Investigating the Effects of Mechanical Stretch on the Physiology of Engineered Human Myometrium

Introduction: : Uterine overdistention is a risk factor for pregnancy complications such as preterm labor, premature fetal membrane rupture, and prolonged labor. All of these disorders are linked to abnormal uterine contractility driven by the myometrium, the smooth muscle layer of the uterus. In healthy pregnancies, the myometrium adapts to increasing mechanical loads while remaining quiescent until labor, but the normal and abnormal mechanisms of mechanosensing and mechanoadaptation are poorly understood. This knowledge gap stems partly from a lack of effective model systems, which has also hindered the development of targeted treatments. Previous studies using human myometrial tissue strips from surgical waste are limited by low reproducibility and low survival and are insufficient for systematic studies. In vitro approaches, such as collagen lattice contraction assays, are often used to measure force generation by cultured myometrial smooth muscle cells, but these models do not recapitulate tissue alignment and are challenging to subject to mechanical stretch. Here, we engineered aligned human myometrial microtissues on stretchable membranes to investigate the effects of stretch on myometrial physiology. We hypothesize that applying physiological levels of static stretch to aligned human uterine smooth muscle cells (HUtSMCs) will enhance oxytocin-stimulated calcium influx and contractile force by modulating the activity of key mechanosensitive ion channels, including TREK-1 (a two-pore potassium channel) and Piezo1 (a mechanosensitive calcium channel).

Materials and

Methods: : Silicone membranes were laser cut, bonded to Curi Bio’s Cytostretcher chambers, and micropatterned with 75 µm fibronectin lanes to induce cell alignment. Engineered chambers were seeded with 25,000 HUtSMCs each and, after 24 h, transferred to 3D-printed frames that apply 20% static stretch for 1 h, 4 h, or 24 h. 20% strain was chosen to simulate physiologic uterine distension in later stages of pregnancy. Control tissues remained unstretched for 48 h. HUtSMCs were incubated with Fluo-4AM 40 min before stretch endpoints, then imaged via fluorescence microscopy at 2 fps for 3 min, with 0.2 mg/mL oxytocin administered during imaging (Fig. 1A). Calcium transients were analyzed using a custom MATLAB script, calculating normalized fluorescence (F/F0), rise slope (at half-max), decay slope (at tau), duration (s), response time (s), decay time (s), amplitude, area under curve (AUC) during rise, AUC during decay, and total AUC. Protein expression of stretch-responsive channels TREK-1 and Piezo1 was assessed via immunostaining with primary anti-TREK-1 (Alomone) and anti-Piezo1 (ThermoFisher) antibodies, followed by a secondary anti-rabbit antibody (ThermoFisher). Cells were also lysed and RNA was isolated (Qiagen RNeasy MiniKit) for real-time reverse transcription polymerase chain reaction (RT-PCR) to evaluate target gene expression of KNCK2 (encoding TREK-1) and Piezo1, normalized to B2M gene.

Results, Conclusions, and Discussions:: Longer durations of stretch progressively increased rise slope and amplitude, and decreased time to peak, indicating faster calcium entry (Fig. 1B). Calcium clearance also improved, reflected by steeper decay slopes and shorter durations. Notably, cells stretched for 1h exhibited the fastest decay and shortest duration and decay time, suggesting a transient early-phase adaptation. At 4h, calcium dynamics appeared intermediate. After 24h, cells showed the highest amplitude and fastest calcium rise, though duration was no longer significantly different from controls (Fig. 1C). Of the nine calcium transient features evaluated, only the AUC parameters remained unchanged across conditions.

Immunocytochemistry confirmed expression of stretch-responsive channels TREK-1 and Piezo1 (Fig. 2A). Quantification of mean fluorescence intensity showed that TREK-1 protein increased significantly after 24h of stretch, while Piezo1 levels were more variable, with no change between unstretched and stretched samples (Fig. 2B). RT-PCR data revealed that KCNK2 (encoding TREK-1) expression remained unchanged at 4 and 24h, whereas Piezo1 was significantly upregulated at 24h. Prior studies have shown that stretch also modulates the activity of Piezo1 and TREK-1, both of which can also be dysregulated in preterm labor. Our data suggest that calcium handling in HUtSMCs may be regulated by both Piezo1 and TREK-1, and that their expression, activity, or both are modulated by prolonged stretch, which will be investigated in future studies.

We established an in vitro system to apply acute durations of stretch to engineered human uterine smooth muscle tissues and obtain functional outputs via calcium imaging. Our results support the hypothesis that mechanical stretch sensitizes the myometrium by enhancing both calcium influx and clearance, potentially increasing contractile strength and responsiveness. Stretch may increase the open probability of L-type calcium channels, amplifying calcium entry and contractile force. Future studies will investigate how the myometrium adapts to pathological stretch, including excessive stretch (40%) and prolonged duration (48 hours), which model uterine overdistention and prolonged labor, respectively. By incorporating stretch and controlling tissue architecture, our model has enhanced physiological relevance and serves as a more reproducible model for investigating myometrial mechanobiology and uterine dysfunction in pregnancy.

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