Novel identification and modulation of the mechanosensitive Piezo1 channel in human myometrium

Abstract

Approximately 10% of US births deliver preterm before 37 weeks of completed gestation. Premature infants are at risk for life-long debilitating morbidities and death, and spontaneous preterm labour explains 50% of preterm births. In all cases existing treatments are ineffective, and none are FDA approved. The mechanisms that initiate preterm labour are not well understood but may result from dysfunctional regulation of quiescence mechanisms. Human pregnancy is accompanied by large increases in blood flow, and the uterus must enlarge by orders of magnitude to accommodate the growing fetus. This mechanical strain suggests that stretch-activated channels may constitute a mechanism to explain gestational quiescence. Here we identify for the first time that Piezo1, a mechanosensitive cation channel, is present in the uterine smooth muscle and microvascular endothelium of pregnant myometrium. Piezo is downregulated during preterm labour, and stimulation of myometrial Piezo1 in an organ bath with the agonist Yoda1 relaxes the tissue in a dose-dependent fashion. Further, stimulation of Piezo1 while inhibiting protein kinase A, AKT, or endothelial nitric oxide synthase mutes the negative inotropic effects of Piezo1 activation, intimating that actions on the myocyte and endothelial nitric oxide signalling contribute to Piezo1-mediated contractile dynamics. Taken together, these data highlight the importance of stretch-activated channels in pregnancy maintenance and parturition, and identify Piezo1 as a tocolytic target of interest. KEY POINTS: Spontaneous preterm labour is a serious obstetric dilemma without a known cause or effective treatments. Piezo1 is a stretch-activated channel important to muscle contractile dynamics. Piezo1 is present in the myometrium and is dysregulated in women who experience preterm labour. Activation of Piezo1 by the agonist Yoda1 relaxes the myometrium in a dose-dependent fashion, indicating that Piezo1 modulation may have therapeutic benefits to treat preterm labour.

Keywords: Piezo1; mechanosensitive channels; myometrium; pregnancy; preterm labour; stretch-activated channels.

figure 1:. Piezo1 protein expression in human myometrium

A) Box and whisker plot (min-max) with overlay of individual values of Western blot data using whole human myometrial tissue (n=5 per state). Piezo1 expression does not significantly increase prior to labor (one-way ANOVA, P=0.103); however, it is upregulated ~3.5-fold during TL (P=0.012) vs. NP tissue and downregulated by ~14-fold in PTL relative to TL (P=0.0028). Piezo1 expression is also significantly different in TL vs. PTNL (P=0.0427), TL vs. TNL (P=0.0126), PTNL vs. PTL (P=0.0020) and TL vs. PTL (P=0.0028). Each sample was normalized to GAPDH expression, and the average value of NP tissue was set to a nominal value of ‘1’ for comparative purposes. NP - non pregnant; TNL - term non-laboring; TL - term laboring; PTNL - preterm non-laboring; PTL - preterm laboring. B) Western blots probed for Piezo1 (~260 kDa) and normalized to GAPDH.

figure 2:. Piezo1 expression in human myometrial phMEC, phUSMC and Piezo1^KO cells

A) Immunofluorescent (IF) imaging of primary phUSMC and phMEC cells from TNL human myometrium and HEK293 Piezo1 KO cells, labelled with DAPI (nuclear), Wheat germ agglutinin (WGA, membrane) and Piezo1-congugated antibodies. Magnified areas of interest (white arrows) presented in the overlay ‘inset’. B) Box and whisker plot (min-max) of Western blot data with overlay of individual values in myometrial phUSMC, phMEC, and Piezo1^KO cell lysates (n=4) reveals a ~2.9-fold increase in Piezo1 expression in phMEC cells over phUSMC (P=0.0008). Piezo1^KO cells expressed insignificant Piezo1 when compared to either phUSMC (P=0.0185) or phMEC (P<0.0001).

Figure 3:. Piezo1-meidiated Ca²⁺ influx in CD31+ and CD31− human myometrial cells

An intracellular calcium flux assay determined Piezo1 activity in phMEC (CD31+) and phUSMC (CD31−) cells. phMEC, phUSMC, and HEK293 Piezo1^KO cells were pretreated with the Ca²⁺ indicator Calbryte^® (ex/em 493/515 nm) followed by exposure to Yoda1 (0.3 or 3 μM) ± the Piezo1 antagonist, Dooku1 (10 μM) and the change in fluorescence (ΔCa_i²⁺) was measured. A) phMECs treated with 3 μM Yoda1 exhibited 4.09-fold increase in Ca²⁺ uptake (ΔCa_i²⁺) over 0.3 μM treated cells (p<0.0001) which decreased by 35.74% when co-treated with Dooku1 (P=0.0327) at 60 minutes. B) phUSMC experienced a 2.64-fold increase in fluorescence when challenged with 3 μM Yoda1 (p<0.0001), with a respective decrease of 22.49% when co-treated with Dooku1 (P=0.0326). C) Piezo1^KO fluorescence did not vary significantly at any dose of Yoda1 or Yoda1 + Dooku1 relative to baseline (Kruskal-Wallis one-way ANOVA, P=0.2622). (D) left panel - 10x bright field images of (top) phMEC, (middle) phUSMC, and (bottom) Piezo1^KO. right panel - Calbryte-induced fluorescence after 3 μM Yoda1 stimulation. 20x fluorescent images of (top) phMEC, (middle) phUSMC, and (bottom) Piezo1^KO. Data presented as ± SD.

figure 4:. EC₅₀ of Piezo1 agonist (Yoda1) in human myometrium

A) TNL human myometrium (n=6) was hung in an organ bath, oxytocin-challenged (8nM), then dosed with the Piezo1 agonist, Yoda1, in 15 minute intervals (0.1, 0.3, 1, 3, 10, 30 μM). Tissue relaxed to Yoda1 exposure in a dose-dependent manner, with an EC₅₀ of 3.02 μM. Relative to untreated control tissue (100%), the AUC for each concentration of Yoda1 was: 100nM (88.03%, SD 13.01), 300nM (86.72%, SD 18.65), 1 μM (70.91%, SD 15.52), 3 μM (52.02%, SD 15.50), 10 μM (35.08%, SD 5.62), 30 μM (29.74%, SD 16.59). Data presented as a box and whisker plot (5-95 percentile) with individual data point overlay. B) Representative traces of Yoda1 (⬤) and control (⬤) tissue.

figure 5:. Inotropic effects of Piezo1 agonism on Ca²⁺-mediated myometrial quiescent pathways

Using an organ bath (n=6), area under the curve (AUC), peak tension, and contractile frequency were determined after co-administration of an EC₅₀ dose (3μM) of Yoda1 + myrPKI_14-22 (PKA_i), FPA-124 (AKT_i), L-NNA (eNOS_i), Paxilline (BK_Ca(i)), or NS1619 (BK_Ca(ex)). A) AUC: In TNL myometrium, co-treatment with myrPKI_14-22 (10 μM) + Yoda1 (P=0.0005) or FPA-124 (10 μM) + Yoda1 (P=0.0067) significantly reduced the effects of Yoda1 returning AUC to baseline. Excitation of BK_Ca (NS1619, 30 μM) + Yoda1 resulted in an additive effect, decreasing AUC to 28.8% of baseline. B) Peak Tension: Co-treatments with myrPKI_14-22 + Yoda1 (P=0.0026), FPA-124 + Yoda1 (P=0.0021), and L-NNA + Yoda1 (P=0.0402) resulted in a significant increase peak tension when compared to treatment with 3 μM Yoda1 alone, while neither NS1619 + Yoda1 (P=0.4770) nor Paxilline + Yoda1 (P=0.1887), significantly altered tension beyond that of 3 μM Yoda1 alone. C) Contractile frequency: None of the treatment conditions, myrPKI_14-22 + Yoda1 (P=0.8343), FPA-124 + Yoda1 (P=0.4077), L-NNA + Yoda1 (P=0.1718), NS1619 + Yoda1 (P=0.8074), or Paxilline + Yoda1 (P=0.7084) significantly deviated contractile frequency when compared to a stand-alone EC₅₀ dose of Yoda1. D) Selectivity of Yoda1 was determined by comparing the AUC (n=5) after a maximum dose of Yoda1 (30 μM) relative to co-treatment of Yoda 1 with Piezo1 antagonist, Dooku1 (30 μM/30 μM). Treatment with 30μM Yoda1 decreased AUC to 17.08% of baseline, while co-treatment with Yoda1 + Dooku1 (30 μM/30 μM) recovered AUC to 90.55% of baseline (P=0.0004).

Figure 6:. Proposed pathway for Piezo1-mediated quiescence in human myometrium

We posit that the stretch experienced by the uterus as the fetus develops activates Piezo1 channels on endothelium and smooth muscle cells of the myometrium. This stretch drives Ca²⁺ influx into the endothelium where PKA and AKT activate eNOS, generating the quiescence-promoting molecule, nitric oxide, which migrates to the myocyte in the form of S-nitrosoglutathione (GSNO). Concurrently, in the smooth muscle, Piezo1 activation may contribute to BK_Ca activation, which polarizes the membrane through K⁺ efflux. Graphic created with BioRender.com.