Isometric force transducer

The mice were euthanized by cervical dislocation. The colon tissue of each group was removed, opened along the mesentery, then rinsed in oxygenated (95% O₂ + 5% CO₂) Kreb’s buffer (NaCl 119 mmol/L, KCl 4.7 mmol/L, NaHCO₃ 25 mmol/L, NaH₂PO₄ 1.2 mmol/L, MgSO₄ 1.2 mmol/L, glucose 11.1 mmol/L, CaCl₂ 2.5 mmol/L, pH 7.30–7.40). The mucosa and submucosa were gently removed, and circular smooth muscle strips (1 cm in length and 3 mm in width) of the distal colon were made. These muscle strips were mounted between the L-shaped tissue hook and the isometric force transducer (AD Instruments, Dunedin, New Zealand) and immersed in a tissue bath filled with 25 mL oxygenated Kreb’s solution at 37 ℃. The muscle strips were then allowed to equilibrate for 30 min under a preload of 1 g to achieve stable spontaneous contraction. Spontaneous smooth muscle activity (frequency, tension, and motility index) was calculated. The neural activation-mediated colonic muscle strip contraction was induced by electric field stimulation (EFS; 20 V, 2–64 Hz, 10 s pulse for an interval of 5 min). Data were analyzed using a polygraph (LabChart software 8.0).

Following the removal of ventricles, nonabsorbable braded silk‐waxed suture (Pearsalls Ltd., Taunton, MA) was used to carefully tie a basic surgical knot around the lateral wings of the two atria. The preparation was then transferred to a 20 mL organ bath containing 10 mL oxygenated PSS, warmed by a water jacket to 37°C. The atria were mounted vertically such that the left atrium was attached to a metal hook positioned at the bottom of the organ bath and the right atrium was attached to an isometric force transducer (AD Instruments, Oxford, UK). The beating rate was calculated from the interval between contractions using Chart 5 software. The beating rate was also calculated using an extracellular electrode placed close to the SAN region to record a compound action potential signal. The beating rate was calculated from the interval between individual extracellular voltage signals associated with compound action potentials using Chart 5 software. Thirty‐minute incubation of the tissue with SR inhibitors, MDL‐12330A, ivabradine, and ZD7288 was required to reach a reduced plateau steady beating rate. The rate (beats per minute) was measured from beats sampled over a period of approximately 20 sec. The preparation was allowed to stabilize for 45 min before an experiment was carried out.