Piezo1 overexpression in the uterus contributes to myometrium contraction and inflammation-associated preterm birth

Abstract

Background: Preterm birth, a leading cause of perinatal mortality and morbidity, is often associated with inflammation and aberrant myometrial contractions. This study investigates the role of Piezo1, a mechanosensitive ion channel, in myometrium contraction and inflammation-associated preterm birth.

Methods: We employed Western blotting, Immunofluorescence, and Quantitative real-time PCR techniques to examine Piezo1 expression in uterine tissues. Functional assays, including myometrial contractility studies and cell contraction assays, were conducted to elucidate the effects of Piezo1 on myometrial contractions. Piezo1 inhibitors and gene knockdown techniques were used to investigate the impact of Piezo1 on inflammation-associated preterm birth, complemented by inflammatory cytokine profiling and calcium imaging to investigate the mechanism.

Results: Our findings reveal that Piezo1 is the predominant mechanosensitive channel in mouse myometrium tissue and mouse primary uterine smooth muscle (pUSMCs), with increased expression during mouse and human pregnancy. Following lipopolysaccharide (LPS) intrauterine injection, Piezo1 mRNA and protein levels were elevated in the mouse uterine smooth muscle layer. Direct pharmacologic activation of Piezo1 by Yoda1 increased the contraction of pUSMCs and shortened the pregnancy duration. In contrast, inhibition with Gsmtx4 or siRNA knockdown of Piezo1 attenuated LPS-induced pUSMCs contraction and spontaneous uterine myometrium contraction. Additionally, blocking or knocking down Piezo1 prolonged the pregnancy in an LPS-induced preterm birth model. Yoda1 stimulation increased intracellular Ca²⁺ levels in pUSMCs, while Gsmtx4 reduced these levels. Gsmtx4 decreased cox-2 expression and inflammation factors in LPS-stimulated pUSMCs.

Conclusions: These results suggest that Piezo1 acts as a critical regulator of uterine function, and its overexpression may predispose to preterm labor through heightened myometrial activity and inflammation. The study underscores the potential of targeting Piezo1 as a therapeutic strategy to mitigate preterm birth associated with uterine inflammation.

Keywords: Mechanosensitive ion channels; Myometrium contraction; Piezo1; Preterm birth.

Fig. 1

Piezo1 expression is higher than other mechanosensitive channels and increases in pregnancy. The relative expression of mechanosensitive channels was determined by qPCR in pUSMCs (A) and myometrium tissue (B), which were isolated from mice at 15.5-day pregnancy (n = 3–4 per group). C Western blotting analysis of Piezo1 protein in myometrium tissue from non-pregnant and pregnant (D18.5) mice (n = 5 per group). D Western blotting analysis of Piezo1 protein in uterine tissue from non-pregnant and pregnant women (n = 5 per group). **P < 0.01, ***P < 0.001, Student’s t-test

Fig. 2

Piezo1 expression increases in the uterine muscle layer of mice after LPS treatment. A Relative Piezo1 mRNA expression in uterine muscle tissue from pregnant mice (E15.5) after LPS intrauterine injection, determined by RT-qPCR (n = 4 per group). B Western blotting analysis of Piezo1 protein in uterine muscle tissue from pregnant mice (E15.5) after LPS intrauterine injection (n = 4 per group). C Representative immunostaining of Piezo1 in myometrial sections from mice. The scale bar represents 100 μm. D Representative Piezo1 immunostaining in pUSMCs after LPS stimulation for 24 h. The scale bar represents 100 μm. E Western blotting analysis of Piezo1 protein levels in pUSMCs after LPS stimulation for 24 h (n = 5 per group). *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test

Fig. 3

Piezo1 promotes contractility in cultured mouse pUSMCs. A qPCR analysis of Piezo1 mRNA expression after pUSMCs were transinfected with Piezo1 si-RNA (n = 5 per group). B The influence of Piezo1 inhibitor Gsmtx4 and agonist Yoda1 on the contraction of cultured mouse pUSMCs, determined by collagen contractility assay (n = 3–4 per group). C Effect of Piezo1 knockdown on the contractility of pUSMCs, determined by collagen contractility assay (n = 3 per group). ^nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t-test and One-way ANOVA

Fig. 4

Effect of inhibiting or knocking down Piezo1 on the contraction of uterine strips in pregnant mice. A Typical recording showing the effect of Gsmtx4 on spontaneous phasic contractions of uterine strips from LPS-reated mice. Bar graphs depicting the effect of Gsmtx4 on the frequency (B), contractile amplitude (C), time between contractions (D), and duration (E) of spontaneous isometric contractions (n = 5–6 per group). F Typical recording showing the effect of Piezo1 knockdown via Piezo1 si-RNA tail injection on spontaneous phasic contractions of uterine strips from LPS-treated mice. Bar graphs depicting the effect of Piezo1 si-RNA on the frequency (G), contractile amplitude (H), time between contractions (I), and duration (J) of spontaneous isometric contractions (n = 5 per group). ^nsP > 0.05, *P < 0.05, ****P < 0.0001, Student’s t-test

Fig. 5

Effect of Piezo1 on the PTB in mice. A Intrauterine injection of LPS induces PTB (n = 7 per group). B Piezo1 inhibitor Gsmtx4 intraperitoneal injection attenuates LPS-induced PTB in mice (n = 6 per group). C Piezo1 agonist Yoda1 intrauterine injection induces PTB in mice (n = 6 per group). D-E Piezo1 si-RNA injection attenuates Piezo1 expression in mice uterine tissues (n = 3 per group) (F) Piezo1 si-RNA injection attenuates LPS-induced PTB in mice (n = 6 per group). ***P < 0.001, ****P < 0.0001, Student’s t-test

Fig. 6

Piezo1 activation increases cytosolic calcium levels and amplifies the inflammatory cascade. A The intracellular Ca²⁺ levels in pUSMCs were measured using a Fluo-4 Calcium Imaging Kit. The scale bar represents 100 μm (n = 4 mice per group). B Graphs showing the change of intracellular Ca²⁺ levels in pUSMCs after different treatment (C) Western blotting analysis detecting COX-2 protein in pUSMCs after LPS and LPS-Gsmtx4 stimulation for 24 h (n = 4 per group). Graphs showing the inflammation factor TNF-α (D), IL-1β (E), and IL-6 (F) from pUSMCs culture medium after LPS and LPS + Gsmtx4 stimulation for 24 h (n = 4 per group). **P < 0.01, ***P < 0.001, ****P < 0.0001; Student’s t-test and One-way ANOVA

Fig. 7

Schematic illustration of Piezo1-mediated molecular processes dominating myometrium contraction and preterm birth. LPS stimulation induced Piezo1 expression in mouse uterine smooth muscle cells. Yoda1 (selective agonist of Piezo1) or LPS stimulated Piezo1 channel. Extracellular Ca²⁺ influxes into uterine smooth muscle cells, and the expression of inflammation factors (IL-β, IL-6, TNF-α, and COX-2) increased. Increased intracellular Ca2 + and inflammatory factors lead to uterine smooth muscle cell contraction