1 μM; TRPV4 blocker) and Ba²⁺ (100 μM) were included in the pipette solution. Amplitude histogram demonstrating the unitary Yoda1-evoked current. In a third group, 30 μM Gd³⁺ was added to the pipette and bath solutions. Each point in the scatter plot was obtained from a single cEC (number of cECs is indicated in the figure). (c) Unitary current-voltage relationship plotted using averaged unitary currents measured at the following voltages (mV): −30 (n = 6 cECs), −50 (n = 7), −70 (n = 7), −90 (n = 7), and −110 (n = 6). Conductance obtained from the slope was 22.7 pS.

Fig. 4:. Brain cortical cECs express functional Piezo1 channels.

(a) Cell-attached recordings at a holding membrane potential of −50 mV. Brain cECs were bathed in a 140-mM K⁺ solution, and the pipette was filled with a 6-mM K⁺ solution containing RR (1 μM) and Ba²⁺ (100 μM). (b) Currents with Yoda1 (5 μM) included in the pipette. (c, d) Traces and summary data showing the open probability (NP₀) of Yoda1-induced currents. Yoda1 (5 μM) was included in the pipette solution in the absence (control; n = 9) or presence of Gd³⁺ (30 μM; n = 6) or GsMTx-4 (2.5 μM; n = 9). *P ≤ 0.05 and **P ≤ 0.01 (Kruskal-Wallis test followed by Dunn’s multiple comparisons test). (e) Traces and plot of unitary current-voltage relationship, used to estimate the single-channel conductance. Currents were recorded at −30 mV (n = 6 cECs), −50 (n = 10), −70 (n = 9), −90 (n = 7), and −110 (n = 4). (f) Representative current traces from a patch exposed to Yoda1 and held at −50 mV. Pressure was stepped from 0 mmHg to −10 mmHg by applying suction via the patch pipette and held for 30 seconds before returning to 0 mmHg.

Fig. 5:. Genetic deletion and RR abolish Piezo1 activity.

(a, b) Representative traces from a cEC from an EC-specific Piezo1-knockout (Piezo1^EC-KO) mouse (a) and a cEC from a control mouse (b). Recordings were obtained in the cell-attached mode at a holding potential of −50 mV before (0 mmHg) and after the application of negative pressure (−10 mmHg) onto the patch. (c) Scatter plots of the open probability (NP_O) of pressure-induced Piezo1-like single-channel openings in Piezo1^EC-KO and control cECs (**P < 0.01, Wilcoxon test; Piezo1^EC-KO, n = 6 cECs/4 mice; Control, n = 9 cECs/3 mice). (d) Scatter plots of the open probability of Yoda1-induced channel openings in Piezo1^EC-KO and control cECs (**P < 0.01, Mann-Whitney test; Piezo1^EC-KO, n = 12 cECs/4 mice; Control, n = 12 cECs/4 mice). ns indicates not significant. Horizontal black lines depict means and error bars are SEM. (e) Representative traces of cell-attached, Yoda1-induced, single-channel inward currents in the presence of 1, 10 or 30 μM RR, recorded at a holding potential of −50 mV. Right insets: Magnified channel openings of the marked segments. Traces are representative of 7 cECs (1 μM RR), 6 cECs (10 μM), and 4 cECs (30 μM). (f) Open-time histograms in the presence of 1 or 5 μM RR in the pipette solution. Lines show a single exponential fit to a Marquardt algorithm, yielding mean open times of 8.9 ms (1 μM) and 3.3 ms (5 μM). (g) Concentration-dependent decrease in open time and estimated IC₅₀ (n = 4, 10, 12, 3, 1 cECs at 0, 1, 5, 10, 30 μM RR, respectively). (h) Reciprocal of mean open time (1/Ƭ_O) plotted against the external [RR].

Fig. 6:. The purported mechanosensors, TRPV4 and G_qPCR, are dispensable for Piezo1 channel activation in brain cECs.

(a) Brain cECs express the Piezo1 channel, TRPV4 channel, and different G_qPCRs. (b) Representative traces illustrating TRPV4 and Piezo1 currents in a brain cEC bathed in a 140-mM K⁺ solution, recorded in cell-attached mode with a pipette filled with a 3-mM K⁺ solution supplemented with Yoda1 (5 μM), GSK101 (3 μM, TRPV4 activator), and Ba²⁺ (100 μM). (c) Representative trace obtained using similar experimental conditions as in b in a different cEC showing overlapping openings of different unitary currents. Blue: Piezo1 channel openings and levels of openings. Orange: a TRPV4 channel opening on top of an open Piezo1 channel. (d) Scatter plots and averaged open probability (NP₀) of Piezo1 channel openings in brain cECs from a) a C57BL/6J mouse in the absence (n = 8) and presence (n = 9) of Yoda1 in the pipette; b) a TRPV4-KO mouse in the presence of Yoda1 alone (n = 7) or together with GSK101 (n = 7); and c) a brain endothelial Gα_q/11-KO (be-Gα_q/11-KO) mouse in the presence of Yoda1 (n = 10 cECs). (e) Unitary currents (scattered data points) and calculated single-channel conductances (text next to data points; means ± SEM) under different conditions. GSK101-induced currents were observed only in C57BL/6J cECs treated with GSK101.

Figure 7.. Mechanically induced endothelial Ca²⁺ transients are driven by Piezo1.

(a, b) Representative micrographs of a retinal preparation from a Cdh5-GCaMP8 mouse, perfused with a solution supplemented with 30 μM Yoda1, showing Ca²⁺ transients at 20 mmHg (left) and after elevating intravascular pressure to 80 mmHg. (a) Whole retina preparation; (b) magnification of boxed region in a. (c) Representative micrographs of endothelial Ca²⁺ responses to a pressure change sequence (20→80→20 mmHg) in capillaries under control conditions and in the presence of Yoda1 (30 μM) or Yoda1+RR (10 μM). (d) Individual capillary line scans from ROIs of the micrographs shown in c. Up and down-arrows represent pressure elevation (20→80 mmHg) and reduction (80→20 mmHg), respectively. Bold lines are averaged scans from the experiment in panel c. (e) Averaged GCaMP8 fluorescence under control conditions (black) and in the presence of Yoda1 alone (blue) or Yoda1+RR (10 μM, red) (n = 3 retinal preparations from 3 Cdh5-GCaMP8 mice). (f) Scatter plots and averaged peak changes in GCaMP8 fluorescence (ΔF/F₀) in response to pressure elevation from 20 to 80 mmHg under the three conditions in c (Friedman test followed by Dunn’s multiple comparisons test; n = 10 ROIs/3 retinas/3 mice). (g, h) Focal application of Yoda1 (30 μM; 3 s, 1 psi) onto retinal capillaries in a pressurized (40 mmHg; g) or unpressurized (0 mmHg; h) ex vivo retina preparation. TRITC-dextran was added to the Yoda1 solution for visualization and confirmation of ejection. (i) Ca²⁺ transients in different capillaries segments under different conditions. Left: Application of Yoda1 onto capillaries of a pressurized retina (baseline: 10 s; ejection: 3 s; post-ejection: 27 s). Right top: Application of Yoda1 onto unpressurized retinal capillaries. Right bottom: Application of aCSF onto pressurized retinal capillaries. Each color represents an independent Cdh5-GCaMP8 preparation. (j) Averaged scatter plots of fold-change in fluorescence under the conditions shown in i. (k) Capillary EC Ca²⁺ responses to a pressure-change sequence (20→80→20 mmHg) under control conditions and with application of GSK219 (1 μM) alone or together with 1 or 10 μM RR. (l) Averaged GCaMP8 fluorescence under control conditions (black) and in the presence of GSK219 (1 μM) alone (yellow) or GSK219+10 μM RR (red) (n = 3 retina preparations from 3 Cdh5-GCaMP8 mice). (m) Scatter plots and averaged peak changes in GCaMP8 fluorescence in response to pressure elevation from 20 to 80 mmHg under the three conditions shown in l (Friedman test followed by Dunn’s multiple comparisons test; n = 9 ROIs/3 retinas/3 mice).