Hank s solution

Noncontact topographic imaging and low‐stress mechanical property measurements were performed using SICM manufactured by ICAPPIC (ICAPPIC Limited, UK). EA.hy926 cells were cultured on 35‐mm Petri dish. Cells were gently washed with Hank's solution (Gibco, USA) before scanning procedure. Nanopipettes with typical radius 35–40 nm were fabricated using a P‐2000 laser puller (Sutter Instruments, USA) from borosilicate glass capillaries (O.D. 1.2 mm, I.D. 0.90 mm, 7.5‐cm length) with the following programs Heat 310, Fil 3, Vel 30, Del 160, Pul 0, Heat 330, Fil 3, Vel 25, Del 160, and Pul 200. For all SICM measurements, a three‐set point mode was used. A noncontact topographic image was obtained at an ion current decrease of 0.5% and further two images were obtained at an ion current decrease of 1% and 2% corresponding to membrane deformations produced by intrinsic force at each setpoint (Kolmogorov et al., Nanoscale, 2021). After two measurements of topography and Young's modulus of single EA.hy926 cell Jasplakinolide were added to Petri dish in final concentration 50 nM. Then, the cell was continuously scanned within 24 min every 6 min. In the end, Pitstop‐2 was added to solution and cell was scanned within 24 min every 6 min.

To investigate the wound-healing ability of the AP-AR42J cells, various gene transfected cells were seeded in 24-well plates, respectively, at a density of 1 × 10⁵ cells per mL. After the cells had grown to 100% of confluence, horizontal lines were drawn at the bottom of each well using a 10 μL pipette tip. Subsequently, all of the cells were washed three times by Hanks solution (Gibco) to remove the unattached cells and supplemented with fresh serum-free medium (Gibco). Then qualitative images were taken by an optical microscope at 0, 24, and 48 h, respectively.

Fetal bovine serum (FBS), trypsin, and Hank's solution were purchased from Gibco (Grand Island, NY, USA). Dulbecco's Modified Eagle's Medium (DMEM), Hoechst 33342, tetramethylrhodamine ethyl ester (TMRE), calcein-AM, and Cyclosporin A (CSA) were brought from KeyGEN Biotech (Nanjing, China). Mito-Tracker Red, Mito-Tracker Green, and Mito-Sox Red were obtained from Invitrogen (Carlsbad, CA, USA). Chir99021 was purchased from Selleck Chemicals (Houston, TX, USA). Edaravone, Mdivi-1, and Mito-TEMPO were from Sigma (St Louis, MO, USA). These reagents were dissolved in dimethyl sulfoxide (DMSO) and the final DMSO concentration was <0.1% v/v. Primary antibodies and secondary antibodies for western blot analyses are listed in Supplementary Tables 1 and 2. Cytochrome c ELISA kit was purchased from Cusabio (Wuhan, China) and the intracellular calcium kit was from Beyotime Biotechnology (Nanjing, China).

Twenty Sprague–Dawley (SD) rats were used for the isolation and culture of primary cardiomyocytes. After whole hearts were extracted aseptically, the pericardium and atrium were removed and washed several times with ice-cold PBS to remove residual blood and debris. The residual ventricular parts of the hearts were minced into 1-2 mm³ tissue blocks under sterile conditions. The tissues were then repeatedly digested with freshly prepared compound digestive enzyme [collagenase II (Gibco) 1 mg/ml, hyaluronidase (Gibco), 0.2 mg/ml in Hank's solution (Gibco), sterile filtered, and pH 7.2] for 20 min in a 37°C incubator until the tissue blocks were completely digested and then filtered through a 200 μm cell strainer. Filtered cardiac cells were cultured in a flask and incubated at 37°C for 1 h, whereas fibroblasts were mostly adherent, the cell supernatant enriched with ventricular myocytes was gently aspirated and the suspensions were centrifuged at 800 rpm for 5 min at 4°C to remove debris. The cell cultures were resuspended in cold DMEM/F12 medium (Gibco) supplemented with 10% FBS (Gibco) and penicillin and streptomycin (Gibco).

The strips of 7 sensitised and 6 non-sensitised guinea pigs were incubated at 37°C for 10 min in 5 ml of Hanks’ solution (Gibco, Gaithersburg, MD, US) with 2 mg cysteine and 0.05 U/ml papain (Sigma Chemical Co., St. Louis, MO, US). The strips were then washed in Leibovitz’s solution (Gibco, Gaithersburg, MD, US) and placed in a physiological saline solution (PSS, mM) containing 118 NaCl, 25 NaHCO₃, 4.6 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄ and 11 glucose (Sigma Chemical Co., St. Louis, MO, US). Smooth muscle strips were cut (0.5 x 5 mm), and fragments weighing 200 mg total were placed in 2.5 ml PSS with collagenase type I (1 mg/ml; Sigma Chemical Co., US) and dispase II (4 mg/ml; Sigma Chemical Co., St. Louis, MO, US) at 37°C. Ten minutes later, the fragments were transferred to similar PSS containing fresh enzymes. Tissue was dispersed mechanically until isolated cells were observed. Leibovitz’s solution was added to stop the enzymatic activity.

Rats (100–160 gram) were euthanized, mandibles were dissected out and the surrounding soft tissues removed. Isolated mandibles were transferred to Hanks solution (GIBCO) containing 1% Antibiotic-Antimycotic and kept on ice at all times. Isolated SSEO and MSEO cells were treated with 0.15% collagenase (Roche, Tokyo, Japan) in PBS for 1 hr at 37 °C in a 5%-CO₂ incubator followed by Trypsin/EDTA (Gibco, Life Technologies, USA) for 10 min at 37 °C in a 5%-CO₂ incubator and manual disruption. Reaction was stopped by adding DMEM (Gibco by Life Technologies, USA) containing 30% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% glutamine. For intracellular Ca²⁺ concentration ([Ca²⁺]_i) measurement, isolated SSEO and MSEO cells were cultured in X-Vivo^TM 15 (Lonza, USA) cell media containing 10% FBS, 1% penicillin/streptomycin, 1% glutamine and maintained at 37 °C in a 5%-CO₂ incubator. Cells were used within 24 hrs after isolation.