Heart failure patients and control subjects

Nine patients (5 men and 4 women) with chronic symptomatic heart failure were studied. Control subjects were seven healthy donors (4 men and 3 women). We cut out parts of the myocardium of heart failure patients when they received heart transplants. In addition, we obtained the control myocardium with the donor’s heart. We described the clinical baseline data of patients in Table 1.

Table 1 Clinical baseline data of the patients involved in this study Mice models

Six to eight weeks of age C57BL/6 J wild-type mice (male) were used in our study. We injected Ang II (1500 ng/kg/min) for 15 days subcutaneously to establish a heart failure model. The CCL24 blocking antibody was administrated intravenous injection to mice beginning 1 day after Ang II injection and continued three times a week during Ang II stimulation. We killed all the mice by cervical dislocation after isoflurane anesthesia (5 vol%).

Reagents and antibodies

CCL24 and TGF-β were acquired in Sigma Chemical Co. Primary antibodies against CCR3, CCL24, Col1a1, Col3a1, CCN2, a-SMA, TGF-β, and CCN2(CTGF) were collected from Cell Signaling Technology (CST). We obtained α-tubulin antibodies from Abcam. We purchased goat IgG-HRP antibodies (anti-rabbit and anti-mouse) from CST. The blocking antibody of CCL24 was purchased from PEPROTECH.

Echocardiographic

We performed an echocardiographic assay using a high-frequency linear array transducer, and we obtain two-dimensional images at mid-papillary and apical levels, and an echocardiographic assay was performed for anesthetized mice. We obtained end-diastolic and end-systolic left ventricular volumes by biplane area–length method. Then, the left ventricular ejection fraction was calculated automatically.

Langendorff-perfused isolated mice hearts

We killed mice humanely through intraperitoneal injection with pentobarbital sodium. The heart was mounted rapidly onto a Langendorff apparatus. Then, perfused the isolated hearts with a Tyrode solution at the flow rate of 3 mL/min at 37 °C.

Electrical mapping of isolated heart

We recorded extracellular potentials (ECP) from epicardium in a multi-electrical array (MEA) mapping system (Mapping Lab Ltd., UK). Left and right atrial simultaneous recordings were performed with two 64-channel MEA. To assess atrial fibrillation (AF) inducibility, the stimulator was placed on the left atrium with burst pacing. The detail of the burst pacing is as follows: 10 s burst pacing at a cycle length of 20 ms with a pulse duration of 5 ms, and then stabilized for 4 min. Repeated burst pacing 8 times with 4 min of stabilization each time. AF was defined as a rapid and irregular atrial rhythm (rapid and irregular ECP) lasting at least 1 s.

High-resolution optical mapping of isolated hearts

We performed high-resolution optical mapping of isolated mice hearts. In brief, to arrest cardiac motion using excitation–contraction uncoupler Blebbistatin (10 μM). Then, load the membrane potential fluorescent dye Rh237 and calcium fluorescent dye Rhod2-AM to optically map membrane potential and calcium signal. We captured fluorescence changes with a high-speed camera (900 frames/s, OMS-PCIE-2002, Mapping Lab Ltd.), and the spatial resolution in our experiments was 128-by-128 pixels. We analyzed related data with O Map Scope 5.0 software (Mapping Lab Ltd.).

Immunohistochemistry assay

We fixed the heart tissues with 4% paraformaldehyde and then embedded the heart tissues in paraffin. An immunohistochemistry assay was carried out with paraffin sections to evaluate hypertrophy, fibrosis, and inflammation of the heart. We dehydrated heart sections in the graded methanol series. Then antigen retrieval was performed using a microwave in citrate buffer. Then, we washed the sections in PBS, and we incubated the sections with 3,3-diaminobenzidine tetrahydrochloride (DAB) for several minutes.

Real-time quantitative polymerase chain reaction (RT-qPCR)

We extracted RNA with TRIZOL as described by the manufacturers’ instructions. We reverse-transcribed 500 ng of RNA with SuperScript VILO DNA Synthesis Kit (Takara). We carried out real-time (RT) qPCR with an ABI RT PCR Detection System with SYBR Green Supermix (Takara). We analyzed the expression of target genes using the ΔΔCt method.

Western blotting

We subjected proteins by sodium dodecyl sulfate–polyacrylamide gels. Then, we transferred the proteins to polyvinylidene difluoride membranes. Followed by blocking with 5% non-fat milk for 60 min, these membranes were incubated with the primary antibodies for 4 h at room temperature (RT). Next, membranes were incubated with secondary antibodies for 60 min at RT. The membranes were visualized by electro-chemiluminescence plus reagent (Merck Millipore Ltd.). The intensity was quantified with Chemiluminescence Imaging System (SHST).

Isolation and cell culture of cardiac primary fibroblasts

We isolated cardiac primary fibroblasts with the cold trypsin digestion method according to our protocol and grew with Fibroblast Medium-2 (FM-2; ScienCell).

In brief, myocardial tissues from neonatal SD rats were incubated with the 4 °C trypsin for 10 h; then, these myocardial tissues were digested with the warm trypsin every 5 min which was performed five times repeatedly. Then, cardiac primary fibroblasts were collected with density gradient centrifuging.

Monolayer wound healing experiments

Cardiac primary fibroblasts in 6-well plates grew to 80–90% confluence and then were serum-starved for 24 h. Then, one scratch in each well was made using a 100–1000 μl tip. After being rinsed twice with phosphate buffer saline, we incubated cardiac primary fibroblasts with FM-2. Then, we took images of the same regions at 0 or 24 h after stimulation and analyzed them.

Cell migration assay with Boyden transwell chamber

We performed migration assay with Boyden transwell chamber (8 µm pore size membrane filter, Corning, NY, USA). Cardiac primary fibroblasts (2 × 104) were grown in the upper chamber. The medium used in the upper chamber is FM-2 containing 0.5% FBS. The medium used in the lower chamber is FM-2 containing 10% FBS. After incubation, we fixed these fibroblasts which migrated to the bottom surface of the filter; then, the membranes on slides were mounted with a mounting medium. Finally, we quantified and analyzed the number of migrating fibroblasts from microscopic images of transwell membranes.

Immunofluorescence staining

We fixed cells with 4% paraformaldehyde for 15 min at room temperature, and rinsed cells twice with PBS. Then, to permeabilize cell membranes, we treated cells with 0.1% Triton X-100 for 5 min. 5% BSA was added to cells and let stand 1 h at room temperature to block nonspecific sites of antibody adsorption. Cells were then incubated with the primary antibody overnight at 4 °C, then rinsed cells three times with PBS. Then, cells were incubated with fluorophore-conjugated secondary antibody for 1 h in the aluminum foil at RT. We washed cells twice with PBS; then, nuclei were stained with DAPI for 10 min. All the samples were rinsed with PBS again and finally observed using a confocal microscope.

RNA-seq

We extracted total RNA from cardiac tissues and cells with RNeasy mini Kit (Qiagen), and then we prepared RNA samples using TruSeq RNA Sample Preparation Kit to construct mRNA library and deep sequencing according to related protocol. Finally, we performed cluster formation and sequencing on the HiSeq X Ten platform on the basis of standard sequencing protocols.

Bioinformatic analysis of RNA-seq data

Bioinformatics analysis was performed of the differentially expressed genes in three groups on the basis of the GSEA (Gene Set Enrichment Analysis) and Gene Ontology (GO) database. The functional assignments were mapped onto Gene Ontology (GO). Genes were compared with the Kyoto Encyclopedia of Genes and Genomes database (KEGG) by using BLASTX4 at E values <  = 1e − 10. Then, a Perl script program was used to retrieve KO information from the blast results, and associations between genes and pathways were established. KEGG pathway and GO enrichments were analyzed in a command-line program KOBAS 2.0. We used the whole genome as the default background distribution to identify the significantly enriched pathways statistically in a set of sequences. For each pathway that occurs in the set of genes, we counted the total number of genes in the set that were involved in the pathway term or the GO term. We calculated the p value using a hypergeometric distribution.

CytoF

Take out the mouse heart tissue from the tissue storage solution and wash twice using a cell 1640 culture medium. Cut the tissue into 1 mm^3 pieces. Collect the pieces into a centrifuge tube and add digestive enzyme mix (C-IV (2 mg/mL) + H (250 µg/mL) + D (20 µg/mL), and fill with cell culture medium up to 5 mL. Incubate the tissue soup in a shaking incubator at 37℃ for 1 h. Filter the dissociated tissue through the 70 μm cell strainer. Centrifuge these cells at 400 g for 5 min at 4℃ to collect the cells. Aspirate the supernatant and resuspend the cells in FACS Buffer and count cell number. Using 36% Percoll to remove debris by centrifuging at speed of 400 g 30 min. Add 103Rh + mouse filler splenocytes (2021-Nature Protocols-Mass cytometry profiling of human dendritic cells in blood and tissues) into mouse heart single-cell suspensions. Prepare Cell-ID Cisplatin Solution in PBS (final concentration of 250 nM 194Pt), resuspend cells in 100 μL Cisplatin Solution, and incubate for 5 min on ice. Wash cells twice by adding 1 mL FACS Buffer to each tube, centrifuge at 400 g for 5 min at 4 ℃, and discard supernatant by aspiration. Add Fc-receptor blocking solution to each tube and incubate for 20 min on ice. Prepare Antibody Cocktail in FACS Buffer (see Antibody Cocktail Table), add 50 μL of the antibody cocktail to each tube, and incubate for 30 min on ice. Following the incubation, wash cells twice by adding 1 mL FACS Buffer to each tube, centrifuge at 400 g for 5 min at 4 ℃, and discard supernatant by aspiration. Prepare cell intercalation solution in Maxpar Fix and Perm Buffer (final concentration of 250 nM 191/193Ir. Add 200 μL of the cell intercalation solution to each tube and gently vortex, leave overnight at 4 ℃. Wash cells by adding 1 ml FACS Buffer, centrifuge at 800 g for 5 min at 4 ℃, and discard supernatant by aspiration. Wash cells with 2 mL of deionized water, centrifuge at 800 g for 5 min at 4 ℃, and discard supernatant by aspiration. Resuspend cells in 1–2 mL deionized water, count cells. Centrifuge at 800 g for 5 min at 4 ℃ and discard supernatant by aspiration. Samples are ready for CyTOF. Turn on the CyTOF, ensemble sample introduction system. Run a Tuning and QC procedures to calibrate the CyTOF using Tuning solution and EQ beads. Resuspend and adjust cell concentration to 1 × 10^6/mL with deionized water containing 20% EQ beads. Transfer cells through a 40-μm filter to a new FACS tube, acquire data on a CyTOF system. Data of each sample were debarcoded from raw data using a doublet-filtering scheme with unique mass-tagged barcodes. Each.fcs file generated from different batches was normalized through bead normalization method. Manually gate data using FlowJo software to exclude to 103Rh + splenocytes, debris, dead cells, and doublets, leaving live single cells. Apply the X-shift clustering algorithm to all cells to partition the cells into distinct phenotypes based on marker expression levels. Adjust clustering parameters to obtain suitable number of clusters. Annotate cell type of each cluster according to its marker expression pattern on a heatmap of cluster vs marker. Use the dimensionality reduction algorithm t-SNE to visualize the high-dimensional data in two dimensions and show distribution of each cluster and marker expression and difference among each group or different sample type. Perform T-test statistical analysis on the frequency of annotated cell population.

Statistical approach

Experimental data were displayed as mean ± SD and p ≤ 0.05 means statistically significant. Means of two groups were compared using Student’s t test (unpaired, 2-tailed), and for comparison of more than two groups, one-way ANOVA was used. We performed statistical analysis with SPSS 20.0 statistical software.

Ethical statement

All mice experiments were performed according to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes, and all animal procedures were approved by the Animal Care and Use Committee of Zhejiang University. Informed consent was obtained from all participants in accordance with the guidelines of the Human Subjects Committee of the Medical Ethical Commission of the First Affiliated Hospital of Zhejiang University (China) and the Declaration of Helsinki.