Animal model and metoprolol treatment

PH and RV hypertrophy was induced in male Wistar rats (317 ± 4 g; bred at the University of Auckland with breeding pairs originally from Charles River Laboratories, USA) by a single intraperitoneal injection of 60 mg kg^-1 monocrotaline in saline (MCT; n = 12). Controls were injected with an equivalent volume of saline (CON; n = 6). Rats were fed normal rat chow and water ad libitum for up to six weeks. Rats were monitored (weight gain, water intake and food consumption) twice weekly for the first two weeks post-injection and then daily once metoprolol dosing began. Two days before commencing metoprolol dosing, the rats were trained to voluntarily ingest sucrose solution (20% (v/v) Ribena and 10% (w/v) sucrose in water) administered by hand from a 1 ml needleless syringe. At day 20 post-MCT injection, dosing of rats with metoprolol commenced. Metoprolol tartrate (1.25 mg mL^-1; Santa Cruz Biotechnology, USA) was dissolved in the sucrose solution and administered (10 mg kg^-1 day^-1) to the treatment group of MCT rats (MCT + BB; n = 6) half an hour before their dark cycle. The dose of metoprolol used was equivalent to that used by Fowler et al. (2018) [21], but in the present study dosing commenced at a later stage of development as older animals were used; these demonstrate a longer time to reach end-stage heart failure based on previous work by our colleagues [37]. Remaining MCT and control rats were fed an equivalent volume of the sucrose solution. In the fourth to fifth week post-injection the MCT and MCT + BB rats were monitored closely for signs of heart failure. Overt signs of heart failure included rats being non-inquisitive, having hunched posture, lack of grooming, dyspnea, cold/pale extremities, pilo-erected fur, porphyrin around the eyes and nose, and significant weight loss. Humane end-points were determined as weight loss of more than: (1) 15% of body weight in 24 hours; (2) 20% of body weight or more plus one other clinical (overt) sign compared with control; or (3) 25% compared with control. All untreated MCT rats were sacrificed when signs of heart failure were observed, and MCT + BB rats were sacrificed at a comparable time point. The use of animals for this study was approved by the University of Auckland Animal Ethics Committee (reference 001403).

On the day of experimentation, animals were weighed, anaesthetised with isoflurane and decapitated before excising the heart. The heart was immediately rinsed in ice-cold Tyrode’s solution prior to blotting and weighing. The wet and dry weights of the liver and lungs were obtained, as well as the tibial length measurement. During the RV tissue dissection, hearts were Langendorff-perfused with oxygenated Tyrode’s solution containing (in mM): NaCl (141.8), KCl (6), MgSO₄.H₂O (1.2), Na₂HPO₄ (1.2), 10 HEPES (10), glucose (10), CaCl₂ (0.25) and 2,3-butanedione monoxime (20) at pH 7.4. Approximately 50 mg of tissue was dissected from the apex of the RV free wall and stored in ice-cold permeabilisation buffer containing (in mM): CaK₂EGTA (2.77; 0.1 μM free Ca²⁺), imidazole (20), taurine (20), K-MES (4-morpholineethanesulfonic acid; 50), dithiothreitol (0.5), MgCl₂ (6.56), ATP (5.77), phosphocreatine (15) at pH 7.1. Another two samples of the free wall were removed. One sample was snap frozen in liquid nitrogen and stored at -80 °C for determination of citrate synthase enzyme activity and soluble protein content. The other sample was fixed in 1% paraformaldehyde for confocal imaging. Measurements of the left and right ventricle free wall thickness were obtained using micro-callipers.