PIEZOs mediate neuronal sensing of blood pressure and the baroreceptor reflex

Blood pressure (BP) is tightly regulated to ensure that the body is prepared to meet varied daily activity demands. Mechanisms that change blood volume control long-term BP regulation. Within seconds and minutes, BP regulation is initiated primarily by baroreceptors, a class of stretch-sensitive neurons within the nodose and petrosal ganglia with peripheral projections in the walls of the aorta and carotid sinus (1, 2). An increase in BP stretches baroreceptor nerve endings to trigger afferent signals that are transmitted to the central nervous system. The consequences of baroreceptor activation are a decrease in heart rate (HR), cardiac output, and vascular resistance that counteract the initial increase in BP (1, 2). Compromised baroreceptor function predicts arrhythmias and premature death in humans with postmyocardial infarction and heart failure (3, 4).

Several ion channels (5–9) have been suggested to contribute to baroreception. However, substantial residual baroreflex is observed when these channels are ablated, implicating the involvement of other sensory systems. None of the candidate ion channels have been directly activated by mechanical stimuli in heterologous systems, which may lack accessory tethering molecules to form a mechanosensory complex. Furthermore, whether these channels are acting as sensors or play a role downstream of mechanotransduction is not clear. PIEZO1 and PIEZO2 are mechanically activated ion channels that play crucial roles in several mechanotransduction processes (10). PIEZO1 is prominently expressed in the cardiovascular system (11, 12), and PIEZO2 is abundant in various populations of sensory neurons (13–15).

We assessed Piezo1 and Piezo2 transcript expression in nodose and petrosal ganglia, where baroreceptor cell bodies are located (1). These ganglia are fused with each other and with the jugular ganglion in mice. Piezo1 and Piezo2 were highly expressed in the nodose-petrosal-jugular ganglion complex (NPJc) (Fig. 1A). Similar numbers of cells were identified that highly expressed either Piezo1 or Piezo2 exclusively (123 and 124 cells with each transcript, respectively, Fig. 1B). A small population of neurons expressed both (43 double Piezo-positive cells, or 14.8% of all Piezo-expressing cells, n = 6 mice, Fig. 1B).

To test whether Piezo1 and Piezo2 are expressed in baroreceptors, we performed retrograde labeling of carotid sensory neurons. We injected fluorescent cholera toxin B (CTB) (16) into the carotid sinus beneath the serosal vessel covering. All CTB-positive neurons detected in the NPJc from eight mice were quantified for the presence of Piezo1 or Piezo2 transcript (Fig. 1, C to F). Six out of 95 retrogradely labeled cells were Piezo1-positive, and eight were Piezo2-positive (Fig. 1B). Piezo-negative cells were likely chemoreceptors, which abundantly innervate the carotid sinus but do not require mechanosensitivity. None of the 95 CTB-labeled cells were double Piezo-positive. These data suggest that a subset of neurons that innervate the carotid sinus (which include mechanoreceptors and chemoreceptors) express either Piezo1 or Piezo2 (Fig. 1G). We hypothesized that these cells could function as baroreceptors.

We therefore crossed Piezo floxed mice to the Phox2bCre line, which express Cre recombinase in epibranchial placode-derived ganglia (e.g., nodose and petrosal) but not in neural crest–derived ganglia (jugular, trigeminal, and dorsal root) (17). We first analyzed the baroreflex in anesthetized mice in response to phenylephrine (PE). PE induces rapid vasoconstriction (6), which elevates BP. Increased BP then triggers baroreceptor activity and induces a reflex decrease in HR. PE-induced baroreflex changes were compared in conditional double-knockout mice (dKO; Phox2bCre⁺;Piezo1^f/fPiezo2^f/f) and Cre-negative wild-type littermates (WT). Infusion of PE into the jugular vein produced a dose-dependent and transient increase in systolic BP and a consequent decrease in HR, reflecting baroreflex control (6) (Fig. 2A). The PE-induced HR reduction [−29 ± 20 versus −234 ± 24 beats per minute (bpm), P < 0.001] and decreased baroreflex sensitivity (−0.6 ± 0.4 versus −5.0 ± 0.5 Δbpm/ΔmmHg, P < 0.001) were essentially abolished in the dKO mice (Fig. 2, A to D). PE-induced systolic BP increase in dKO mice was significantly higher than in WT littermates (55.7 ± 3 versus 45.7 ± 6 mmHg, P <0.05) (Fig. 2, A and B). HR response to sodium nitroprusside–induced acute baroreceptor unloading was also absent in dKO mice (fig. S1, A to C). By contrast, Phox2bCre⁺;Piezo1^f/f (P1^cKO) and Phox2bCre⁺;Piezo2^f/f (P2^cKO) single-knockout mice showed no difference in PE-induced change of baroreflex compared with WT littermates (Fig. 2, B to D). We focused remaining analyses primarily on dKO mice.

We next measured aortic depressor nerve (ADN) activity during a rise of BP induced by PE. We observed a lack of drug-induced ADN activity in dKO mice compared to WT mice (−131.9 ± 163.9 versus 5558 ± 1234 normalized area under curve of integrated ADN activity, P < 0.001; Fig. 2, E to G). The dKO mice had no appreciable responses during both phasic and tonic phases of PE-induced ADN activity (fig. S1, D and E). This is not due to gross anatomical deficits, because we observed comparable baroreceptor ending densities within the aortic arch of dKO and WT mice (fig. S2).

Impaired baroreceptor function leads to dysregulation of BP, including volatile hypertension and increased BP variability in humans (18–20). We examined daily BP variability in freely moving, conscious mice using a telemetric sensor (6). The dKO mice showed significantly increased mean arterial pressure (MAP) during their active time (gray shading, 6 p.m. to 6 a.m.) compared with WT littermates (112 ± 0.4 versus 95 ± 0.5 mmHg, P < 0.001) (Fig. 3, A and B, and figs. S3, A and B, and S4). The HR of dKO mice was slightly increased during active times compared with that of WT mice (583 ± 3 versus 566 ± 3 bpm, P < 0.001), whereas the HR remained unchanged during inactive times (6 a.m. to 6 p.m., 532 ± 3 versus 536 ± 3 bpm, not significant) (Fig. 3B). No difference in locomotor activity was observed between dKO and WT mice (fig. S3C), ruling out the possibility that activity caused the increased BP and HR in dKO mice.

We scanned telemetry data for spontaneous changes in systolic BP and pulse interval (PI) consistent with a baroreflex relationship. This method (sequence technique) is used to noninvasively assess baroreflex function (21, 22). The spontaneous baroreflex sensitivity (sBRS) is defined as the slope of changes in systolic BP versus PI from 1 hour of recording. sBRS was severely reduced in dKO mice (2.0 ± 0.1 versus 3.8 ± 0.2 ms/mmHg for WT, P < 0.001, Fig. 3C). Sinoaortic baroreceptor denervated mice also show residual sBRS (22), and this may be due to compensation from other sensory systems.

We compared the BP variability of WT and dKO mice. The systolic BP values of dKO mice were distributed in a broader range than those of WT littermates (Fig. 3D). Variability was greatly enhanced in dKO mice (7.9 ± 0.3 versus 6.1 ± 0.3 mmHg in WT, P < 0.001, Fig. 3E). We quantified the range of BP variability of mean, systolic, and diastolic BP within each group in a 72-hour period. Maximum values of BP from dKO mice were significantly higher than those from WT littermates, whereas minimum values were significantly lower (Fig. 3F). Lastly, homovanillic acid concentrations in dKO mouse urine were significantly higher than those in WT urine (13.9 ± 0.05 versus 12.1 ± 0.06 μg/ml, fig. S3D), suggesting an increase in hormone norepinephrine concentration, as in human baroreflex failure patients (18). There were no significant BP variability and sBRS differences in P1^cKO (fig. S5) and P2^cKO (fig. S6) single-knockout mice compared with WT littermates. P2^cKO mice showed a subtle hypotensive BP distribution (fig. S6).

We next investigated whether stimulating Piezo2-positive neurons can induce the baroreflex in adult mice. We crossed Piezo2^GFP-IRES-Cre (Piezo2Cre) knockin mice with Cre-dependent channelrhodopsin-2 (ChR2) reporter mice to generate Piezo2Cre⁺;ChR2-eYFP mice (13) and recorded the cardiovascular response to activating different regions of Piezo2-positive vagal sensory nerves by optogenetics (Fig. 4A; eYFP, enhanced yellow fluorescent protein gene). We did not observe cardiovascular changes during long optogenetic stimulation (5-ms pulses, 50 Hz, 10 s) of the vagal nerve trunk (area 1 in Fig. 4A; BP, −5.3 ± 1.0%, and HR, −2.0 ± 1.0%; not significant) (Fig. 4, A to C). Next, we focused on specifically activating baroreceptor afferents. For aortic baroreceptors, we stimulated the superior laryngeal nerve branch, which carries afferent inputs from the ADN (area 2 in Fig. 4A). For carotid baroreceptors, we exposed the carotid sinus region and directly stimulated the local nerve terminals (area 3 in Fig. 4A). Light stimulations at both locations caused an immediate decrease in both BP and HR (area 2: BP, −55.6 ± 2.0%, and HR, −50.5 ± 2.0%; area 3: BP, −37.5 ± 3.5%, and HR, −32.3 ± 3.7%; P <0.001) compared with the unstimulated baseline (Fig. 4, B and C). A prominent consequence of baroreceptor activation is rapid inhibition of efferent sympathetic activity (1). We found that light-induced decrease in HR was markedly attenuated after administration of the β-adrenergic receptor–blocker propranolol, indicating that the reflex bradycardia was mediated primarily by inhibition of cardiac sympathetic nerve activity (fig. S7). Piezo2Cre⁻;ChR2-eYFP mice (WT) did not show any changes during optogenetic stimulation in all three regions (BP, −0.6 ± 0.6%, and HR, 0.4 ± 0.8%; not significant; Fig. 4, B and C).

This study demonstrates that the mechanically activated ion channels PIEZO1 and PIEZO2 are together required for arterial baroreceptor activity and function. Baroreflex is critical to maintain short-term BP homeostasis in mammals. The long-term changes observed in HR and BP that accompany baroreflex failure are complex. Acute elimination of baroreceptor function (e.g., sino-aortic denervation) causes immediate, large increases in BP and HR (23, 24). Over time, the mean BP decreases but remains labile hypertensive, and BP variability is markedly increased and persists (18–20, 24, 25). We observed a significant increase in MAP during the active period of the Piezo dKO mice that falls just under the designation for hypertension (26), and dKO mice also developed increased blood pressure variability. These data show that losing PIEZO1 and PIEZO2 function recapitulates the phenotype observed in animal models (24, 25) and humans with baroreflex failure (18–20). However, we cannot exclude the possibility that sensory mechanisms beyond the baroreceptors within the vagus contribute to the observed increased blood pressure.