Establishment of an AMI injury model in mice
Male C57BL/6 J mice aged 15 months (weighing 28 ~ 35 g) were obtained from the Experimental Animal Center of Xinjiang Medical University. All animal experiments were performed following a protocol approved by the Institutional Animal Care and Use Committee at the First Affiliated Hospital of Xinjiang Medical University. The experimental mice were housed in an SPF environment (12-h:12-h day and night cycle) with free access to food and water. Two weeks later, after the mice were acclimated to the new environment, the mice were randomly assigned to five experimental groups.
Mice were assigned to the following groups. In the control group (sham operation group, n = 15), mice received tail vein injections of dsAVV9-GFP (1 × 1011 vg/mice) for 5 weeks. Then, the chest was opened, but the left coronary artery (LCA) was not ligated. In the M3 group (n = 28), mice received tail vein injections of dsAVV9-GFP for 5 weeks and were subsequently subjected to LCA ligation following the methods described previously [12,13,14]. Mice in this group were sacrificed 3 days after the LCA ligation. In the M3 + Sfrp1 group (n = 30), mice received tail vein injections of dsAVV9-Sfrp1 (1 × 1011 vg/mice) for 5 weeks, were subjected to LCA ligation and were sacrificed 3 days later. In the M7 group (n = 27), mice received tail vein injections of dsAVV9-GFP for 5 weeks, underwent LCA ligation and were sacrificed 7 days later. In the M7 + Sfrp1 group (n = 28), mice received tail vein injections of dsAVV9-Sfrp1 for 5 weeks and were then subjected to the LCA ligation and sacrificed 7 days later. The experimental procedures are shown in Fig. 1. Successful modeling was characterized as follows: under the surgical microscope, the left coronary artery innervation area was paler and had segmental dysplasia compared with the surrounding myocardial tissue; the electrocardiogram suggested ST-segment elevation or QRS wave widening malformation and persisted.
Echocardiography
Echocardiography analysis was performed using a Sonos 5500 ultrasound system (Hewlett-Packard, Palo Alto, CA, USA). Mice were anesthetized by injection of compound anesthetic containing 0.1 g of ketamine, 1 mg of atropine, and 5 mg of xylazine in 10 ml of physiological saline (0.1 ml/10 g weight). Mice could spontaneously breathe and were placed on a table with a thermostatic pad. Then, posterior wall thickness in diastole (Pwdth), left ventricular end-diastolic dimension (LVEDd), and left ventricular short-axis shortening rate (FS) were measured. All measurements were measured by double-blind methods by two physicians.
Histological analysis
Hematoxylin-eosin (HE) staining and Masson’s staining was applied to visualize cardiomyocyte morphological changes and cardiac fibrosis area according to previous description [15, 16].
Immunoblotting
Proteins extracted from tissues or cells were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA), and then incubated with the primary antibody followed by appropriate secondary antibody (Abcam, Cambridge, MA, USA). The following primary antibodies were used: anti-TrkB (ab18987), anti-Akt (ab32505), anti-p-Akt (ab81283), anti-Erk (ab54230), anti-p-Erk (ab50011), anti-ADRB2 (ab182136), anti-PKA (ADI-KAS-PK017-F; Enzo, Hong Kong, China), anti-p-PKA (ab75991), and anti-caveolin-3 (ab2912). All antibodies were obtained from Abcam unless otherwise stated.
TUNEL staining
Cardiomyocyte apoptosis was examined using a TUNEL kit (Beijing Zhongshan Biotechnology Co.) following the instructions. Under a 200× light microscope, five visual fields were randomly selected and counted to calculate the apoptosis index of cardiomyocytes. Cardiomyocyte apoptosis index = number of apoptotic cells/total number of cardiomyocytes × 100%.
Immunohistochemical (IHC) assay
IHC was performed to detect the locations and protein levels of Col-1 according to previous description [15]. Optical microscopy was performed to visualize section images. ImageJ software was used to analyze the data.
Statistical analysis
Data are expressed as the mean ± SD. Statistical analysis was performed using one-way analysis of variance (ANOVA)) following Tukey’s tests (GraphPad Prism 6) were applied for multiple comparisons between groups. P < 0.05 was considered statistically significant.