Piezo channels in stretch effects on developing human airway smooth muscle

Abstract

The use of respiratory support strategies such as continuous positive airway pressure in premature infants can substantially stretch highly compliant perinatal airways, leading to airway hyperreactivity and remodeling in the long term. The mechanisms by which stretch detrimentally affects the airway are unknown. Airway smooth muscle cells play a critical role in contractility and remodeling. Using 18-22-wk gestation human fetal airway smooth muscle (fASM) as an in vitro model, we tested the hypothesis that mechanosensitive Piezo (PZ) channels contribute to stretch effects. We found that PZ1 and PZ2 channels are expressed in the smooth muscle of developing airways and that their expression is influenced by stretch. PZ activation via agonist Yoda1 or stretch results in significant [Ca²⁺]_i responses as well as increased extracellular matrix production. These data suggest that functional PZ channels may play a role in detrimental stretch-induced airway changes in the context of prematurity.NEW & NOTEWORTHY Piezo channels were first described just over a decade ago and their function in the lung is largely unknown. We found that piezo channels are present and functional in the developing airway and contribute to intracellular calcium responses and extracellular matrix remodeling in the setting of stretch. This may improve our understanding of the mechanisms behind development of chronic airway diseases, such as asthma, in former preterm infants exposed to respiratory support, such as continuous positive airway pressure (CPAP).

Keywords: asthma; contractility; extracellular matrix; lung; preterm birth.

Graphical abstract

Figure 1.

In vivo expression of Piezo1 and Piezo2 in human fetal and neonatal lung tissue. Representative IHC staining for ASM cells positive for piezo1 (PZ1, A) and piezo2 (PZ2, B). Magenta staining with yellow arrows presented in control vs. case groups. Control samples were from patients with no history of exposure to mechanical ventilation before demise (intrauterine fetal demise, stillbirth, preterm birth without intervention, and sudden infant death syndrome), whereas case samples were from patients with a history of preterm birth and exposure to respiratory support via mechanical ventilation or CPAP. Patient ages ranged from 20 wk gestational age (GA) to 5 mo postnatal. Quantification of positive staining is presented as percentage in ASM bundles in the control (n = 8) vs. case (n = 8) groups. Data are expressed as means ± SE. *P < 0.01. ASM, airway smooth muscle; CPAP, continuous positive airway pressure; IHC, immunohistochemistry.

Figure 2.

Expression of Piezo1 and Piezo2 in fetal vs. adult human airway smooth muscle cells. A and B: Western blot was performed to determine the relative expression of PZ1 and PZ2 in fASM vs. adult human airway smooth muscle cells (HASM). Both PZ1 and PZ2 were present in fASM and HASM, but protein expression was significantly lower for both in HASM (n = 7) compared with fASM (n = 9, 6 female, 3 male). C: the difference in PZ1 and PZ2 expression between fASM and HASM had similar ratios (i.e., there was not a differential drop-off in expression of one channel vs. the other observed with age). Data are expressed as means ± SE. *P < 0.05. fASM, fetal airway smooth muscle; Rel, relative.

Figure 3.

mRNA expression of piezo channels is impacted by stretch. fASM cells grown on Flexcell plates were exposed to either 12 h of 5% oscillatory stretch (control) or 5% oscillatory stretch superimposed on 5% static stretch (CPAP). Additional cells were treated with 10 µM Yoda1 or 1 µM GsMTx4 before exposure to stretch. Cells were harvested for PCR analysis via standard techniques. A and B: static (CPAP) stretch increased both PZ1 and PZ2 mRNA expression, whereas PZ2 inhibitor GsMTx4 blunted these effects. Administration of Yoda1 did not change PZ mRNA expression (n = 6 or 7, 4 female, 3 male). Data are expressed as means ± SE. *Difference from oscillatory control, P < 0.05. #Difference from CPAP stretch, P < 0.05. CPAP, continuous positive airway pressure; fASM, fetal airway smooth muscle; Osc., oscillatory; PCR, polymerase chain reaction; PZ1, Piezo1, PZ2, Piezo2.

Figure 4.

Piezo channels modulate [Ca²⁺]_i responses to shear stress and histamine in fASM. A and B: fASM cells were loaded with fura-2 before exposure to rapid perfusion (5 mL/min) with Hanks balanced salt solution (HBSS) to induce membrane stretch. Rapid perfusion resulted in [Ca²⁺]_I response, suggesting functional PZ channels. The [Ca²⁺]_i amplitude response to rapid perfusion was increased with PZ1 agonist Yoda1 (0.625 µM) and blunted by pretreatment with PZ1 inhibitor GsMTx4 (2.5 µM) (n = 5, 3 female, 2 male). *Significant difference from slow perfusion control. #Significant difference from vehicle with rapid perfusion. Data are expressed as means ± SE, P < 0.05. C and D: fASM cells were loaded with fura-2 before exposure to slow or rapid perfusion with Hanks balanced salt solution (HBSS). After baseline was established, cells were treated with 10 µM histamine. [Ca²⁺]_i responses to histamine were enhanced if preceded by rapid perfusion. Pretreatment with PZ1 inhibitor GsMTx4 (2.5 µM) blunted this effect. Pretreatment with PZ1 agonist Yoda1 had no significant effect on histamine response with rapid perfusion (n = 4, 2 female, 2 male). Data are expressed as means ± SE. *Significant difference from histamine with slow perfusion group, #significant difference from histamine with rapid perfusion group, P < 0.05. fASM, fetal airway smooth muscle; PZ, Piezo.

Figure 5.

Yoda1 and GsMtX4 modulate [Ca²⁺]_i responses in fASM. A: in fura-2-loaded fASM cells, perfusion with 1.25–5 µM Yoda1 resulted in dose-dependent increases in [Ca²⁺]_i. This response was similar to the magnitude of response seen with agonist histamine, though the duration of response with Yoda1 was much more prolonged. B: pretreatment with 2.5 µM GsMtX4 blunted [Ca²⁺]_i response to rapid perfusion and Yoda1 (n = 7, 4 female, 3 male). fASM, fetal airway smooth muscle.

Figure 6.

Yoda1 increases fASM ECM deposition. A–D: ECM deposition was assessed via a modified in-cell Western technique. Exposure of fASM cells to 10 µM Yoda1 for 72 h significantly increased deposition of collagen I and collagen III. Fibronectin deposition was decreased with Yoda1 exposure. Pretreatment with GsMTx4 had no impact on baseline ECM deposition and did not mitigate the increased ECM deposition seen with Yoda1 exposure. D-GsMtX4 (PZ2 antagonist) did significantly decrease the increase in collagen 1 expression induced by Yoda1 (n = 9, 6 female, 3 male). Data are presented as means ± SE. *Significant difference from control, #Significant difference from Yoda1. P < 0.05. ECM, extracellular matrix; fASM, fetal airway smooth muscle; PZ2, Piezo2.

Figure 7.

Static stretch increases ECM expression in fASM cells. A–D: fASM cells were grown on Flexcell plates and pretreated with 10 µM Yoda1 or 1 µM GsMTx4 before 48 h of 5% oscillatory stretch (control) vs. 5% oscillatory stretch superimposed on 5% static stretch (CPAP). Collagen I, III, and fibronectin expression were increased with static/CPAP stretch. GsMTx4 pretreatment prevented the observed increase in collagen III and fibronectin expression (n = 6, 3 female, 3 male). Data are presented as means ± SE. *Significant difference from control, #Significant difference from CPAP/vehicle, P < 0.05. CPAP, continuous positive airway pressure; ECM, extracellular matrix; fASM, fetal airway smooth muscle; Osc., oscillatory.

Figure 8.

Model of Piezo channels in developing airway. Static stretch of CPAP superimposed on oscillatory breathing is important in more compliant airways of premature infants. In developing ASM, stretch activates mechanosensitive Piezo (PZ) channels that promote [Ca²⁺]_i (toward contractility) and remodeling: effects that can be blunted by PZ inhibitors. ASM, airway smooth muscle; CPAP, continuous positive airway pressure. [Image was prepared using ScienceSlides from VisiScience.]