Experimental animals
Eight-week-old 20 ± 2 g C57BL/6 J male mice (n = 31) were supplied by Ensville Biological Technology Co., Ltd. (Chengdu, Sichuan, China). The mice were housed at a constant temperature (23 ± 1 °C) under a 12/12-h light–dark cycle with free access to food and water. All procedures were approved by the Ethics Committee for Animal Care and Use of Sichuan University, and all procedures were conducted in accordance with the guidelines of the National Institutes of Health Animal Care and Use Committee (No. 2020267A).
Animal groups
Mice were randomly allocated to the sham-operated group (Sham, n = 13), the myocardial infarction group (MI group, n = 9), and the acupuncture group (MI + PC6 group, n = 9, Neiguan acupoint). The surgical procedures were performed as previously described [20, 21]. In brief, mice were anaesthetized by 3% isoflurane with 99.5% O2 and maintained under anaesthesia by 1–1.5% isoflurane. The left anterior descending (LAD) coronary artery was permanently ligated using a 7/0 monofilament suture (Shanghai Pudong Jinhuan Medical Instrument Co., Ltd.) to induce myocardial ischaemia. The ligation was confirmed by the pale appearance of the apex and anterior wall of the left ventricle. After the surgery, the mice were placed on a warm cushion until they were awake. Mice in the sham group were also subjected to the same procedure except for LAD ligation. The Lead II electrocardiogram was monitored before and after the operation.
Acupuncture intervention
The MI + PC6 group mice underwent acupuncture intervention awake at PC6 acupoint bilaterally after 24 h of MI operation once a day for 28 days. The acupuncture needles were folded into an “L” shape by hemostatic forceps and inserted into PC6 acupoint about 5 mm. Then the needles were fixed with adhesive tape. We also made sure that the needles would not fall off and strengthen the stimulation of acupuncture intervention every 5 min. The needles were removed after 15 min of intervention. Details for animal handling and fixation procedures are the same as our previous study [22]. PC6 was located at a point 1.5 cm proximal to the palm crease just above the median nerve (the experimental protocol and acupoint locations are depicted in Fig. 1).
Electrocardiogram recording
Electrocardiograms were performed after 4 weeks of treatment. All mice were anaesthetized in the chamber at 3% isoflurane, carefully positioned on the electrocardiogram (ECG) recording platform and attached to a mask under 1.5% isoflurane. Surface lead II electrocardiogram was obtained. To minimize stress, we accomplished the electrode setup and system adjustment within 5 min, and thus, the first 5 min for each mouse were not included in our ultimate analysis. The next 5 min of ECG recordings were analysed by LabChart 8.2.3 (AD Instruments, Australia).
Echocardiography analysis
After finishing the 28-day acupuncture treatment, all mice underwent transthoracic echocardiography under 1% isoflurane anaesthesia to characterize the effects of PC6 on cardiac structure and function using an ultrasound system (Vevo 3100, FUJIFILM Visual Sonics, Inc., Canada) equipped with an MX550D detector (25–55 MHz) of a wide-band frequency-fusion phase-array transducer. The heart was visualized in B mode from a long axis view. Left ventricle ejection fraction (EF) and fractional shortening (FS) were calculated from the measurements of wall thickness and chamber diameters. Left ventricle posterior wall thickness at diastole (LVPWd) and left ventricle anterior wall thickness at diastole (LVAWd) were measured in M-mode.
Biochemical analyses
The serum levels of cTnT (JM-11710M1), TNF-α (JM-02415M1), IL6 (JM-02446M1), IL-10 (JM-02459M1), renin (JM-02627M1) and brain natriuretic peptide (BNP, JM-02343M1) were measured using commercially available kits (Jiangsu Jingmei Biological Technology Co. Ltd., China) in accordance with the manufacturer's instructions. All the methods and procedures strictly followed the protocols of the test kits.
Haematoxylin and eosin staining
The ischemic heart tissues were dissected and then fixed with 4% paraformaldehyde immediately. Haematoxylin and eosin staining was performed on serial sections (4 μm thick) of paraffin-embedded heart tissues. Briefly, the sections were dewaxed in xylene, rehydrated in descending grades of ethanol and washed in distilled water. Excess water was blotted from the slides before haematoxylin staining. The tissues were stained in haematoxylin solution for 3–5 min, differentiated in acid alcohol, and then dipped in ammonia solution. The sections were washed in distilled water, re-hydrated in descending grades of alcohol, stained in eosin solution for 5 min and washed in distilled water for 1 min. Each mount was allowed to spread beneath the coverslip, covering all the tissues. Images were acquired using a microscope (NIKON Eclipse Ci, NIKON, Japan) and analysed with an image analysis system (NIKON Digital Sight DS-FI2, NIKON, Japan).
Masson’s trichrome staining
All heart tissues from the area equidistant at the papillary muscle level between the ligation point and the apical section were subjected to 4% paraformaldehyde fixation and paraffin embedding. The sections were dewaxed in xylene, rehydrated in descending grades of ethanol and washed in distilled water. The sections were stained in iron haematoxylin solution for 3 min, differentiated in acid alcohol solution, washed in distilled water (kits from Beijing G-CLONE Biological Technology Co., Ltd., China, RS3960). The sections were stained in Ponceau acid fuchsin for 5–10 min and rinsed in distilled water. The slides were placed in phosphomolybdic acid solution for 1–3 min and then stained with aniline blue solution for 3–6 min. The sections were differentiated in 1% glacial acetic acid and dehydrated in ethanol, followed by xylene for 5 min. Each mount was allowed to spread beneath the coverslip by covering all the tissues. Images were acquired using a microscope (NIKON ECLIPSE E100, Japan) with an image analysis system (NIKON DS-U3, Japan). Fibrosis was analysed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
Immunohistochemistry
Mice were euthanized and perfused with PBS or fixative. All heart tissues from the area equidistant at the papillary muscle level between the ligation point and the apical section were immersion-fixed in 4% paraformaldehyde. Tissues were trimmed, embedded, sectioned and stained for TNFα (1:200, sc-52746, Santa Cruz Biotechnology, USA). Goat polyclonal secondary antibody to rabbit IgG (H&L) was purchased from BioVision (1:1000, 6927-100, California, USA).
Western blotting
Protein samples were extracted from the area equidistant at the papillary muscle level between the ligation point and the apical section in cold RIPA lysis buffer (MB-030–0050, Multi-Sciences Biotech, Hangzhou, China) containing a complete protease inhibitor cocktail (11697498001, ROCHE, Switzerland) and phosphatase inhibitor (4906837001, ROCHE, Switzerland). Protein samples were separated by SDS–polyacrylamide gel electrophoresis and transferred to a 0.22 μm PVDF membrane (PI88520, Millipore, USA), which was detected using specific primary antibodies (anti-TH, 1:1000, 2792, Cell Signaling Technology, USA, anti-p-TH, 1:1000, 2791, Cell Signaling Technology, USA, anti-ACHE, 1:1000, PA5-95250, Invitrogen, USA). Bound antibodies were detected using rabbit peroxidase-conjugated secondary antibody and visualized by enhanced chemiluminescence (RK-18-8816-31, Multi-Sciences Biotech, China) in a chemiluminescence imaging system (Chemi Scope 6100, Clinx Science Instruments, China). The band intensity was quantified by using Image J (National Institutes of Health, USA).
RNA-seq and computational analysis for RNA-seq data
Extracted RNA from the area equidistant at the papillary muscle level between the ligation point and the apical section was qualified by using an Agilent 2100 Bioanalyzer (Agilent, 1309, Agilent Technologies, Inc. CA, USA) according to the manufacturer’s protocols. The RNA library was prepared according to the TruSeq RNA Sample Preparation v2 (Illumina, 15025062) protocol, followed by cluster generation and sequencing using a cBot Multiplex rehybridization plate and TruSeq SBS kit V3 (Illumina, 15021668). Sequencing was performed using an Illumina HiSeq 2000 (Illumina, USA). Data analysis was performed as previously described [22]. Before read mapping, clean reads were obtained from the raw reads by removing the adaptor sequences, reads with > 5% ambiguous bases (noted as N) and low-quality reads containing more than 20% of bases with qualities of < 20. The clean reads were then aligned to the mouse genome (version: mm10 NCBI) using hisat2 [23]. We applied the EBSeq algorithm to filter the differentially expressed genes [24] after the significance analysis, P value and FDR analysis under the following criteria [25]. mRNA under the following criteria: i) fold change > 2 or < 0.5 and ii) FDR < 0.05. The gene functional annotation and pathways were analysed using DAVID Bioinformatics Resources.
qPCR
Total RNA was isolated from the area equidistant at the papillary muscle level between the ligation point and the apical section using a Fast Pure Cell/Tissue Total RNA Isolation Kit (RC101-01, Vazyme, Nanjing, China), and the concentration of isolated RNA was determined with a Qubit RNA BR assay (Invitrogen, Q10211, California, USA). Then, cDNA was prepared using HiScript Q RT Super Mix for qPCR (+ gDNA wiper) (R123-01, Vazyme, Nanjing, China) according to the manufacturer’s instructions. The mRNA levels were assessed on an ABI QuantStudio6 Q6 Real-time PCR system (ABI, USA) by qPCR using ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme, Nanjing, China). The relative expression of mRNA was calculated by △△Ct according to standard methods (The primer sequences were as follows: Cd84, forward: 5′-TTCCTCAGTGCAGCTTTCT-3′, reverse: 5′-CCTTGTGTCCTTCGTGGT-3′, Cd180, forward: 5′-GCAAGCCACTAATCTGAGC-3′, reverse: 5′-GTCCCCAGCCAAAGAGA-3′, Ccr2, forward: 5′-AAGGGTCACAGGATTAGGAAG-3′, reverse: 5′-ATGGTTCAGTCACGGCATA-3′, Ccr5, forward: 5′-GTGCCTGACTGCCAACA-3′, reverse: 5′-GAGACTACCTTCCCGGCTA-3′, Cx3cr, forward: 5′-TGTGCGGTCATCCTGTC-3′, reverse: 5′-CATCTCCCTCGCTTGTGT-3′, Tnf-α, forward: 5′-CGCTGAGGTCAATCTGC-3′, reverse: 5′-GGCTGGGTAGAGAATGGA-3′, Il6, forward: 5′-GCCTTCTTGGGACTGATGCT-3′, reverse: 5′-TGCCATTGCACAACTCTTTTC-3′, Scn5a, forward: 5′-GGAGGGTTGTGGTTCCTGT-3′, reverse: 5′-GTCCCTGCGGCCTATGT-3′, KChIP2, forward: 5′-AATCCCGGCAGCGCCTA-3′, reverse: 5′-CCCGGCGCTCACACA-3′, Kcne1, forward: 5′-ACATACCACACAGCAAGGGG-3′, reverse: 5′-CGATGTACTGGTGGTACGGG-3′, Cyp11b2, forward: 5′-TGAACTGAAGACAGGAGGATGG-3′, reverse: 5′-GGTATGGCTTCAAAGGGCTG-3′.)
Statistical analysis
All data are presented as the mean ± SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey’s test using GraphPad Prism 7.0 (GraphPad Software, Inc., CA, USA) and SPSS 20.0 software (IBM, Chicago, USA). A value of P < 0.05 was considered to be statistically significant.