Tongguan capsule‐derived herb reduces susceptibility to atrial fibrillation by inhibiting left atrial fibrosis via modulating cardiac fibroblasts

Abstract Tongguan capsule is a compound Chinese medicine used to treat ischaemic heart diseases. This study aimed to investigate whether Tongguan capsule‐derived herb (TGD) has a preventive effect on atrial fibrillation (AF) in post‐myocardial infarction (MI) rats and to determine the underlying mechanisms. MI was induced by ligation of the left anterior descending coronary artery. TGD was administered to the post‐MI rats over a 4‐week period. The TGD‐treated rats had lower rates of AF inducibility and shorter AF durations than the MI rats. TGD improved the left atrial (LA) conduction velocity and homogeneity. It reduced the fibrosis‐positive areas and the protein levels of collagen types I and III in the left atrium. In vitro, it inhibited the expression of collagen types I and III by inhibiting the proliferation, migration, differentiation and cytokine secretion of cardiac fibroblasts (CFs). In conclusion, the current study demonstrated that TGD reduces susceptibility to AF and improves LA conduction function in rats with post‐MI by inhibiting left atrial fibrosis and modulating CFs. Targeting the CF population may be a novel antiarrhythmic therapeutic approach.

Atrial fibrillation has a complex pathogenesis that is associated with underlying cardiovascular diseases, including myocardial infarction (MI) and heart failure (HF). 3,4 Increased fibrosis is observed in the extracellular matrix of the atrial myocardium during HF in both animal models 5 and human biopsies. 6 Fibrosis in the left atrium is a key substrate component for maintaining AF. 7 Scientists have been investigating an upstream approach that can reduce atrial profibrotic signals, thereby preventing AF. 8,9 Accumulating evidence suggests that decreasing left atrial (LA) fibrosis in animal models can effectively inhibit AF susceptibility. [10][11][12] A growing number of studies have demonstrated the protective cardiovascular effects of Chinese herbal medicines. [13][14][15] Tongguan capsule (Chinese national patent number: CN101647856A) has long been used to treat ischaemic heart diseases, including acute coronary syndrome and angina pectoris in clinical practice. [16][17][18][19][20] Previous data have shown that Tongguan capsules decrease infarct size, mitigate ventricular remodelling and prevent ischaemia/reperfusion arrhythmia occurrence in MI animal models. 18,19,21 Tongguan capsule-derived herb (TGD) consists of a Tongguan capsule formula (ie, Astragalus mongholicus, Salvia miltiorrhiza, Hirudo medicinalis) and adds Ophiopogon japonicus. Based on the results of ultra high-performance liquid chromatography-tandem mass spectrometry, TGD contains a variety of established cardioprotective ingredients, such as salvianolic acid B, salvianolic acid A and rosmarinic acid (Supporting Information), most of which demonstrate significant antifibrotic effects on different organ fibrosis models, including cardiac fibrosis. [22][23][24] The present study aimed to investigate whether TGD can inhibit AF in post-MI stage by reducing LA fibrosis and to explore the underlying mechanisms.  Briefly, after drying, these herbs were mixed in proportion and macerated overnight at room temperature with distilled water 10 times (v/w). Then, the entire mixture was decocted twice for 2 hours each time. The filtrates were mixed and condensed at −0.09 Mpa, 60°C to a final concentration of 1 g/mL (weight of the original herbs/ vole of solution). The filtrate solution was slowly added to 95% ethanol to a final concentration of 70% ethanol, precipitated for 24 hours under refrigeration (4°C to 8°C), and then dried through spray drying at 60°C. The mean yield of TGD was 17.2% (w/w), according to the original herbs. The resulting powder was stored in sealed containers at −20°C and diluted to the concentrations needed with distilled water and filtered before use. The quality control of the TGD and compounds identification is described in the Supporting Information.

| Animal model and drug administration
The MI model was established using the method previously described. 25 Male Sprague-Dawley rats (weight 250-280 g, SPF, Guangdong Medical Experimental Animal Center) were anaesthetized. Intubation and mechanical ventilation were subsequently performed with a small-rodent ventilator (Harvard Apparatus, Holliston, MA, USA). 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 to be successfully established when myocardial blanching and cyanosis were visualized within the downstream myocardium.
Thorax closure was performed 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. The mortality rate during and 7 days after the MI surgery was 30%. No deaths were observed in the sham animals. In the infarcted groups, 20% of the animals died from intraoperative arrhythmia during the surgery, and 10% of the animals died during the recovery period. One week after the MI surgery, only the survived rats with LV ejection fraction (EF) <45% performed further experiments. The rats were randomly grouped into MI group (MI, n = 15), low-dose TGD group (TGD-L, n = 15) and high-dose TGD group (TGD-H, n = 15). There was no significant difference in cardiac function between the three groups (Supporting Information). TGD rats were gavaged at a dose of 0.6 g/kg/d in the TGD-L group or 1.2 g/kg/d in the TGD-H group 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 Tongguan capsules and according to a previous study of Tongguan capsules in rats. 26 Other rats were gavaged with an equivalent volume of water. No deaths were observed after 4 weeks of treatment in any of the groups.

| Programmed electrical stimulation technique
After 4 weeks of TGD or water treatment, induction of AF measurement was performed, as we described previously. 27 AF was defined as irregular, rapid atrial activation with varying electrogram morphology lasting ≥2 seconds. The atrial rates were typically >1500 bpm in rats, as described previously. 28,29 The trachea was intubated via the mouth and mechanically ventilated with a small-rodent ventilator (Harvard Apparatus, Holliston, MA, USA). For atrial stimulation, a 4-French quadripolar catheter was advanced through the esophagus and placed at the site with the lowest threshold for atrial capture.
Atrial pacing was performed at twice the diastolic threshold using two electrodes on the pacing catheter. Inducibility of atrial arrhythmias was tested by applying 35-second bursts, using the automated stimulator. The burst had a cycle length of 20 milliseconds and pulse width of 5 milliseconds. This series of bursts was repeated once. All rats were allowed 5 minutes of recovery in the sinus rhythm between stimulations for respiratory and circulatory recovery. AF was considered inducible if one or more bursts induced an episode of AF. Otherwise, AF was considered to be non-inducible. The longest record time was 30 minutes after the burst pacing. Data were sampled using Labchart software (AD Instruments, Sydney, Australia).
Heart rate, RR interval, the duration and probability of inducible AF episodes were analysed using Labchart software (AD Instruments).
Mean AF duration was based on the duration of rats with inducible AF.

| Multielectrode array measurement
After the induction of AF measurement, multielectrode array (MEA) measurement was performed as we described previously. 27 The heart was removed rapidly, and the left atrium from the isolated heart was separated and then immersed in Tyrode's solution. For

| Histology
Masson's trichrome staining of the Paraffin section prepared from bouin's fixed samples was performed as previously described. 31 After the induction of AF measurement, the heart was arrested in diastole by injecting 1.0 mL of 10% potassium chloride intraperitoneally and fixed in 4% paraformaldehyde for 24 hours. Cryosections (5 μm) were stained with Masson trichrome. Images were digitized using a digital camera (DP 72; Olympus, Tokyo, Japan) under a BX53 microscope (Olympus). Images were quantified by the CellSens Dimension percentage of red-positive stained area to the total tissue area using the software.

| Cell culture
Cardiac fibroblasts (CFs) were obtained from the adult rats' atrium using the enzymatic dissociation and characterized as previously described. 31 After rinsing in cold phosphate-buffered saline (PBS), the heart was minced and digested with trypsin and collagenase type II (Gibco BRL Co. Ltd., Grand Island, NY, USA) at 37°C. After a 1.5 hours period of attachment to uncoated culture plates, the cells that were weakly attached or unattached were discarded, and the attached cells (mainly fibroblasts) were washed and grown in Dulbecco minimum essential medium (DMEM) supplemented with 10% foetal bovine serum (FBS). The TGD could dissolve in water. We used the 4 and 5 mg/mL TGD for the cellular experiment. Formazan was quantified at 570 nm using an Epoch multi-volume spectrophotometer system (BioTek, Winooski, VT, USA).

| Cell cycle analysis
To determine the effect of TGD on the cell cycle, CFs were seeded and incubated at 37°C overnight. Then, the cells were exposed to indicate concentration of TGD. Cells were collected and fixed with ice-cold 70% ethanol and kept at 4°C overnight. Thereafter, the cells were collected and washed with PBS. The cell pellets were resuspended and stained in PBS containing RNase A and PI for 30 minutes at room temperature. Cell distribution across the cell cycle was determined with a BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA).

| Migration assay
Cell migration capacity was determined using a Transwell chamber

| Hydroxyproline assay
The total collagen content in equal amounts of culture supernatants was analysed with a hydroxyproline assay using a commercially available colorimetric assay kit (BioVision Inc., Milpitas, CA, USA). After treatment with TGD for 48 hours, the expression of hydroxyproline was performed, according to the manufacturer's instructions. Absorbance was measured at 550 nm, using an Epoch multivolume spectrophotometer system (BioTek). The results were compared with a standard curve constructed using titrating standards.

| Data analysis and statistics
Data were expressed as the mean ± SD, except for the AF duration, which was expressed as the median and interquartile range (25%-

| TGD reduces AF susceptibility in rats with
post-MI Figure 1A shows a representative example of induced AF electrocardiogram. AF occurred after the termination of the burst pacing (Figure 1Ab). The AF terminated spontaneously, and sinus rhythm resumed ( Figure 1Ac). However, the non-induced AF rats also displayed sinus rhythm after the burst (Figure 1Bb). AF inducibility was higher in the rats with MI (12/15, 80%) than in the sham rats (1/15, 6.7%, Figure 1C). Treatment with TGD decreased the inducibility (7/15, 46.7% in TGD-L vs 80% in MI; 33.3% in 5/15, TGD-H vs 80% in MI). The mean duration of the AF episode was longer in the rats with MI than in the sham rats (P < 0.05, Figure 1D). TGD decreased the AF duration in the TGD-H group (P < 0.05). The atrial effective refractory period (ERP) was shorter in the rats with MI than in the sham rats (P < 0.01, Supporting Information). The ERP in TGD-treated rats was longer than in rats with MI (TGD-L, P < 0.05; TGD-H, P < 0.01).

| TGD improves the cardiac function and LA conduction function in rats with post-MI
Echocardiogram showed that TGD-treated rats significantly increased EF (P < 0.01, Supporting Information) and fractional short-  Figure 2C). This decrease was reversed after administering TGD, with a recovery of conduction velocity observed in both the TGD-L and TGD-H groups (P < 0.01). These results indicated that TGD improved cardiac function and LA conduction function after MI.

| TGD inhibits LA fibrosis and increases the expression of connexin 43 in rats with post-MI
The Masson trichrome-stained heart sections confirmed that MI led to increased fibrosis in the left atrium (P < 0.01, Figure 3A).

| TGD inhibits the expression of collagen and proliferation of cardiac fibroblasts in vitro
Collagen synthesis in CFs is the most important step in the development of cardiac fibrosis. TGD significantly decreased the protein levels of collagen I and collagen III in CFs (P < 0.05, Figure 4A,B).
The MTT assay showed that TGD inhibited CFs proliferation (Figure 4C, P < 0.05). The effect of TGD on cell cycle progression was evaluated to determine the mechanism underlying TGD-regulated CFs proliferation ( Figure 4D). The result indicated that TGD treatment induced cell cycle arrest in the G0/G1 phase in CFs (P < 0.05, Figure 4E). The CFs apoptosis was determined through the Annexin V-propidium iodide double staining method, using a flow cytometer ( Figure 4F). Treatment with TGD increased the proportion of apoptotic cells in CFs (TGD 4 mg/mL, P < 0.05; TGD 5 mg/mL, P < 0.01). Together, these results indicated that TGD inhibited proliferation and induced apoptosis in CFs.

| TGD inhibits FBS-induced CFs migration
The inhibitory effects of TGD on CFs migration were examined using the Transwell assay. Representative pictures are shown in Figure 5A. Conduction was more heterogeneous in the rats with MI compared with the sham and TGD groups. C, Conduction velocities in the four groups. TGD improves the conduction velocity (n = 5 rats/group). **P < 0.01 vs sham group; ## P < 0.01 vs MI group F I G U R E 1 Tongguan capsule-derived herb (TGD) inhibits atrial fibrillation (AF) inducibility and duration in post-myocardial infarction (MI) rats. A, An example of an induced AF episode. Before the burst (a), the rat was in sinus rhythm. After termination of the burst (b), the rat displayed an irregular atrial rhythm, with an irregular ventricular response. After seconds (c), the AF episode terminated spontaneously, and the sinus rhythm resumed. B, An example of a non-induced AF episode. After termination of the burst (b), the rat also displayed sinus rhythm. C, TGD inhibits AF inducibility in post-MI rats (n = 15 rats/group). D, TGD decreases AF duration in post-MI rats (n = 15 rats/group). HR, heart rate; RR, R-wave to R-wave interval; AR, atrial rate. *P < 0.05 vs sham group; # P < 0.05 vs MI group

| TGD inhibits the TGF-β1-induced CFs differentiation into myofibroblasts
Fibroblasts may respond to mechanical loading through a switch to a myofibroblastic phenotype in which they express α-SMA. 32 The transformation of myofibroblasts from CFs is a critical event in the initiation of myocardial fibrosis. 33 The total cells were identified using

TGD (mg/mL)
Western blot analysis also showed that TGD reduced the expression of α-SMA protein (P < 0.05, Figure 6B). The TGF-β1 pathway was vital in the transformation of myofibroblasts. Western blot analysis showed that the protein levels of TGF-β1 were significantly greater in the TGF-β1 group than in the control group (P < 0.01, Figure 6C).
TGD reduced the expression of TGF-β1 protein compared with the TGF-β1 group (P < 0.05). Smad3 showed no differences between the four groups (P > 0.05, Figure 6D). Figure 6E shows that the expression of MMP-9 significantly increased in the TGF-β1 group compared with the control group (P > 0.05). TGD decreased the level of MMP-9 compared with the TGF-β1 group (TGD 4 mg/mL, P < 0.05; TGD 5 mg/mL, P < 0.01). Conversely, the expression of TIMP-1 decreased in the TGF-β1 group (P < 0.05). TGD increased the level of TIMP-1 (P < 0.05, Figure 6F). The data demonstrated that TGD inhibited the myofibroblast differentiation induced by TGF-β1.

| TGD inhibits CFs secretion in vitro
As collagen is a key component of the extracellular matrix regulated by myocardial fibroblasts, the secretion of collagen by CFs was investigated using the hydroxyproline assay. Figure 7A shows that the exposure of Fluorescence immunohistochemistry (using a specific α-SMA first antibody, followed by a secondary antibody conjugated with FITC) was performed to demonstrate TGF-β1-induced myofibroblast transformation. Nuclei were stained with DAPI. B, Western blot analysis of the protein expression of α-SMA. TGD reduces the expression of α-SMA protein in cardiac fibroblasts (n = 5 independent samples/ group). C, Western blot analysis of the protein expression of TGF-β1. TGD reduces the expression of TGF-β1 protein in cardiac fibroblasts (n = 5 independent samples/group). D, Western blot analysis of the protein expression of Smad3. TGD has no effect on the protein level of Smad3 (n = 5 independent samples/group). E, Western blot analysis of the protein expression of MMP-9. TGD reduces the protein level of MMP-9 (n = 5 independent samples/group). F, Western blot analysis of the protein expression of TIMP-1. TGD increases the protein level of TIMP-1 (n = 5 independent samples/group). *P < 0.05, **P < 0.01 vs the control group; # P < 0.05, ## P < 0.01 vs the TGF-β1 group systematically investigated the anti-AF effect of a Chinese herb, using fibroblasts as the target. The findings indicated that TGD reduces susceptibility to AF and improves LA conduction function in rats with post-MI by inhibiting LA fibrosis via modulating CFs.
Atrial fibrillation is generally considered to be a complicated and progressive condition. Ectopic (triggered) activity and re-entry are major and necessary arrhythmogenic mechanisms in AF. 34,35 Each AF episode requires initiation by triggers on a vulnerable substrate. The ion channel determinants govern AF initiation, and the structural remodelling stabilizes AF-maintaining re-entrant mechanisms. 36 Development and progression of LA fibrosis are the hallmark of structural remodelling in AF and are considered to be the substrate for AF perpetuation. 37 Fibrotic transformation of atrium results in the deterioration of atrial conduction. This increases anisotropy of impulse propagation and builds boundaries that promote re-entry in the atrial walls, which may be directly related to the mechanisms responsible for maintaining AF. 36,38 Pharmacologic therapy targeted at the fibrotic substrate itself may be important in managing AF. Drugs that prevent the LA fibrosis, such as pirfenidone, 10 pioglitazone 11 and Irbesartan 12 can effectively inhibit AF susceptibility in different animal models. In the present study, TGD significantly decreased fibrotic areas and the deposition of collagen type I/III in the left atrium, compared with rats with MI. This finding strongly proved that TGD can reduce LA fibrosis.
TGD also improved the LA conduction velocity and homogeneity, decreased the AF incidence and shortened the duration, particularly in the TGD-H group, similar to the effects of other antifibrotic drugs. 10,11 Therefore, it was concluded that TGD decreased the AF susceptibility in post-MI rats by improving atrial conduction function via preventing fibrosis in the left atrium.
After HF, the effect of improving the ventricular remodelling or cardiac function on AF is still inconclusive. 27 CFs, the largest cell population in the heart, are known as the main effector cells responsible for cardiac fibrosis. After injury, various peptide growth factors stimulate fibroblasts to migrate to the wound site and proliferate to reconstitute the various connective tissue components. 39 Fibroblasts are activated and differentiated into myofibroblasts that proliferate and cause fibrosis as a component of the wound-healing response. 39 In most organs, active fibroblasts undergo apoptosis and disappear following the completion of tissue repair, while in the heart, myofibroblasts have been shown to persist in atrium or mature infarct scars. 27,40 Myofibroblasts are responsible for excessive deposition of the extracellular matrix. Myofibroblasts also form electrical coupling (through connexin) with cardiomyocytes in the heart. 41 Existing myofibroblasts may actively contribute to the structural and electrical remodelling leading to the development of cardiac arrhythmias. 9 Therefore, a novel antiarrhythmic therapeutic F I G U R E 7 Tongguan capsule-derived herb (TGD) inhibits cardiac fibroblasts secretion in vitro. A, Hydroxyproline assay in the cell culture supernatants. TGD inhibits collagen secretion (48 h, n = 5 independent samples/group). B-E, ELISA of MCP-1, IL-6, TGF-β1 and TNF-α in the cell culture supernatants. TGD reduces the level of MCP-1, IL-6 and TGF-β1, without TNF-α (48 h, n = 5 independent samples/ group). *P < 0.05, **P < 0.01 vs the control group approach targeted at the CF population is needed. 40,42 We tested the expression of myofibroblasts using a specific marker α-SMA in vitro. TGD decreased the expression of α-SMA, collagen I and collagen III in CFs. These findings demonstrated that TGD inhibited the differentiation of fibroblasts into myofibroblasts in vitro.
Fibroblasts and myofibroblasts produce and secrete a number of chemical mediators, which help to maintain the inflammatory response to injury. 43,44 The effects of chemical mediators have been studied by treating cultured myocytes with fibroblast-conditioned media. 44 LaFramboise has found that neonatal fibroblast chemical mediators affect the size, contractile capacity and phenotype plasticity of cardiac myocytes in cultures. 45 Paracrine factors also demonstrate direct electrophysiological effects, contributing to conduction slowing. 46 Vasquez has reported cardiac injury significantly alters the profile of paracrine factors released by fibroblasts, potentially increasing arrhythmogenesis. 47 The present study found that TGD decreased the level of MCP-1, IL-6 and TGF-β1 in the cell culture supernatants, indicating that TGD inhibited the cytokine secretion of CFs in vitro.
Several limitations of the present study should be highlighted.
First, we provided no information on the exact mechanism through which TGD reduced arrhythmia in our study. Ion channels in cardiomyocytes are as important as atrial fibrosis in AF occurrence after MI. We could not exclude the effect of TGD on ion channels in our model. TGD increases the protein expression of Cx43 in the left atrium (Supporting Information), which may be another mechanism through which TGD reduces the susceptibility of AF. Second, TGD is a complex mixture of herbs, and little is known about the effects of the individual components and their interaction with one another. As TGD is composed of diverse herbal medicine ingredients, the compound(s) in TGD that are responsible for the effects observed in this study must to be further investigated.

| CONCLUSION
In recent years, cardiovascular medicine has undergone a major paradigm shift, transforming its focus from symptom-based therapy to prevention of the elements in the cascade of pathogenesis in cardiovascular disease. 48 Chinese herbs have been widely used in Chinese clinical cardiovascular therapy, with the notable advantages of being multi-component and multi-target. 49 In conclusion, the present study showed that TGD decreases AF occurrence after MI by preventing LA fibrosis by inhibiting the proliferation, migration, differentiation and cytokine secretion of CFs. Hence, targeting the CF population may be a novel antiarrhythmic therapeutic approach, and Chinese medicine, particularly TGD, could play a beneficial role.