Alzet micro osmotic pumps model 1004

Serotype 9 adeno‐associated virus (AAV9) carrying the target sequence that overexpresses Bmi‐1‐RING1B or the corresponding control virus was designed and produced (Hanbio Biotechnology, China). Previous reports considered AAV9‐cytomegalovirus (CMV) viral packaging as an efficient and safe tool for cardiac gene transfer due to its consistent transduction efficiency and established cardiac tropism.³⁰, ³¹, ³² It has been used in multiple preclinical studies, for example, in the first‐in‐human cardiac gene therapy trial because of its robust expression.³³, ³⁴ Because of the large base number of Bmi‐1 (975 bp)–RING1B (1, 011 bp) complex, we used the universal promoter CMV promoter to better express Bmi‐1–RING1B complex. Eight‐week‐old male mice were injected with 100 μl AAV9‐CMV‐Bmi‐1‐RING1B (1 × 10¹² v.g/ml) by caudal vein. After 2 weeks of AAV9‐CMV‐Bmi‐1‐RING1B treatment, these mice were anesthetised with 0.02 g/ml pentobarbital sodium (50 mg/kg body weight, intraperitoneal injection). Subcutaneous infused with angiotensin II (Ang II, #A9525, Sigma‐Aldrich, USA) for 4 weeks at a dose of 1.3 mg/kg/day was to induce cardiac hypertrophy using ALZET® Micro‐Osmotic Pumps (Model 1004) (#10370‐16, DURECT Corporation, USA).

ApoE-deficient mice (ApoEKO; C57BL/6 background) were purchased from the Jackson Laboratory (Bar Harbor, Me). Mice were housed in pathogen-free conditions and had access to sterilized food and water ad libitium. All the animal experiments were performed at the department of Physiology and Pharmacology of Cumming School of Medicine at the University of Calgary. Alzet micro-osmotic pumps (Model 1004, Durect Corp., Cupertino, CA, USA) were implanted subcutaneously in 8–10-week-old male mice to deliver Ang II (1.44 mg/kg/day) or saline (control) for 4 weeks [22 (link)]. A subgroup of ApoEKO mice receiving Ang II was also implanted with osmotic pumps to deliver Ang 1-7 (0.576 mg/kg/day) intraperitoneally. The study was approved by the Institutional Ethics Committee of the University of Calgary (AC17-0055 & AC21-0029). All experiments were conducted as per the guidelines of the University of Calgary Animal Care and Use Committee and the Canadian Council of Animal Care.

The Bmi‐1 heterozygous (Bmi‐1^+/−) mouse line with a C57BL/6J background was from McGill University.¹³Bmi‐1 overexpression in mice with a C57BL/6J background under the control of the 2.4‐kb Prx1 promoter (Bmi‐1^Tg) was generated at Nanjing Medical University (Nanjing, China).²², ²³ All experiments on animals followed the guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, 8th Edition, 2011) and uses of mice were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (Permit Number: IACUC‐1706001).
For chronic induction of cardiac hypertrophy, male mice were anesthetised with 0.02 g/ml pentobarbital sodium (50 mg/kg body weight, intraperitoneal injection) and implanted with ALZET® Micro‐Osmotic Pumps (Model 1004) (#10370‐16, DURECT Corporation, CA, USA) in the subcutaneous skin on the back for infusing angiotensin II (Ang II, #A9525, Sigma‐Aldrich, St. Louis, MO, USA), and the dose was 1.3 mg/kg/day for 4 weeks. For tissue collection, mice were anaesthetised using overdose anesthesia (50 mg/kg body weight, intraperitoneal injection) and then sacrificed by cervical dislocation and hearts were used for ex vivo experiments.