2.1 Animal preparation
All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health, the Animal Experiment Regulations of the University of Tsukuba, and the Basic Guidelines for the Proper Conduct of Animal Experiments in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Animal experiments were performed with approval from the University of Tsukuba Animal Experiment Committee.
Male C57BL/6 mice (eight weeks old; Japan Charles River Kanagawa, Japan) were randomly assigned to three groups: control, AngII-infused (AngII), and AngII and isorhamnetin-treated (AngII+isorhamnetin). AngII (1,000 ng/kg/h) was administered continuously for two weeks using an osmotic minipump (Alzet, model 2002; Cupertino, United States). Normal saline was injected into the mini-osmotic pump in the control group. Isorhamnetin (5 mg/kg) was intraperitoneally administered daily for three weeks to mice in the AngII+isorhamnetin group one week prior to AngII administration. Isorhamnetin was suspended in 0.1% dimethyl sulfoxide (DMSO) and 1% polypropylene glycol. In addition, the same amount of vehicle solution (0.1% dimethyl sulfoxide, 1% polypropylene glycol, and saline) was administered intraperitoneally as a sham injection to the mice in the control and AngII groups. After three weeks, the mice were euthanized by CO2 inhalation and their hearts were harvested for further experiments. The mice used to obtain biochemical and histological data were not assigned to the AF induction study to avoid causing mechanical effects associated with catheters.