2.2. Animals and Treatments

All animal care and experimental procedures were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University, which is guided by the Guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication, revised 2011). All the animal treatments and subsequent analysis were performed in a blind fashion for all groups. Male C57BL/6 mice (8 weeks old; body weight: 25.5 ± 2 g) were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijing, China). The mice were allowed free access to food and water under a 12 h light-dark cycle with controlled temperature (20–25°C) and humidity (50 ± 5%) in the Cardiovascular Research Institute of Wuhan University (Wuhan, China). A total of 40 male C57BL/6 mice were randomly divided into four groups: control group (CON, n = 10), control+levosimendan group (CON+L, n = 10), doxorubicin group (DOX, n = 10), and doxorubicin+levosimendan group (DOX+L, n = 10). The mice were injected intraperitoneally with DOX (5 mg/kg, once a week; the total cumulative dose was 20 mg/kg) or the same dose of saline for 4 weeks. Meanwhile, levosimendan (1 mg/kg) or the same volume of saline was administered through oral gavage once daily for 4 weeks. At the endpoint of the treatment, all the mice were anesthetized using 1.5% isoflurane and euthanized by cervical dislocation. The mouse hearts were dissected and weighed to assess heart weight/tibial length (HW/TL) and then snap-frozen in liquid nitrogen for further analysis.