Animal care
All experiments with animals were conducted strictly following the University's guidelines for the Care and Use of Laboratory Animals (publicationNo.85 − 23, revised1996; National Institutes of Health, Bethesda, MD, United States) and were approved by the Committee on Use and Care of Experimental Animals of Nanjing Medical University (Nanjing, China).
Wild-type C57BL/6J male mice (4–6 weeks old) were purchased from Model Animal Research Center of Nanjing University (Nanjing, China) and housed in a temperature-controlled room with a 12 h/12 h light/dark cycle. The standard chow and tap water were given ad libitum.
Study design and Grouping
After random assignment into groups according to body weight, the mice were implanted with an ALZET 2004 osmotic mini-pump (Durect Corp, Cupertino, CA) filled with either AngII (2.5 mg · kg− 1 · d− 1) or phosphate-buffered saline (PBS) subcutaneously under inhalation anesthesia with isoflurane, and the contents were delivered at the rate of 0.25 µl/h for 4 successive weeks. PBS was administered in control groups, and the infusion rate of AngII was determined according to a previous report ([35]). The treatment group and the LIPUS control group underwent LIPUS irradiation (described below) for 20 minutes under isoflurane anesthesia every 2 days from 1 week before surgery to 4 weeks after surgery. The sham group and model group underwent the same anesthesia but without LIPUS irradiation. After evaluating cardiac function by echocardiography at the end of 4 weeks, all the mice were euthanized, and heart samples were excised and weighed for follow-up studies. Thereafter, the atrium was removed, and the left ventricle was isolated. Frozen tissue samples were employed for WB and qPCR analysis, while formalin-fixed and paraffin-embedded tissue samples were used for Sirius Red staining, hematoxylin and eosin staining, Masson staining, and immunofluorescence staining.
Cell isolation and culture
Fresh neonatal rat cardiomyocytes (NRCMs) and neonatal rat cardiac fibroblasts (NRCFs) were isolated from 1 to 3-day-old newborn Sprague–Dawley rats (Vital River Biological Co., China) as previously described ([36, 37]). Then, approximately 4 × 105 NRCMs were seeded and cultured in 60-mm cell culture dishes with DMEM (Gibco Co., USA) containing 10% (v/v) horse serum (HS, Gibco Co., USA), 5% (v/v) fetal bovine serum (FBS, HyClone Co., USA), and 1% (v/v) penicillin-streptomycin (P/S, HyClone Co., USA).
On the second day of culture, NRCMs were incubated in serum-free DMEM overnight starvation before further experiments. Then, the treatment group and LIPUS control group were exposed to LIPUS irradiation for 20 minutes every 6 hours for a total of 2 times. After that, the model group and treatment group underwent induction with 1 mmol/l of AngII (Sigma-Aldrich Co., USA) for 48 hours. In some experiments, cells were preliminary incubated with 10 µmol/l of pyrazolopyrimidine 2 (pp2, S7008, Selleck Co., China) and/or 1 µM TLR4 inhibitor TAK-242 (soluble in 1‰ DMSO, Selleck Co., China) for 2–3 hours or 1 ng/ml reconstitute the lyophilized recombinant Rat Interleukin-1β (rRtIL-1β) / recombinant Rat Interleukin-6 (rRtIL-6) for 12 hours prior to LIPUS irradiation.
NRCFs were also seeded in 60-mm cell culture dishes but were cultured in fibroblast medium (FM, Sciencell Co., China); they were treated identically to NRCMs in subsequent experiments.
All these cells were subjected to WB, qPCR, ELISA, and immunofluorescence analyses.
Echocardiography
Transthoracic echocardiographic images of mice hearts were acquired at the end of 4 weeks after surgery with an ultrasound system (Vevo 3100, VisualSonics, Toronto, Canada) under isoflurane anesthesia. Then, LV fractional shortening (LVFS), LV ejection fraction (LVEF), LV internal diameters at end systole (LVIDs), and LV internal diameters at end diastole (LVIDd) were calculated by the operator who was blinded to the grouping design of this animal experiment.
Western blotting (WB)
The isolated tissues or lysed cells were sonicated and then homogenized in RIPA lysis buffer (Beyotime Biotechnology Co., China). After centrifugation, the protein concentration of the supernatant was quantified with the BCA assay (Pierce Biotechnology, Inc., Rockford, IL, USA).
Equal amounts of proteins (30 µg) mixed with loading buffer were separated by electrophoresis using 10% SDS–PAGE and then transferred to PVDF membranes. After blocking with 5% bovine serum albumin for 2 hours at room temperature, the membranes were incubated overnight at 4℃ with primary antibodies (1:1,000 dilution) against collagen I (Beyotime Biotechnology, China), α-smooth muscle actin (α-SMA, Cell Signaling Technology, USA), TGF-β (Beyotime Biotechnology, China), VEGF (Cell Signaling Technology, USA), caveolin-1 (Cell Signaling Technology, USA), and GADPH (Beyotime Biotechnology, China). Following incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000 dilution) for 2 hours at room temperature and washing in tris-buffered saline, the blots were detected with enhanced chemiluminescence reagent (ThermoFisher Co., USA) with optimal exposure time. The intensity of protein bands was analyzed and normalized to that of GAPDH by ImageJ software.
Quantitative PCR analysis
Total RNA was extracted from the isolated specimens or lysed cells by homogenizing in TRIzol (Invitrogen Life Technologies, Carlsbad, CA) following the manufacturer’s instructions. A total of 0.5 mg of RNA was reverse transcribed to single-strand cDNAs by PrimeScript™ RT reagents kit (TaKaRa, Japan). Then, q-PCR was performed with Power SYBR green PCR Master Mix using a 7900HT fast real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). The relative mRNA levels were expressed according to the 2−ΔΔCt method and normalized to those of the endogenous control (GAPDH). The primer sequences are listed in Supplemental Table 1 (Additional file 1).
Low-intensity pulsed ultrasound stimulation
LIPUS irradiation was performed using an ultrasound machine including an ultrasonic generator (Agilent Technologies, Santa Clara, CA, USA), a broadband power amplifier (Verasonics, Inc., USA), and a planar transducer (Haifu, Chongqing, China). LIPUS stimulation was applied for 20 minutes at a planar transducer frequency of 0.5 MHz and an intensity of 19.30-120.63 mW/cm2 in 10-ms pulse bursts (Additional file 2: Supplemental Table 2). The number of cycles was 100, and the spatial-temporal average sound pressure was 0.3 MPa. The 60-mm culture dishes seeded with cells were placed on the top of the transducer filled with deaerated water. In addition, we used a temperature test paper (TMCHallcrest, USA) to test the temperature of the culture media in the dishes, and the results showed that there were hardly any changes in temperature under our ultrasound conditions during LIPUS procedures (Additional file 3: Fig. S1).
Flow cytometry
After different treatments, the collected viable cells and cell debris were incubated with propidium iodide (PI) and Annexin-V (Fcmacs Biotech Co., China) in the binding buffer away from light at room temperature for 25 minutes following the manufacturer's use instructions. Then, the flow cytometry method was applied to analyze the relative ratio of cell apoptosis.
Masson’s and Sirius Red staining
After dewaxing and rehydration, 5-µm-thick formalin-fixed, paraffin-embedded tissue sections were stained with Masson’s and Sirius Red staining (Service Biological Technology Co., Ltd, Wuhan, China) using standard procedures. Then, more than 3 randomly selected whole-section images were taken with a Zeiss fluorescence upright microscope (Carl Zeiss, Jena, Germany) and measured to assess the percentage of myocardial fibrosis with Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Bethesda, MD, USA).
Wheat Germ Agglutinin (WGA) and Hematoxylin–Eosin (HE) staining
To determine the area of cardiomyocyte cross-sections, the deparaffinized and rehydrated paraffin-embedded sections were stained with WGA Alexa Fluor 647 (1:500 dilution; Invitrogen Life Technologies, Carlsbad, CA) in combination with the secondary antibody and examined by HE staining (Service Biological Technology Co., Ltd, Wuhan, China) according to the provided protocols. A minimum of 5 random fields were observed and chosen by a Zeiss fluorescence upright microscope (Carl Zeiss, Jena, Germany); they were further analyzed using ImageJ software in a blinded manner.
Immunofluorescence analyses
NRCMs seeded in culture dishes were blocked with PBS containing 5% goat serum, 5% BSA, and 0.5% Triton for 1 hour at room temperature after fixation with 4% paraformaldehyde and permeabilization with 1% Triton. Then, cells were incubated with anti-actinin antibody (1:150; Sigma-Aldrich Co., USA) and caveolin-1 (Cell Signaling Technology, USA) overnight at 4 °C, followed by incubation with rabbit IgG antibody (Alexa Fluor 488) and dilution with DAPI at room temperature. After that, a Zeiss fluorescence inverted microscope was used to digitize more than 3 fields of view in each group at random. The images were blindly analyzed with Image J software.
Statistics
Data are presented as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was applied to determine statistical significance for experiments with more than two groups, followed by Bonferroni’s post hoc tests (NS indicates not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Statistical analyses were performed using GraphPad Prism 7.0 software (GraphPad software Inc., CA, USA).