Bladder Smooth Muscle Contractility Assay

Bladders were pinned on a small Sylgard block, and the muscle was dissected free of the mucosal tissue. BSM strips were then cut longitudinally (two mm wide and seven mm long). Muscle strips were mounted in an SI-MB4 tissue bath system (World Precision Instruments, FL, USA). Force sensors were connected to a TBM 4M transbridge (World Precision Instruments), and the signal was amplified by PowerLab (AD Instruments, CO, USA) and monitored through Chart software (AD Instruments). BSM strips were gently prestretched to optimize contraction force and then pre-equilibrated for at least 1 h. All experiments were conducted at 37 °C in physiological saline solution (in mM: 120 NaCl, 5.9 KCl, 1.2 MgCl₂, 15.5 NaHCO₃, 1.2 NaH₂PO₄, 11.5 glucose, and 2.5 mM CaCl₂) with continuous bubbling of 95% O₂/5% CO₂. Contraction force was sampled at 2000/s using Chart software. BSM tissue was treated with agonists or antagonists and/or subjected to electrical field stimulation (EFS). BSM strip EFS was carried out using a Grass S48 field stimulator (Grass Technologies, RI, USA) using previously described standard protocols (Rajandram et al., 2016 (link); Chen et al., 2020 (link)).