Anne-Marie Koop

7 255 MATERIAL AND METHODS Experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committees and authorized by the Animal Experimental Committee of the local government in accordance to the Dutch law (DEC 2012-172) and European Directive 2010/63/EU. In addition, the investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). Animal models and Pulmonary Artery Banding Surgery Mice harboring a floxed allele of Hand2 ( Hand2 F/F ) in a B6129F1 background were described previously, 20 and crossbred with mice harboring a tamoxifen-regulated form of Cre recombinase (MerCreMer) under control of the murine Myh6 promoter (MHC–MerCreMer; MCM mice) 21 in a B6129F1 background to generate MCM– Hand2 F/F mice. Hand2 F/F and MCM– Hand2 F/F were treated with either vehicle (10/90 v/v ethanol/peanut oil, Sigma P2144) or tamoxifen (45 mg kg −1 per day) by daily intraperitoneal injections for 5 consecutive days. Both male and female, adult MCM– Hand2 F/F mice and Hand2 F/F mice (10-12 weeks of age) were used for functional and histological analyses. Pulmonary artery banding (PAB) was performed, as described below, in mice older than 8weeks from both genders. Animals were anesthetizedwith isofluorane/air mixture (5% induction; 2–3% maintenance), and subsequently analgesia with buprenorphine (0.01 mg/kg s.c.) was injected. Animals were placed in a supine position on a heating pad (37°C) and intubated with a 20G catheter and ventilated with room air using a Harvard mini-ventilator (model 687, Hugo Sachs, Germany; respiratory rate 180 breaths per minute and a tidal volume of 125 μL). The pulmonary artery was approached by a left lateral thoracotomy and banded with a 7-0 suture by tying over a 23G needle. Post- operative pain relief was provided with buprenorphine (0.01 mg/kg s.c.) twice daily for 2-3 consecutive days if necessary. Sham-operated animals underwent the same procedure without PAB. All protocols were reviewed and approved by the Animal Care and Use Committee of the University of Maastricht and Animal Experiments Committee of the University of Groningen and were performed according to the rules formulated in the Dutch law on care and use of experimental animals. Hemodynamic analyses Hemodynamic functionwas assessed at day 5 andweek 6 after shamor PAB surgery, by cardiac magnetic resonance imaging (MRI) during anesthesia with isoflurane (induction 5%; 1.5-3% maintenance) and warming at 37°C. Echocardiography to assess the pulmonary artery banding gradient was performed using a Vivid Dimension 7