Reagents and antibodies
Rapamycin (≥ 99.93% purity, as determined by high-performance liquid chromatography) was obtained from MCE (Shanghai, China). Primary antibodies against STING (1:1000) and Beclin1 (1:1000) were bought from Abcam (Cambridge, UK). Antibodies forAtg7 (1:1000), Atg12 (1:1000), LC3B (1:1000), LC3I//II (1:1000), GAPDH (1:1000), total ULK1 (1:1000), and phosphorylated-ULK1ser757 (p-ULK1ser757, 1:1000 ) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-rabbit/mouse EnVisionTM+/HRP reagent for immunohistochemical staining was purchased from Gene Technology (Shanghai, China) and secondary antibody used for immunoblotting was obtained from LI-COR Biosciences.
Animals and treatment
The STING target gene was integrated into the genome of wild type (WT) C57/B6 mice using microinjection to construct the STING transgenic (TG) mice. Male C57/B6 mice were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijing, China). The WT mice and TG mice used in this study were 8 ~ 10 weeks old with body weight 25.3 ± 6 g. All animal experimental procedures were approved by the Animal Experimentation Ethics Committee of Wuhan University (Protocol No. 00013274) and comply with the Guide for the Care and Use of Laboratory Animals by the US National Institutes of Health (NIH Publication No. 85 − 23, revised in 1996).
The mice were subjected to aortic banding (AB) surgery or sham operation as described previously13. The mice hearts and lungs of the were harvested and weighed after they were sacrificed using sodium pentobarbital. Then data involving the ratios of heart weight/body weight (HW/BW, mg/g), lung weight/body weight (LW/BW, mg/g), and heart weight/tibia length (HW/TL, mg/mm) was collected.
To activate autophagy, Rapamycin (2.0 mg/kg) was used via intraperitoneal injection every other day for 4 weeks before AB surgery.
Echocardiography
On the basis of the previous study, transthoracic echocardiography was used to collect the mean echocardiographic parameters of 3 to 5 cardiac cycles, including heart rate (BPM), ejection fraction, ejection fraction, left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) 13.
Histological analysis
Simply put, the mouse heart was fixed with 4% formaldehyde overnight, then embedded in paraffin to make 5-µm sections. Next, according to our previous study, hematoxylin eosin (H&E) staining and Picrosirius Red (PSR) staining were carried out. 14, aiming to observe the sectional area of cardiomyocytes and collagen volume in cardiac tissue.
Immunohistochemical staining was carried out to evaluate the content of inflammatory cells infiltration and LC3B. Endogenous peroxidase was inhibited by 3% hydrogen peroxide and the nonspecific binding of the antibody were blocked with 10% goat serum. Then, the sections were incubated with anti-CD45 (1:100), anti-CD68 (1:200), or anti LC3B (1:100) overnight at 4 °C as described previously12.
Cell culture and treatment
Neonatal rat cardiomyocytes as well as cardiac fibroblasts were isolated and cultured based on our previous description15, 16. To overexpress STING, cardiomyocytes and cardiac fibroblasts were transfected with Ad-STING (MOI = 10) or adenovirus harboring no overexpression sequence (Ad-Ctrl) for 6 hours. When the cells reached 75% confluence, cardiomyocytes were treated with AngII (1 µM) while cardiac fibroblasts were treated with TGF-β (10 ng/ml) for 24 hours. Experiments were performed at least 3 times in duplicate.
Western blot and real-time PCR
Western blot was performed as our previously described17. Extracting the total proteins of fresh ventricle tissues or iced cell lysates and quantified, approximately 50 µg of total proteins were loaded on an SDS/PAGE gel. The resolved proteins were subsequently transferred onto polyvinylidene fluoride (PVDF) membranes. After that, membranes were put into 5% non-fat milk with PBS/0.1% Tween and blocked for 1 h. Next, membranes containing target proteins were incubated with primary antibodies overnight at 4 °C. The next day, after washing with PBS/0.1% Tween, membranes were incubated with secondary antibodies conjugated to IRDye 800CW for 50 minutes. Finally, images of the bands with target proteins were acquired and quantified by the Odyssey infrared imaging system (Odyssey, LI-COR, Lincoln, NE).
Real-time PCR was carried out as previously described12. The total RNA of the frozen ventricle tissues or iced cells was extracted by using TRIzol reagent (Invitrogen, 15596-026) and cDNA was synthesized using a Transcriptor First Strand cDNA Synthesis Kit (04896866001, Roche, USA). Quantitative real-time PCR was performed using Light Cycler 480 SYBR Green I Master Mix (Roche, 04707516001). The expression levels of target genes were uniformly normalized to GAPDH. The primers in real-time PCR are listed in Table 1.
Table 1
Gene
|
Species
|
Forward primer
|
Reverse primer
|
IL-1β
|
Mouse
|
AATGAAGGAACGGAGGAGCC
|
CTCCAGCCAAGCTTCCTTGT
|
IL-6
|
Mouse
|
GTTGCCTTCTTGGGACTGATG
|
ATACTGGTCTGTTGTGGGTGGT
|
MCP-1
|
Mouse
|
TGGCTCAGCCAGATGCAGT
|
CCAGCCTACTCATTGGGATCA
|
HMGB1
|
Mouse
|
CCGGCAAGTTTGCACAAAGA
|
TTGGGAGGGCGGAGAATCAA
|
GAPDH
|
Mouse
|
ACTCCACTCACGGCAAATTC
|
TCTCCATGGTGGTGAAGACA
|
ANP
|
Mouse
|
ACCTGCTAGACCACCTGGAG-
|
CCTTGGCTGTTATCTTCGGTACCGG
|
Collagen1
|
Mouse
|
AGGCT TCAGTGGTT TGGATG
|
CACCAACAGCACCATCGTTA
|
Collagen3
|
Mouse
|
CCCAACCCAGAGATCCCATT
|
GAAGCACAGGAGCAGGTGTAGA
|
Fibronectin
|
Mouse
|
CGGTGGCTGTCAGTCAGA
|
TCCCACTGCTGATTTATC
|
CTGF
|
Mouse
|
TGTGTGATGAGCCCAAGGAC
|
AGTTGGCTCGCATCATAGTTG
|
STING
|
Mouse
|
ATCTATGCTAGTCGTAGTTTA
|
CGTAGTGCTAGTGATTAGTC
|
ANP
|
Rat
|
ATGGGCTCCTTCTCCATCAC
|
TCTTCGGTACCGGAAGCTG
|
BNP
|
Rat
|
TTCCTTAATCTGTCGCCGCTGG
|
CAGCAGCTTCTGCATCGTGGAT
|
Collagen1
|
Rat
|
GAGAGAGCATGACCGATGGATT
|
TGGACATTAGGCGCAGGAA
|
Collagen3
|
Rat
|
CAGACGGGAGTTTCTCCTCGGA
|
GACCAGGAGGACCAGCAACTCC'
|
GAPDH
|
Rat
|
GACATGCCGCCTGGAGAAAC
|
AGCCCAGGATGCCCTTTAGT
|
Immunofluorescent staining
To further access cardiomyocyte hypertrophy and the transformation of cardiac fibroblast to myofibroblast, immunofluorescent staining for α-actin and α-SMA was performed according to our published article[16]. Briefly, the cardiomyocytes or cardiac fibroblasts were fixed using 4% paraformaldehyde and permeabilized using Triton X-100 (0.2%). Cardiomyocyte hypertrophy was assessed by anti-α-actinin staining while the transformation of cardiac fibroblast to myofibroblast was evaluated by anti-α-SMA staining. DAPI dye was used to visualize cell nucleus.
Statistical Analysis
All data are presented as mean ± SD and analyzed with the software SPSS 23.3. Two-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons while Student’s unpaired t-test was applied to compare 2 groups. P-value < 0.05 was regarded as statistically significant.