2 3 butanedione monoxime bdm

Immediately after killing, hearts were excised and placed in ice‐cold Krebs–Henseleit (KH) solution (mM): NaCl, 108; NaHCO₃, 25; KCl, 4.0; KH₂PO_4, 1.2; MgCl_2, 1.0; CaCl_2, 1.8; glucose, 10; and Na‐pyruvate, 2.0; pH adjusted to 7.4 and bubbled with 95% O₂/5% CO₂ (British Oxygen Company, Manchester, UK). The aorta was cannulated with a trimmed and filed 21‐G hypodermic needle, and then secured onto the perfusion cannula of a Langendorff perfusion system by an aneurysm clip and a 5‐0 braided silk suture. Retrograde perfusion under constant flow (2.1 ml/min; Watson‐Marlow, Falmouth, UK, peristaltic pump) perfused 75 ml of a KH solution to which 10 mM 2,3‐butanedione monoxime (BDM) and 10 μM blebbistatin (Cayman Chemical Company, Ann Arbor, MI) were added to give a KH‐BDM/blebbistatin solution to achieve electromechanical uncoupling. The heart was then immediately placed in ice‐cold KH‐BDM/blebbistatin solution for dissection of the right ventricle from the rest of the heart. The right ventricle was then mounted on Sylgard (Dow Chemical Company, Staines, UK) and placed in a temperature monitored experimental chamber filled with filtered KH buffer solution at 27°C.

Krebs–Henseleit (KH) solution was prepared (mmol/L: NaCl, 119; NaHCO₃, 25; KCl, 4.0; KH₂PO_4, 1.2; MgCl_2, 1.0; CaCl_2, 1.8; glucose, 10; and Na‐pyruvate, 2.0; pH adjusted to 7.4 and bubbled with 95% O₂/5% CO₂ (British Oxygen Company, Manchester, UK)) for all tissue preparations. Chemical agents were purchased from Sigma‐Aldrich (Poole, UK) unless otherwise stated. A modified KH solution containing 10 mmol/L 2,3‐butanedione monoxime (BDM) and 10 μmol/L blebbistatin (Cayman Chemical Company, Ann Arbor, MI, USA) was also prepared for Langendorff perfusion of isolated hearts to electromechanically uncouple the heart prior to isolation of right ventricular preparations. KH solution containing 8‐(4‐chlorophenylthio)adenosine 3′,5′‐cyclic monophosphate sodium salt (8‐CPT, 1 μmol/L) and/or dantrolene (10 μmol/L) (LKT Laboratories Inc, St Paul, MN, USA) was prepared and filtered to remove particles greater than 10 μm in diameter with standard filtration paper (Millipore, Bedford, MA, USA).