Mouse model and adeno-associated virus (AAV) injection

A total of 50 C57BL/6 mice (20-25 g; 8 weeks old; male; Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used in the present study and were grouped as follows: Sham group (n=15), DOX+AAV-NC group (n=15) and DOX+AAV-miR-133b group (n=20). Mice were raised with ad libitum access to water and food in a temperature-controlled room (22±2°C) with a 12-h light/dark cycle and a relative humidity of 40-60%. A cardiac injury mouse model was generated by chronic intraperitoneal injections (on days 0, 2, 4 and 6) of 4 mg/kg doxorubicin. The mice in the sham group were treated with the same dose of PBS (Invitrogen; Thermo Fisher Scientific, Inc.). All mice were euthanized 4 weeks after the first injection of doxorubicin or PBS. For AAV (serotype 9) injection, mice received a single-bolus injection of AAV-miR-133b or empty AAV (AAV-NC) resuspended in PBS via the tail vein at 1×10¹¹ viral genomes per animal. AAV vectors were purchased from Han Heng Biotechnology (Shanghai) Co., Ltd. One week after injection of AAVs, the mice were treated with doxorubicin or PBS. The mice were anesthetized with 1.5% pentobarbital sodium (60 mg/kg) by intraperitoneal injection and then sacrificed by cervical dislocation under anesthesia. The whole experiment lasted 5 weeks, beginning from AAV injection and ending up with the euthanasia of mice.