2. Methods
2.1 Cell culture and reagents
Cultured H9C2 cell lines (immortalized rat cardiomyocytes) were obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). The cells were cultured in Dulbecco’s modified Eagle 110 medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin at 37°C in a humidified 5% CO2 incubator.
DOX (D107159) was purchased from Aladdin (L. A, USA). LCZ696 (S7678) was obtained from Sellerk (Shanghai, China). Antibodies against IκBα (4812S), NF-κB (p65) (8242S) and GADPH (5174) were purchased from Cell Signaling Technology (CST, USA), and antibodies against TLR2 (12276), TLR4 (ab22048), MyD88 (ab2064), COL-1 (ab34710), TGF-β (ab92486), TNF-α (ab6671) and LaminB (ab133741) were purchased from Abcam (Shanghai, China).
2.2 DOX-induced chronic cardiac injury in mice
All animal care and experimental procedures were approved by the Animal Policy and Welfare Committee of Wenzhou Medical University (Approval Document No. wydw2016-0124), and all animals received humane care according to the National Institutes of Health (USA) guidelines. All studies followed the ARRIVE guidelines for reporting experiments involving animals(Lilley et al., 2020; Percie du Sert et al., 2020).
C57BL/6 male mice were obtained from the Animal Center of Wenzhou Medical University, and male TLR2KO mice (B6.129-TLR2tm1Kir/J) backcrossed to C57BL/6 were provided by the Jackson Laboratory of America (Bar Harbor, ME, United States). The mice were housed with a 12:12 h light–dark cycle at a constant room temperature and fed a standard rodent diet. The mice were acclimatized to the laboratory for at least 2 weeks before initiating the studies. Detailed methods for the model are presented below. Sample sizes were defined by a priori power calculations with G-Power 3.1.9 software (http://www.gpower.hhu.de/), considering a statistical power of 80% and α=0.05.
Eight-week-old C57BL/6 mice and TLR2KO mice were randomly divided into five groups: (I) untreated C57BL/6 mice receiving PBS (WT-Ctrl, n=7); (II) DOX-injected C57BL/6 mice receiving PBS (WT-DOX; n=7); (III) DOX-injected C57BL/6 mice treated orally with LCZ696 (60 mg/kg/day) (Suematsu et al., 2016) (WT-DOX+LCZ696; n=7); (IV) uninjected TLR2 KO mice receiving PBS (TLR2KO-Ctrl; n=7); and (V) DOX-injected TLR2 KO mice receiving PBS (TLR2KO-DOX; n=7). DOX (5 mg/kg, once a week, total cumulative dose of 15 mg/kg) was administered 3 times via intraperitoneal injection as described previously(Pop-Moldovan et al., 2017). LCZ696 treatments were initiated one day after starting the DOX injections and continued throughout the 6-week follow-up. Six weeks after DOX treatment, cardiotoxicity was evaluated.
The animals were sacrificed using sodium pentobarbital anesthesia. Heart tissues were snap frozen in liquid nitrogen for gene and protein expression analyses or fixed with 4% paraformaldehyde for histological analysis.
2.3 Cardiac function evaluation
Cardiac function was determined noninvasively by transthoracic echocardiography in anesthetized mice one day before sacrifice as described previously (Kandalam et al., 2010). The mice were anesthetized using isoflurane, and echocardiography was performed with a SONOS 5500 ultrasound (Philips Electronics, Amsterdam, Netherland) with a 15-MHz linear array ultrasound transducer.
2.4 Real-time PCR
RNA was isolated from cultured H9C2 cells and heart tissue by using TRIZOL (Thermo Fisher, USA). Reverse transcription and quantitative PCR were carried out using a two-step PrimeScript™ RT reagent kit (Perfect Real Time) (TAKARA), Eppendorf Mastercycler ep realplex detection system (Eppendorf, Hamburg, Germany) for reverse transcription and QuantStudio3 Real-Time PCR Systems (Applied Biosystems, Thermo Fisher Scientific, USA) for real-time PCR. Primers for the genes were synthesized and obtained from Thermo Fisher. The primer sequences are presented in supplementary Table S1. mRNA levels of the target genes were normalized to Actb gene mRNA.
2.5 Western blot analysis
Fifty micrograms of total protein from cell or tissue lysates was separated by 10% SDS-PAGE and electrotransferred to PVDF membranes. Membranes were blocked in Tris-buffered saline containing 0.05% Tween 20 and 5% nonfat milk for 1.5 h. The PVDF membranes were then incubated with specific primary antibodies. Immunoreactive bands were detected by incubating the membranes with secondary antibodies conjugated to horseradish peroxidase and an enhanced chemiluminescence reagent (Bio-Rad). Densitometric quantification was performed using ImageJ analysis software version 1.38e and normalized to their respective controls (GAPDH for cytosolic proteins, Lamin B for nuclear fractions, and total protein for phosphorylated-form detection).
2.6 Heart histology and immunostaining
Hearts were fixed in 4% paraformaldehyde and embedded in paraffin. Five-micrometer-thick sections were stained with hematoxylin and eosin (H&E) (C0105, Beyotime Biotechnology) for histological analysis and Sirius Red and Masson’s trichrome (G1340-7×100 ml, Solarbio Life Sciences) to evaluate cardiac fibrosis. The sections were observed under a light microscope (Nikon, Japan).
For immunohistochemical staining, the sections were deparaffinized and rehydrated. The sections were treated with 3% H2O2 for 30 min to block endogenous peroxidase activity and then with 1% BSA in PBS for 30 min. The slides were incubated overnight at 4°C with the primary antibody (TNF-α, 1:50; both from Santa Cruz). Peroxidase-conjugated secondary antibodies were used for detection (Santa Cruz; 1:100 dilution; 1 h incubation). The slides were counterstained with hematoxylin for 5 min, dehydrated, and mounted. Images were viewed by a bright field microscope (Nikon).
2.7 ELISA
The TNF-α (70-EK382/3-96) levels in heart tissue were measured using ELISA kits (eBioscience, San Diego, CA). All experiments followed the manufacturer’s instructions.
2.8 siRNA transfection and gene silencing
Gene silencing in cells was achieved using specific siRNA sequences. TLR2 siRNAs were purchased from Gene Pharma Co., Ltd. (Shanghai, China). Custom siRNAs were synthesized for rat TLR2, TLR4 and MD2. The sequences are presented in supplementary Table S1. The H9C2 cells were transfected with siRNA using LipofectAMINE™ 2000 (Thermo Fisher, Carlsbad, CA).
2.9 Immunoprecipitation
Following treatments, cell lysates or heart tissues were prepared and incubated with an anti-TLR2 or MyD88 antibody overnight. Complexes were retrieved with protein G-Sepharose beads at 4°C for 4 h. The TLR2 and MyD88 levels were further detected by immunoblot using anti-TLR2 and MyD88 antibodies (IB), respectively.
2.10 Statistical analysis
The data presented in this study are representative of 5 independent experiments and are expressed as the mean ± SEM. The exact group size (n) for each experiment is provided, and ‘n’ refers to independent values. Statistical analysis was performed with GraphPad Prism 8.0 software (San Diego, CA, USA). We used one-way ANOVA followed by Dunnett’s post hoc test when comparing more than two groups of data and one-way ANOVA and nonparametric Kruskal–Wallis tests followed by Dunn’s post hoc test when comparing multiple independent groups. A P value ˂ 0.05 was considered to be statistically significant. Post-tests were run only if F achieved P < 0.05 and there was no significant variance in homogeneity.