Stg 100

To image calcium flux, the spheroids were loaded with freshly dissolved, cell-permeable 1 µM Fluo 4-AM (F14201, Thermo Fisher Scientific), a green fluorescent, intracellular Ca²⁺ indicator, for 30 min at 37 °C. Then, the spheroids were moved to a glass bottom dish (P50G-0-30-F, MatTek, Ashland, MA, USA) containing media with 1 × 10⁶ microbubbles. A coverslip was placed above the spheroids. The presence of microbubbles on the spheroids was microscopically confirmed. Using an in-house designed cone housing and a water immersion transducer (A303S-SU, 0.5-inch diameter, Olympus NDT), US was delivered (frequency 1 MHz; PNP 250 kPa; pulse length 10 µs; pulse interval 10 ms; treatment duration 10 s), and green fluorescence was measured for up to 1 min (Supplementary Videos S3, 4). The involvement of mechanosensitive channels in UTMC-induced Ca²⁺ influx was confirmed by pretreating spheroids with 1 µM GsMTx4 (STG-100, Alomone labs, Jerusalem, Israel) for 30 min. The occurrence of sonoporation was confirmed by loading spheroids with 120 µM PI (P3566, Thermo Fisher Scientific) in the same setup (Supplementary Video S2). The videos were quantified using Fiji.

HH Stage 18 hearts were isolated and cultured as previously described [55] (link). Briefly, the inflow, atria, atrioventricular junction, ventricle and 3/4 of the outflow tract were isolated and transferred to DMEM/F12 high glucose media supplemented with 1% penicillin/streptomycin. Hearts were then cultured for 8 hrs at 37°C in 95% O₂/5% CO₂. To stretch atrial myocardium, silicone oil was microinjected into isolated hearts prior to culture. Injection needles backloaded with silicone oil (GFS Chemical, 8297) were fashioned from borosilicate glass capillaries (World Precision Instruments, Inc., KTW100-3) with a vertical pipette puller (David Kopf Instruments, Model 720). A small incision was made where the right common cardinal vein meets the sinus venosus, and the micropipette was passed through this incision into the lumen of the atria. A pressure injector (Eppendorf, Femtojet) was then used to deliver a bead of silicone oil into the atrial lumen (beads were ∼512 um in diameter). Following 8 hrs of culture atria were isolated and RNA extraction was performed. To block stretch activated responses hearts were cultured with 5 uM GsMTx4 (Alomone Labs, STG-100) dissolved in Tyrode's solution (pH 7.4).