Danqi soft capsule prevents infarct border zone remodelling and reduces susceptibility to ventricular arrhythmias in post-myocardial infarction rats

2.1 Ethics statement

All animal procedures were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and the National Standard of the People's Republic of China for Laboratory animal Guidelines for ethical review of animal welfare.

2.2 DQ preparation and quality control

The DQ extraction procedure and ultra-high performance liquid chromatography (UPLC) are described in the supplementary file.

2.3 Animal model of myocardial infarction and drug administration

The MI model was carried out as our previously described.18 Male Sprague-Dawley rats (with the weight of 250-280 g, SPF, Guangdong Medical Experimental Animal Center) were anaesthetized. With the heart exposed by a 1.2 cm lateral thoracotomy, the left coronary artery was permanently ligated 2 mm below the tip of the left auricle with 6-0 nylon silk. The MI model was considered successfully established when myocardial blanching and cyanosis were visualized within the downstream myocardium. Thorax closure was done with three layers of sutures. The sham-operated animals (Sham, n = 15) underwent the same procedure except that the silk suture was placed around the left coronary artery without being tied.

One week after MI surgery, all survival rats underwent echocardiography and were randomly grouped into MI group (MI, n = 15), low-dose DQ group (DQ-L, n = 15), middle-dose DQ group (DQ-M, n = 15) and high-dose DQ group (DQ-H, n = 15). DQ rats were orally gavaged with DQ (batch number 14060108; Beijing Changcheng Pharmaceutical Factory, Beijing, China) at a dose of 0.6 g/kg d in the DQ-L group, 0.9 g/kg d in the DQ-M group and 1.2 g/kg d in the DQ-H group started from 1 week after MI surgery for 4 weeks, which was derived from the equivalent conversion between animals and human by body surface area, based on the recommended daily human dosage of DQ.19 Other rats were gavaged with an equivalent volume of water. After 4 weeks of treatment, the rats in each group underwent echocardiographic and electrophysiological examinations as described below. Following these procedures, rats were killed and their hearts and blood removed for weighing and other assessments, as also described below.

2.4 Echocardiogram

All rats were lightly anaesthetized with 2% sevoflurane. Transthoracic echocardiography was performed using a 21-MHz phased-array probe (Vevo 2100, VisualSonics Inc, Canada). Echocardiography was performed in the parasternal short axis B- and M-modes at the papillary muscle level to obtain left ventricular (LV) wall thickness and ejection fraction (EF), and in the parasternal long-axis view to measure LV end-systolic and end-diastolic volumes (LVESV and LVEDV). Each echocardiographic variable was determined in at least four separate LV images acquired from the same heart. The observer was unaware of grouping and treatment.

2.5 Programmed electrical stimulation (PES)

Programmed electrical stimulation (PES) in this study was performed essentially as described by Nguyen T20 and Nguyen DT.4 Induction of ventricular arrhythmias was attempted by ventricular stimulation at a basic cycle length of 120 ms (S1) for eight beats, followed by one to three extra-stimuli (S2, S3 and S4) at shorter coupling intervals. The end-point of PES was induction of a VT consisting of at least six consecutive non-driven ventricular extra-stimulus beats. A preparation was considered to be non-inducible when PES produced less than six beats. Distinction was not made between ventricular tachycardia and fibrillation. A VT was considered to be non-sustained if lasting for 15 beats, and sustained if lasting for >15 beats. A PES-induced ventricular arrhythmias scoring system was used: 0, non-inducible preparation; 1, non-sustained tachyarrhythmias induced with three extra-stimuli; 2, sustained tachyarrhythmias induced with three extra-stimuli; 3, non-sustained tachyarrhythmias induced with two extra-stimuli; 4, sustained tachyarrhythmias induced with two extra-stimuli; 5, non-sustained tachyarrhythmias induced with one extra-stimulus; 6, sustained tachyarrhythmias induced with one extra-stimulus; 7, tachyarrhythmias induced during the 20 paced beats at a basic cycle length of 100 ms. If the heart stopped before PES, an arrhythmia score of 8 was assigned.

2.6 Histological analysis

Several sections of each heart (5-µm thick) were prepared, and stained with haematoxylin and eosin for histopathology. Masson's trichrome staining was performed as previously described.21 Images were digitized using a digital camera (DP 72, Olympus) under a BX53 microscope (Olympus, Tokyo, Japan). Images were quantified by the CellSens Dimension 1.16 software (Olympus, Tokyo, Japan). The infarct area was expressed as a percentage of the fibrotic scar circumference to the total circumference; the fibrotic area was expressed as a percentage of red-positive stained area to total tissue area in the IBZ, which was defined a 3-mm zone adjacent to the infarcted area22 using the software.

2.7 Immunohistochemical assay

Immunohistochemical staining was performed as described previously.23 Heart sections were stained with anti-connexin (Cx) 43 antibody (1:200, Cell Signaling Technology), with rabbit IgG or serum used instead of primary antibody in negative controls. Peroxidase activity was visualized with the use of diaminobenzidine, and sections were counterstained with haematoxylin. Images were obtained using a DP 72 camera attached to a BX53 microscope (Olympus, Tokyo, Japan). Five fields were selected randomly from each slide for quantitation with the CellSens Dimension software (Olympus, Tokyo, Japan). The percentage of Cx43-positive staining area represented Cx43 expression.

2.8 Quantitative real-time polymerase chain reaction

After extraction of total ribonucleic acid (RNA) from the mouse hearts using the TRIzol reagent (Invitrogen Life Technologies, USA), RNA was reverse transcribed to produce complementary DNAs (cDNAs) using the Prime Script RT reagent kit (TaKaRa, Japan). cDNA was used as a template for quantitative real-time polymerase chain reaction (qRT-PCR) and gene expression was normalized to glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The specific forward and reverse primers used were as follows: rats atrial natriuretic peptide (ANP) forward: 5’-CCTGGACTGGGGAAGTCAAC-3’ and reverse 5’-GTCAATCCTACCCCCGAAGC-3’; rats GAPDH forward: 5’-ATCCGTTGTGGATCTGACATG-3’ and reverse 5’-CAAAGGTGGAAGAATGGGAGT.

2.9 Western blot

Protein concentration was determined using a BCA Protein Assay Kit (Novagen, Darmstadt, Germany). Low-molecular weight marker (Cell Signaling Technology, Beverly, MA) and 50 μg of total protein from samples were separated by 10% SDS-PAGE. Separated proteins were transferred to a polyvinylidene difluoride membrane, which was blocked at room temperature for 1 hour in Tris-buffered saline with 0.2% Tween 20 containing 5% skim milk and probed with primary antibodies overnight at 4°C. The diluted concentrations of the primary antibodies (Abcam,�Cambridge,�UK or Cell Signaling Technology, Beverly, MA, USA) were as follows: collagen I (1:500), collagen III (1:500), connexin (Cx) 43 (1:1000), α-smooth muscle actin (α-SMA, 1:500), transforming growth factor β1 (TGF-β1, 1:1000), mothers against decapentaplegic homolog 3 (Smad3, 1:500), phospho-Smad3 (p-Smad3, 1:500) and GAPDH (1:1000). Secondary antibodies (Cell Signaling Technology, Beverly, MA) included horseradish peroxidase-labelled were diluted 1:1000 and incubated for 1h at room temperature. Protein bands on Western blots were visualized with ECL Plus (Millipore, Billerica, MA). After immunoblotting, the film was scanned and the intensity of immunoblot bands was detected with the ImageJ (NIH imaging) analysis software. Relative protein band intensities were based on GAPDH signals. The final results were expressed as fold changes by normalization to control values.

2.10 Enzyme-linked immunosorbent assay

Blood was taken from the abdominal aorta. The serum was separated by centrifugation and immediately stored at −80°C until further analysis. The serum levels of brain natriuretic peptide (BNP), monocyte chemoattractant protein (MCP)-1 and TGF-β1 were determined by ELISA using commercially available kits (RD, RD Systems, Minneapolis, USA), according to the manufacturer's instructions. Absorbance was measured at 450 nm with background reading at 570 nm on a microplate reader. The concentration of each sample was calculated according to the standard curve. All concentrations were expressed in pg/mL.

2.11 Data analysis and statistics

Data are mean ± standard deviation. All differences were normally distributed variables except arrhythmia scores and were examined by one-way anova; post hoc analyses were performed by Bonferroni correction for multiple comparison. Arrhythmia scores were examined by Kruskal-Wallis anova, with post hoc Dunn's test. The SPSS statistical software (SPSS, IL) was used for statistical analyses, and P < 0.05 indicated statistical significance.

3.1 DQ improves cardiac function after MI

Five weeks after MI modelling, echocardiography showed clear anterior wall motion abnormality (Figure 1A), and ventricular systolic dysfunction was significantly reduced in the MI group (Figure 1B,C). EF in MI rats decreased from 54.1 ± 3.7% at baseline to 29.5 ± 3.1% compared with the Sham group (P < 0.01, Figure 1B). Left ventricle fraction shortening in MI rats decreased from 34.4 ± 3.6% to 15.1 ± 1.3% compared with the Sham group (P < 0.05, Figure 1C). However, rats administered DQ at low, middle and high doses, respectively, showed significantly increased EF (P < 0.05, Figure 1B) and FS (P < 0.05, Figure 1C) compared with the MI group. Both diastolic LV internal dimension (LVIDd, P < 0.01, Figure 1D) and end-systolic LV internal dimension (LVIDs, P < 0.01, Figure 1E) were elevated in the MI group, but decreased after DQ treatment (both P < 0.05).

3.2 DQ inhibits susceptibility to VT in rats with MI

Figure 2A shows typical examples of no arrhythmia (a), non-sustained VT (b) and sustained VT (c) induced by programmed stimulation in rats. The rates of VT induction were 80% (12/15, including six sustained and six unstained VT) in the MI group, and 13.3% (2/15, both unstained VT) in the Sham group. VT induction rates in the DQ groups were significantly decreased (9/15, 60% in the DQ-L group; 7/15, 46.7% in the DQ-M group; 6/15, 40% in the DQ-L group; Figure 2B). Arrhythmia scores in the DQ-M and DQ-L groups were also lower than those of the MI group (P < 0.05, Figure 2C). The conduction velocity was lower in the MI group than in the Sham group (P < 0.01, Supporting Information). This decrease was reversed after administering DQ, with a recovery of conduction velocity observed in DQ-treated groups. These results suggested that DQ could reduce susceptibility to PES VT and increase conduction velocity after MI.

3.3 DQ inhibits cardiac fibrosis in IBZ

Histology confirmed large infarcted areas (Figure 3A), with an infarct percentage of 38% ± 3.6% in MI rats (Figure 3B); the fibrotic tissue within the IBZ accounted for 42.3 ± 4.5% of total area (Figure 3C). DQ effectively decreased the total infarct areas (P < 0.05, Figure 3B) and fibrotic areas in the IBZ (P < 0.05, Figure 3C). To quantify the impact of DQ on collagen amounts, type I and III collagen were detected by Western blot. MI resulted in significantly increased type I and III collagen deposition (Figure 3D and Figure 3E). DQ treatment markedly reduced type I (P < 0.05 in the DQ-L group, and P < 0.01 in the DQ-M and DQ-H groups; Figure 3D) and III (P < 0.05 in the DQ-L group, and P < 0.01 in the DQ-M and DQ-H groups; Figure 3E) collagen deposition.

3.4 DQ reverses Cx43 expression and distribution in the IBZ

Immunohistochemistry showed the Cx43 staining in LV sections of sham-operated rats, predominantly localized to the myocyte-myocyte junction corresponding to intercalated discs. Cx43 in the MI model group was clearly decreased and had a disordered and uneven distribution (Figure 4A). The immunohistochemical levels of Cx43 were significantly reduced in the IBZ in MI rats compared with Sham animals (P < 0.05, Figure 4B). Meanwhile, Western blot showed that Cx43 protein levels in the IBZ were markedly decreased in MI rats compared with Sham animals (P < 0.01, Figure 4C). The expression and distribution of Cx43 in the DQ group were significantly reversed compared with the MI group (P < 0.05 in the DQ-L and DQ-M groups; P < 0.01 in the DQ-H group).

3.5 DQ inhibits myocyte hypertrophy in the IBZ

Five weeks post-MI, rats developed significantly increased heart weight (HW)/bodyweight (BW) ratios (P < 0.05, Figure 5A) and LV weight (LVW)/HW ratios (P < 0.05, Figure 5B); meanwhile, DQ inhibited the above indexes (P < 0.05 in the DQ-M and DQ-L groups). Haematoxylin and eosin staining was used to assess myocyte morphology and transverse diameters (Figure 5C). Five weeks post-MI, animals developed a higher degree of hypertrophy with a sparser myocyte distribution and increasing myocyte area; DQ treatment inhibited MI-induced myocyte hypertrophy and showed a denser myocyte distribution and reduced myocyte area (Figure 5D). Results of the qRT-PCR also showed that the enhanced expression levels of the hypertrophic markers natriuretic peptide ANP were significantly decreased in DQ rats in comparison to MI rats (Figure 5E). Collectively, our results suggest that DQ inhibited myocyte hypertrophy in IBZ.

3.6 DQ inhibits myofibroblast differentiation in MI rats and TGF-β1/Smad3 pathway

In MI rats, α-SMA was markedly increased in the IBZ compared with Sham rats (P < 0.01, Figure 6A). DQ treatment resulted in reduced protein expression of α-SMA compared with MI rats (P < 0.05 in the DQ-L and DQ-M groups, and P < 0.01 in the DQ-H group; Figure 6A). TGF-β1 levels were markedly increased in the MI group compared with the Sham group (P < 0.01, Figure 6B). DQ attenuated the TGF-β1 level increase after MI (P < 0.05 in the DQ-L group, and P < 0.01 in the DQ-M and DQ-H groups; Figure 6B). Smad3 amounts showed no difference among the five groups (P > 0.05, Figure 6C). But DQ decreased Smad3 phosphorylation (p-Smad3) after MI (P < 0.05 in the DQ-L group, and P < 0.01 in the DQ-M and DQ-H groups; Figure 6D).

3.7 DQ decreases the serum level of BNP, MCP-1 and TGF-β1

As shown in Figure 7, serum levels of BNP, MCP-1 and TGF-β1 were all increased in the MI group compared with Sham animals (P < 0.01). After 4 weeks of DQ treatment the increased levels of BNP, MCP-1 and TGF-β1 were reversed in the three different dose groups compared with the MI group (BNP, P < 0.01, Figure 7A; MCP-1, P < 0.05, Figure 7B; TGF-β1, P < 0.01, Figure 7C).