Inducing Cardiac Hypertrophy in Mice

To induce LVH, 8-week-old male mice were fed a high phosphorus diet, which contained modified AIN-93G, lactose 20.0%, sucrose 2.023%, β-corn starch 20.3486%, α-corn starch 7.0%, CaCO₃ 0.55%, Ca(H₂PO₄)₂ 5.05%, and phosphate 1.5g/100g (Oriental Yeast, Tokyo, Japan) 1 day after left heminephrectomy (removal of the whole left kidney). This diet was provided for the induction of FGF23 and was administered with a continuous subcutaneous dose of 100 mg/kg IS (I3875; Sigma-Aldrich, St. Louis, MO) or 28 μl/day of NS (Otsuka Pharmaceutical Factory, Tokushima, Japan) for 4 weeks using a micro-osmotic pump (2002-0000296; Alzet, Cupertino, CA). Half of the mice were treated with a continuous intraperitoneal dose of 7.5 mg/kg H3B-6527 (H3B), which is an FGFR4 inhibitor (S8675; Selleck Biotech, Tokyo, Japan), or 3.6 μl/day of NS using a micro-osmotic pump (1004-0009922; Alzet) for 4 weeks. The 2,002-pumps were replaced biweekly. To examine the effect of IS administration on cardiac hypertrophy and fibrosis, the mice were divided into the five following groups: sham; control; IS + NS; NS + H3B; and IS + H3B. Sham mice were treated with anesthesia, skin incision, and laparotomy, but did not have pumps inserted and were fed a normal phosphorus diet (CRF-1LID10; Oriental Yeast, Tokyo, Japan). The mice were operated on day 0, observed for 4 weeks, and euthanized on day 28. The mice were anesthetized intraperitoneally with medetomidine hydrochloride (0.3 mg/kg body weight; Wako, Osaka, Japan), midazolam (4 mg/kg body weight, Sandoz, Tokyo, Japan), and butorphanol tartrate (5 mg/kg body weight, Wako). The body temperature of the mice was maintained at 37°C during the whole procedure.