4 chamber tissue organ bath chambers with isometric force transducers 0 25g

tHIO and tHIO+S grafts were harvested and placed in ice-cold Hank’s Balanced Salt Solution (HBSS). Human surgical samples were maintained overnight at 4°C in HBSS prior to assaying in order to minimize the effects of anesthetics. Muscle strips (4–6 mm in length and 1–2 mm in width) were dissected from the samples. Strips were then suspended vertically in an organ bath chamber (Radnoti) filled with freshly prepared Krebs-Ringer solution (Sigma; supplemented with 2.5 mM CaCl₂ and 15 mM NaHCO₃; pH 7.4), warmed to 37°C and gassed with 95% O₂ + 5% CO₂. Segments were allowed to equilibrate for an hour at an initial tension of 0.5 g. The contractile response of the muscle was continuously recorded, using 4-chamber tissue-organ bath chambers with isometric force transducers (0–25g; AD Instruments) coupled to a multi-channel bridge amplifiers and data recorder (AD Instruments; PowerLab 4/35) linked to a computer equipped with LabChart Pro software (AD Instruments). A logarithmic dose response to Carbamyl-β-methylcholine chloride (Bethanechol; Sigma-Aldrich) was obtained through the administration of exponential doses with concentrations of 1 nM to 10 mM at 2 minute intervals before the administration of 10 µM scopolamine (Tocris Bioscience).