Wenxin Keli suppresses atrial substrate remodelling after epicardial ganglionic plexi ablation
The Chinese herb extract Wenxin Keli is made by a mixture of several different herbs. A study published in early 2012 investigated claims that the Chinese herb Wenxin Keli was effective in suppressing AFib. It now holds the title of the first state-sanctioned traditional Chinese medicine-based antiarrhythmic drug.
The chronic effects of ganglionic plexi (GP) ablation on atrial fibrillation (AF) inducibility have not been elucidated.
To investigate the effect of Wenxin Keli (WK) on the inducibility of AF and atrial substrate remodelling after epicardial GP ablation.
Twenty dogs were randomly divided into a sham-operated group, a GP ablation group and a WK-treated group. All animals underwent a left thoracotomy at the fourth intercostal space. AF inducibility was assessed by burst rapid pacing at the right atrium. Both the GP ablation group and the WK-treated group received four major GP ablations. In the WK-treated group, dogs were treated with oral WK once per day, and all animals were allowed to recover for eight weeks, after which AF inducibility and AF duration were measured again.
After eight weeks of WK treatment, AF inducibility was lower than in the GP ablation group, and was similar to that of the sham-operated group. Compared with the sham-operated group, the levels of atrial natriuretic peptide (ANP), tumour necrosis factor-alpha (TNF-α) and interleukin (IL)-6 in right atrial tissues were increased in GP ablation group (143.6±33.7 pg/mg versus 206.2±41.4 pg/mg, P=0.02; 75.3±12.1 pg/mg versus 141.3±64 pg/mg, P=0.03; and 175.1±42.5 pg/mg versus 351.7±101 pg/mg, P<0.01, respectively). There were no significant differences in levels of ANP, TNF-α and IL-6 in atrial tissues between the sham-operated group and WK treated group. Expression of connexin 43 in atrial tissues was increased after eight weeks of GP ablation, while WK administration inhibited connexin 43 remodelling.
Epicardial GP ablation can induce atrial substrate remodelling, including Cx43 upregulation and increased levels of ANP, TNF-α and IL-6. These changes may be suppressed by long-term oral WK administration.
The influence of the autonomic nervous system on the triggering and perpetuation of atrial fibrillation (AF) is well established . Ganglionic plexi (GP) ablation appears to be a safe and efficacious method to improve pulmonary vein (PV) isolation in patients with AF . However, the effectiveness of GP ablation in patients with AF is ambiguous and the chronic efficacy is not clear. Oh et al reported that radiofrequency (RF) fat pad ablation may not achieve long-term suppression of AF induction in canine models, and GP ablation is associated with inferior clinical outcomes compared with circumferential ablation . In our previous study, we also found that AF was easily induced by rapid atrial pacing eight weeks after anterior right GP (ARGP) and inferior right GP (IRGP) ablation, and that partial atrial denervation can induce atrial inflammatory responses .
Wenxin Keli (WK) is a Chinese herbal extract reported to be beneficial in the treatment of cardiac arrhythmias and cardiac inflammation. The extract is composed of five components: Nardostachys chinensisBatal extract, Codonopsis, Notoginseng, amber and Rhizoma polygonati. One study showed that WK reduces inflammation in patients with unstable angina. However, the effect of WK on the inducibility of AF and the atrial inflammatory response after atrial GP ablation is unknown. Therefore, in the present study, we examined the effects of WK on the inducibility of AF and atrial substrate remodelling induced by GP ablation in a canine model.
Twenty mixed-sex mongrel dogs (body weight between 16 kg and 21 kg) were used in the present study. The present study conformed to the current Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication number 85 – 23, revised 1996). The protocol of the study was approved by the Ethics Committee at Wuhan University (Wuhan, China). Animal handling was performed according to the Wuhan Directive for Animal Research. All dogs were premedicated with pentobarbital sodium (30 mg/kg intravenously), intubated and ventilated with room air supplemented with oxygen using a respirator (MAO01746, Harvard Apparatus, USA). Normal saline was infused at a rate of 50 mL/h to 100 mL/h to replace spontaneous fluid losses. Standard surface electrocardiographic leads (I, II and III) were monitored continuously throughout the procedure. After left thoracotomy at the fourth intercostal space, the heart was exposed in a pericardial cradle.
After surgery, the superior left GP and inferior left GP were ablated. The fat pads that contain the ARGP and IRGP were exposed by lifting the pericardium, and the ARGP and IRGP were ablated through the left incision. Briefly, after the GP was identified, RF energy was delivered (50°C, 30 W to 50 W, 30 s to 80 s) at the sites where high-frequency stimulation induced evoked vagal reflexes. Complete vagal denervation was defined arbitrarily as the abolition of all vagal reflexes evoked by high-frequency stimulation. After RF ablation, the chest was closed in layers, negative pressure was re-established in the pleural cavity and the animals were allowed to recover for eight weeks. Antibiotics were administered for five days after surgery.
Study protocol and electrophysiological measurements
Twenty dogs were used for the present study and were divided into three groups (a sham-operated group, a GP ablation group and a WK-treated group). The sham-operated group consisted of seven dogs that underwent a thoracotomy without GP ablation. The GP ablation group comprised seven dogs that underwent GP ablation after the left thoracotomy. In the WK-treated group, 0.25 g/kg/day of oral WK (Buchang Group, China) was administered after GP ablation. All animals were allowed to recover for eight weeks.
The effective refractory period (ERP) was determined at each electrode pair along the catheters positioned at the right atrium (RA). The induction of AF was assessed three times in each dog. An S1S1 (120 ms, 100 ms and 75 ms cycle length, 5 s, respectively) programmed stimulus method was used to induce AF. AF was defined as irregular atrial rates faster than 500 beats/min associated with irregular atrioventricular conduction lasting >5 s. In the sham-operated group, ERP and AF were measured immediately following thoracotomy and after eight weeks. In the GP ablation and WK-treated groups, ERP and AF were measured before GP ablation, immediately after GP ablation and eight weeks after GP ablation.
Biochemical tests and Western blot studies
Four millilitres of venous blood was collected in EDTA-containing vacutainers and centrifuged (Beckman Coulter, USA) at 2310 g for 10 min at 4°C before and after pacing. The serum was separated and stored in microtubes at −80° until assays were performed. The levels of atrial natriuretic peptide (ANP), tumour necrosis factor-alpha (TNF-α) and interleukin (IL)-6 were measured using ELISAs. Tissues were obtained from the RA and left atrium (LA) in all animals after eight weeks. Western blotting was used to assess the relative protein levels of connexin (Cx)-43 using anti-Cx43 antibodies (Abcam Incorporated, United Kingdom). The density of the bands was determined using image analyzer software (AlphaEase FC, USA).
Values are presented as mean ± SD. Statistical comparisons were made using ANOVAs. Paired and unpaired comparisons were conducted using Student’s t test. Unpaired t tests were used to compare the means of nerve densities. P<0.05 was considered to be statistically significant.
ERP and induction of AF
There was no change in the ERP at baseline and eight weeks after surgery in the sham-operated group (132±12 ms versus 130±14 ms; P=0.8). Furthermore, there were no significant differences in the ERP immediately before and after GP ablation in the GP ablation group and WK-treated group, respectively (129±13 ms versus 136±15 ms, P=0.5; 128±14 ms versus 134±13 ms, P=0.6). After eight weeks, however, the ERP was shorter compared with immediately after GP ablation in the GP ablation group (136±15 ms versus 114±13 ms; P=0.03). There were also no significant differences in the ERP before and after eight weeks in the WK-treated group (128±14 ms, 134±13 ms versus 131±12 ms; P>0.05) .
In the sham-operated group, AF was induced in three dogs at baseline and in four dogs eight weeks after surgery. The mean duration of AF was 9.1±10.3 s and 16.9±17.4 s, respectively. In the GP ablation group, AF was induced in three dogs at baseline, in three dogs immediately after GP ablation and in six dogs eight weeks after GP ablation. The mean duration of AF was 8.4±8.7 s, 8.3±7.2 s and 110.0±63.5 s, respectively. In the WK-treated group, AF was induced in three dogs at baseline, in three dogs immediately after GP ablation and in three dogs eight weeks after GP ablation. The mean duration of AF was 10.4±11.2s, 4.2±3.1 s and 7.2±8.4 s, respectively. Compared with the WK-treated group, the mean duration of AF was higher in the GP ablation group after eight weeks (110.0±63.5 s versus 7.2±8.4 s; P<0.05)
Figure 5 shows the plasma ANP levels in the three groups of dogs. There was an increase in ANP levels eight weeks after GP ablation in the GP ablation group (142.2±21.4 pg/mL versus 259.3±34.5 pg/mL; P<0.01). However, there was no change in ANP levels in the sham-operated group and the WK-treated group at baseline and after eight weeks (167.4±27.1 pg/mL versus 155.8±25.3 pg/mL, P=0.1; 185.6±24.7 pg/mL versus 157.3±19.8 pg/mL, P=0.07). Furthermore, serum levels of TNF-α and IL-6 were not significantly different among the three groups before thoracotomy and after eight weeks.
Figure 6 shows the levels of ANP, TNF-α and IL-6 in atrial tissues in the three groups. When compared with that in the sham-operated group, levels of ANP, TNF-α and IL-6 in atrial tissues were greatly increased in the GP ablation group (ANP in RA tissues: 143.6±33.7 pg/mg versus 206.2±41.4 pg/mg, P=0.02; TNF-α and IL-6 levels in LA tissue: 91.3±35 pg/mg versus 180.3±72 pg/mg, P=0.02, and 125.3±42 pg/mg versus 273.3±83 pg/mg, P<0.01; TNF-α and IL-6 levels in RA tissue: 75.3±12.1 pg/mg versus 141.3±64 pg/mg, P=0.03, and 175.1±42.5 pg/mg versus 351.7±101 pg/mg, P<0.01). However, there was no significant difference in levels of ANP, TNF-α and IL-6 in atrial tissues between the sham-operated group and the WK-treated group.
Western blot studies
Figure 7 compares the results of Western blot analysis in the atria in the three groups. The band density was determined and the measurement was normalized to the RA, enabling a relative quantification of Cx43 band densities. The densities of the Cx43 bands in the RA and LA were increased in the GP ablation group compared with the sham-operated group (2.17, 1.86 versus 1, 1.15; P<0.01). There was no significant difference in Cx43 band densities in the RA and LA between the GP ablation and WK-treated groups (1.21, 1.19 versus 1, 1.15; P>0.05).
AF was easily induced by rapid atrial pacing eight weeks after GP ablation, and WK administration decreased the inducibility of AF after GP ablation. GP ablation induced atrial substrate remodelling including Cx43 upregulation and increased levels of ANP in the RA, and TNF-α and IL-6 in the atrium. WK effectively inhibited atrial Cx43 remodelling and significantly attenuated levels of ANP, TNF-α and IL-6 after GP ablation.
GP ablation and AF
Ablation of the major atrial GP suppressed the inducibility and maintenance of AF in animals . Recent clinical studies also demonstrated that GP ablation as an adjunct therapy to PV isolation was an efficient treatment for AF. Pokushalov et al indicated that regional ablation at the anatomical areas of GP is an effective and easily reproducible method for the treatment of paroxysmal AF.
However, ablation-induced atrial or ventricular proarrhythmia has been reported with both endocardial and epicardial approaches. Anatomical GP modification appears to carry a higher risk of iatrogenic left atrial tachycardia than PV isolation. Oh et al showed that RF fat pad ablation did not achieve long-term suppression of AF induction in a canine model. Furthermore, Hirose et al reported that partial right atrial vagal denervation facilitated rather than prevented initiation of vagally mediated AF. In our previous study, we found that partial atrial denervation induced an increase in ANP, TNF-α and IL-6 levels in atrial tissues . We hypothesize that the autonomic effect was eliminated after atrial GP ablation, but the increase in inflammatory factors in the atrium may provide a new causative factor in terms of AF vulnerability.
In the present study, we further investigated changes in atrial substrates and the effects of WK on the inducibility of AF and atrial substrate remodelling after GP ablation. We found that Cx43 expression increased significantly after GP ablation. An association among AF, increased Cx expression and remodelling of gap junction distribution has been documented . The mechanism underlying this observation remains unknown. Previous studies have demonstrated that sympathetic nerve stimulation promotes the degradation of Cx43 protein after myocardial ischemia, suggesting that sympathetic activity is correlated with the life cycle of Cx43. Our previous study found that densities of adrenergic nerves decreased in the RA after ARGP and IRGP ablation . We hypothesize that the Cx43 remodelling is associate with atrial denervation after GP ablation.
WK and AF
WK is a traditional Chinese medicine developed for treating cardiac arrhythmia, as demonstrated in previous studies. However, the mechanisms remained poorly understood. A recent study suggested that WK possesses potent anti-AF properties owing to its ability to depress sodium channel-dependent parameters; WK effectively terminates persistent acetylcholine-mediated AF and prevents its induction . In another study, Fan et al found that the average levels of high-sensitivity C-reactive protein, IL-6 and TNF-α decreased significantly after treatment with oral WK for approximately 10 weeks, suggesting that WK could reduce inflammation.
Some studies have investigated components of WK, such as Nardostachys chinensis Batal extract andNotoginseng, and have found that they significantly block sodium current (INa) and transient outward potassium current (Ito) in experimental animal models . Very limited data are available in the literature concerning the effect of WK or its individual components on atrial substrates. The results of the current study demonstrate that WK is effective in preventing the induction of AF after GP ablation. Furthermore, our data indicated that WK could inhibit atrial substrate remodelling including suppressing the overexpression of Cx43 and increase in inflammatory factors. Our data provide a novel mechanism for the effective management of AF and suggest that WK possesses potent anti-AF properties owing to its ability to suppress atrial substrate remodelling after GPs ablation.
The present study has several limitations. First, previous studies have suggested that WK possesses potent anti-AF properties owing to its ability to depress sodium channel-dependent parameters. In the present study, we did not measure changes of INa and Ito currents and did not investigate the effect of WK on INa and Ito currents after GP ablation. Whether there is ionic remodelling and effects of WK on ionic currents after GP ablation should be further investigated. Second, previous studies have shown that the cardiac autonomic nervous system forms an interconnected neural network composed of multiple GPs and the ligament of Marshall. In the present study, we used high-frequency electrical stimulation to identify GP locations, and the four major GPs were targeted for ablation without the ligament of Marshall. Third, previous work confirms that alterations of intercellular communication through gap junctional connections are likely contributing factors to the occurrence of AF. Recently, Igarashi et al showed that overexpression of Cx43 improves conduction and prevents AF. In the present study, we found Cx43 expression increased significantly after GP ablation. Because current transfer occurs only at gap junctions, the spatial distribution and biophysical properties of gap junction channels are important determinants of the conduction properties of cardiac muscle. We hypothesized that the overexpression induces the heterogeneity of Cx43 expression and contributes to reentry and AF. Unfortunately, we did not measure the conduction velocity before and after GP ablation. Further studies should investigate changes of atrial conduction velocity after GP ablation.
Epicardial RF GP ablation can induce atrial substrate remodelling including Cx43 upregulation and increased levels of ANP, TNF-α and IL-6. These changes may be suppressed by long-term oral WK.
This research was supported by the Natural Science Foundation of China (NSFC, 81070144).
DISCLOSURES: The authors have no conflicts of interests to declare.