Piezo1 channel activation facilitates baroreflex afferent neurotransmission with subsequent blood pressure reduction in control and hypertension rats

Abstract

Mechanosensitive cation channels such as Piezo1 and Piezo2 are activated by mechanical force like a starched wall of the aorta while blood pressure (BP) rising, which helps to elucidate the underlying mechanism of mechanotransduction of baroreceptor endings. In this study we investigated how Piezo1 channel activation-mediated gender- and afferent-specific BP regulation in rats. We established high-fat diet and fructose drink-induced hypertension model rats (HFD-HTN) and deoxycorticosterone (DOCA)-sensitive hypertension model rats. We showed that the expression levels of Piezo1 and Piezo2 were significantly up-regulated in left ventricle of HFD and DOCA hypertensive rats, whereas the down-regulation of Piezo1 was likely to be compensated by Piezo2 up-regulation in the aorta. Likewise, down-regulated Piezo1 was observed in the nodose ganglion (NG), while up-regulated Piezo2 was found in the nucleus tractus solitarius (NTS), which might synergistically reduce the excitatory neurotransmitter release from the presynaptic membrane. Notably, microinjection of Yoda1 (0.025-2.5 mg/ml) into the NG concentration-dependently reduced BP in both hypertensive rat models as well as in control rats with similar EC₅₀; the effect of Yoda1 was abolished by microinjection of a Piezo1 antagonist GsMTx4 (1.0 μM). Functional analysis in an in vitro aortic arch preparation showed that instantaneous firing frequency of single Ah-fiber of aortic depressor nerve was dramatically increased by Yoda1 (0.03-1.0 μM) and blocked by GsMTx4 (1.0 μM). Moreover, spontaneous synaptic currents recorded from identified 2nd-order Ah-type baroreceptive neurons in the NTS was also facilitated over 100% by Yoda1 (1.0 μM) and completely blocked by GsMTx4 (3.0 μM). These results demonstrate that Piezo1 expressed on Ah-type baroreceptor and baroreceptive neurons in the NG and NTS plays a key role in a sexual-dimorphic BP regulation under physiological and hypertensive condition through facilitation of baroreflex afferent neurotransmission, which is presumably collaborated by Piezo2 expression at different level of baroreflex afferent pathway via compensatory and synergistic mechanisms.

Keywords: Yoda1/GsMTx4; aortic depressor nerve; baroreflex afferent pathway; blood pressure; mechanosensation; piezo channels.

Fig. 1. Changes of BP and cardiac structure, Piezos mRNA and protein in HFD-HTN model rats.

a Systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean blood pressure (MBP) difference are shown in control and HFD-HTN model of male and age-matched female rats, n = 8 rats/group. b, c SAX and PSLAX-M type cardiac ultrasound results of HFD-HTN and control rats; the scale bar in the figure is 2.5 mm. The diastolic left intraventricular diameter (LVIDd, mm) and cardiac function was calculated of HFD-HTN vs. control (n = 10). The ratio of heart weight to body weight (HW/BW index, %) of HFD-HTN vs. control (n = 6). d, e Expression profiles of mRNA and protein for Piezo1 and Piezo2 in the left ventricle and aorta of control and HFD-HTN model groups, n = 6. Summarized results were presented by mean ± SD, *P < 0.05 or **P < 0.01 vs. control (Ctrl).

Fig. 2. Immunofluorescence and tissue distribution of Piezo channels in the NG and NTS.

a Fluorescent distribution of Piezo1 and Piezo2 protein (green) in the tissue sections (7 μm) of NG, DAPI (blue) and HCN1 (red) were also applied for staining nuclei and myelinated (orange arrowhead) neurons vs. unmyelinated neurons (white arrowhead) of adult male and age-matched female rats. b, c Expression profiles of mRNA and protein for Piezo1 and Piezo2 in the NG of control and HFD-HTN model groups (male), n = 6. d, e Results of PCR and WB mean expression of Piezo1 and Piezo2 in the NTS of control and HFD-HTN model groups (male), n = 6. Averaged data were presented by mean ± SD, *P < 0.05 or **P < 0.01 vs. control (Ctrl).

Fig. 3. Effects of Yoda1 by microinjected into the NG on BP in HFD-HTN and control rats.

a The reduction of BP after microinjection of Yoda1 (0.025, 0.075, 0.25, 0.75 mg/ml) in male control (M-Ctrl) and male HFD-HTN (M-HFD) rats, respectively. The scale bars applied to all recordings. b, c The summarized data of ΔMAP and recovery time after injection of different concentrations of Yoda1 in control and HFD-HTN rats. d, e ΔMAP reduction was monitored in the presence of Yoda1 alone and GsMTx4 on top of Yoda1, and summarized data of ΔMAP after treatments. Averaged data were expressed by mean ± SD, n = 6. **P < 0.01 vs. solvent; ^##P < 0.01 vs. Yoda1.

Fig. 4. Piezo1 activation significantly facilitates discharge of Ah-type fibers of aortic depressor nerve.

a Single-fiber discharge was recorded in the presence of Yoda1 (0.03–1.0 μM) along with 100 nM capsaicin (Cap) and sodium nitroprusside (NP) to block vascular effect and unmyelinated C-type discharge activity that was quantified as averaged power as the function of computer-controlled ramped pressure. b The discharge profile of myelinated Ah-fiber activated by Yoda1 (1.0 μM) was partially blocked by Piezo1 channel antagonist (GsMTx4, 3.0 μM). Averaged data were presented by mean ± SD from four preparations (n = 4).

Fig. 5. Piezo1 activation significantly facilitates baroreflex afferent neurotransmission.

The 2nd-order Ah-type baroreceptive neurons were verified by electrophysiological property (delay excitation) conjugated with fluorescence (cardiac modality) and chemosensitivity (negative to capsaicin) in the NTS. Spontaneous synaptic currents (miniature) of myelinated Ah-type baroreceptive neurons were recorded from two separate neurons. a, a1 Representative recordings of miniatures (T_1-5) from two different neurons before Yoda1 as the control (Ctrl); b, b1 Representative recordings of miniatures (T_1-5) from two different neurons after 1.0 μM Yoda1; c, c1 Representative recordings of miniatures (T_1-5) from two different neurons after 3 μM GsMTx4 on top of Yoda1; the scale bars as shown in (c1) applied for all other recordings.

Fig. 6. Schematic illustration: Piezo1 channel activation facilitates baroreflex (BRx) afferent neurotransmission and plays a fundamental role in blood pressure (BP) homeostasis and hypotensive action in hypertension rats.

(1) Down-regulated Piezo1 of aorta and nodose ganglia (NG) is compensated by up-regulated Piezo2 leading to baroreceptor (BR) resetting under hypertensive conditions; (2) NG microinjection of Piezo1 agonist Yoda1 directly mediates BP reduction in control and hypertension rats; (3) Instant firing frequency (IFF) of single Ah-fiber discharge of aortic depressor nerve (ADN) and spontaneous synaptic currents (SSCs) of Ah-baroreceptive neurons of nucleus tractus solitary (NTS) are significantly increased in the presence of Yoda1, which is a great contributor in sexual-dimorphic baroreflex afferent function; (4) Up-regulated microRNA (miR)-101-3p under hypertensive condition down-regulates Piezo1 expression at post-transcriptional level.