BASIC SCIENCE
Europace (2015) 17, 647–654 doi:10.1093/europace/euu212
Effect of renal sympathetic denervation on the progression of paroxysmal atrial fibrillation in canines with long-term intermittent atrial pacing Xule Wang 1,2, Congxin Huang 1,2*, Qingyan Zhao1,2*, He Huang 1,2, Yanhong Tang 1,2, Zixuan Dai 1,2, Xiaozhan Wang 1,2, Zongwen Guo 1,2, and Jinping Xiao 1,2 1 Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan City 430060, People’s Republic of China; and 2Cardiovascular Research Institute of Wuhan University, Wuhan City 430060, People’s Republic of China
Received 25 March 2014; accepted after revision 3 July 2014; online publish-ahead-of-print 27 October 2014
Aims
The aim of the present study was to explore the effect of renal sympathetic denervation (RSD) on the progression of paroxysmal atrial fibrillation (AF) in canines with long-term intermittent atrial pacing. ..................................................................................................................................................................................... Methods Nineteen beagles were randomly divided into sham-operated group (six dogs), control group (six dogs), and RSD group and results (seven dogs). Sham-operated group were implanted with pacemakers without pacing; control group were implanted with pacemakers with long-term intermittent atrial pacing; and RSD group underwent catheter-based RSD bilaterally and were simultaneously implanted with pacemakers. Atrial pacing was maintained for 8 h a day and a total of 12 weeks in the control group and RSD group. Echocardiography showed that the left atrial structure and function were significantly improved in the RSD group compared with the control group (P , 0.05). Compared with the control group, the RSD group had fewer incidences of AF and a shorter duration of AF (P , 0.05) after long-term intermittent atrial pacing. In addition to increased atrial effective refractory period (AERP) and AF cycle length, AERP dispersion and P-wave duration and dispersion were significantly decreased in the RSD group compared with the control group (P , 0.05). Atrial morphological evaluation suggested that fibrosis and ultrastructural changes induced by long-term intermittent atrial pacing were markedly suppressed in the RSD dogs compared with controls (P , 0.05). Immunohistochemistry results showed that connexin 43 distribution in RSD mid-myocardial was significantly fewer heterogeneous than that in control midmyocardial (P , 0.05). ..................................................................................................................................................................................... Conclusion Renal denervation inhibits the progression of paroxysmal AF, which might be related to the suppression of atrial electrophysiology and structural heterogeneity.
----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords
Renal sympathetic nerve † Ablation † Atrial fibrillation † Atrial electrophysiology † Heterogeneity
Introduction In the recent few years, renal sympathetic denervation (RSD) applied to the treatment of cardiovascular disease has become a hot spot in the clinical trials and animal experiments. The SYMPLICITY HTN-11 and HTN-2 studies2 have suggested that RSD has achieved a breakthrough in the treatment of resistant hypertension, which might have related to the inhibition of chronic sympathetic nervous overactivation. Although optimism has recently been tempered with the broadcast alert from Medtronic that the SYMPLICITY HTN-3 did
not meet its primary efficacy endpoint,3 RSD has great potential in the treatment of other cardiovascular diseases, such as heart failure, left ventricular hypertrophy, refractory atrial fibrillation (AF), and post-myocardial infarction cardiac remodelling.4 – 9 In recent reports from our laboratory,10,11 we demonstrated that RSD not only could suppress AF in canines with 7 h rapid right atrial pacing (RAP), but also could effectively suppress the incidences of AF and atrial remodelling in canines during sustained RAP for 5 weeks, which might be related to the inhibition of renin–angiotensin –aldosterone system (RAAS). Whether renal denervation can effectively
* Corresponding author. No. 238 Jiefang Road, Wuchang District, Wuhan City, Hubei Province, China. Tel: +86 13871329139; fax: +8627 88042292. E-mail address: huangcongxin@vip. 163.com (C.H.) or [email protected] (Q.Z.) Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2014. For permissions please email: [email protected].
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What’s new? † This study showed that the induced atrial fibrillation produced by long-term intermittent atrial pacing could be reduced by catheter-based renal sympathetic denervation (RSD) in ambulatory canines, suggesting that renal denervation inhibits the progression of paroxysmal atrial fibrillation. † Furthermore, we found that the increase of right atrial pressure, mean pulmonary artery pressure, and pulmonary capillary wedge pressure produced by long-term intermittent atrial pacing was prevented by RSD. † In addition to increased atrial effective refractory period (AERP) and atrial fibrillation cycle length, AERP dispersion, P-wave duration and dispersion were significantly decreased in the RSD group compared with the control group, suggesting that RSD suppresses the heterogeneity of atrial electrophysiology after long-term intermittent right atrial pacing. † We also found that atrial structural remodelling including left atrial dilatation, atrial fibrosis, and ultrastructural changes together with the heterogeneity of connexin 43 were markedly suppressed in the RSD dogs compared with controls.
suppress the progression of paroxysmal AF is unknown. The aim of the present study was to explore the effect of RSD on the progression of paroxysmal AF in canines with long-term intermittent atrial pacing, and further observe the effect of RSD on functional and morphological changes induced by pacing in the canine model.
Methods Experimental animals and study protocol The study protocol was approved by the Ethical Committee of the Wuhan University School of Medicine, and all animal handling was performed in accordance with the Wuhan Directive for Animal Research and the current Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 85 – 23, revised 1996). We studied 19 healthy adult beagles (weight, 11.2 – 14.5 kg) assigned randomly to three groups. The sham-operated group consisted of six dogs that were implanted with pacemakers without pacing; the control group consisted of six dogs that were implanted with pacemakers with long-term intermittent atrial pacing; and the RSD group consisted of seven dogs that underwent catheterbased RSD and simultaneously were implanted with pacemakers. Anaesthesia was induced by administering 30 mg kg21 pentobarbital sodium intravenously and was maintained by isoflurane. All of the dogs were intubated and ventilated with room air supplemented with oxygen from a respirator (MAO01746, Harvard Apparatus). Normal saline at 50 – 100 mL h21 was infused to replace spontaneous fluid losses. Standard surface 12-lead electrocardiograms (ECGs) (MAC1200, GE) were monitored continuously throughout the procedure. Under fluoroscopy, an atrial endocardial pacing electrode (1642T, St Jude Medical, Inc.) was delivered to the RA appendage via the right external jugular vein and connected to a high-rate cardiac pacemaker (Fudan University, Shanghai, China), which was implanted in a subcutaneous pocket of the neck. After 3 days for recovery, RSD group and control group dogs were paced at 400 b.p.m. for 8 h a day and a total of 12 weeks (Figure 1).
Beagles (n = 19)
Control group (n = 6)
Sham-operated group (n = 6)
RSD group (n = 7)
Echocardiography, haemodynamic study, electrophysiological measurements, venous blood collected
Pacemakers without pacing, with feed for 12 weeks
Atrial pacing in 400 b.p.m. for 8 h a day and 12 weeks
After RSD, atrial pacing in 400 b.p.m. for 8 h a day and 12 weeks
Echocardiography, haemodynamic study, electrophysiological measurements, venous blood and atrial tissue collected
Figure 1 Flowchart of the study.
In the RSD group dogs, a tailor-made quadrupole radiofrequency ablation catheter was inserted into each renal artery via femoral artery under fluoroscopy. (When the quadrupole ablation catheter was introduced into each renal artery, its four tips were expanded automatically and applied to the wall of renal artery.) We applied radiofrequency ablations of 6 W or less and lasting up to 90 s within each renal artery. The catheter system monitored tip temperature and impedance, altering radiofrequency energy delivery in response to a predetermined algorithm. In sham-operated group and control group dogs, the ablation catheter was inserted into each renal artery without ablation.
Echocardiography The structure of left atrium (LA) and left ventricle were assessed via transthoracic echocardiographic examinations (IE33, S5-1, Philips) at both the baseline and endpoint of the study in all dogs. The LA diameter (LAD) and left ventricle end-diastolic dimension (LVEDD) were measured as the maximum dimension along the parasternal long-axis view using twodimensionally guided M-mode tracings. The LA volume (LAV) was measured using a single-plane Simpson’s method in the apical view showing the largest and smallest LA area. All of the volumes were measured in triplicate, and the averages were reported. Left atrial ejection fraction (LAEF) was calculated as (LAVmax 2 LAVmin)/LAVmax. An independent echocardiography expert reviewed the images and the parameters.
Haemodynamic study A 6F haemostatic sheath was inserted into the femoral artery, and femoral systolic and diastolic blood pressure (BP) was measured at the baseline and at the endpoint of the protocol in each dog. Furthermore, RA pressure, mean pulmonary artery pressure (MPAP), and pulmonary capillary wedge pressure (PCWP) were monitored by a balloon wedge pressure catheter (REF AI-07126, Arrow International, Inc.) through a 6F sheath placed in the femoral vein.
Electrophysiological measurements P-wave duration was measured from the surface ECG at baseline and after pacing. Twelve-lead ECG was recorded at the speed of 200 mm/s in supine position. P-wave duration was measured from the onset to the offset of the P-wave in the lead with the longest P-wave. The onset
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and offset were defined as the junction between the P-wave pattern and the isoelectric line. P-wave dispersion was defined as the difference between the maximum and minimum P-wave duration in 12 leads. A multipolar electrode catheter was introduced into the femoral veins through a 6F haemostatic sheath and placed in the high RA. Atrial vulnerability and atrial effective refractory period (AERP) were determined at the RA at the baseline and after pacing. Once intermittent atrial pacing up to 12 weeks, the chest was opened by means of a median sternotomy and the heart was exposed and suspended in a pericardial sling. Four quadripolar plaque electrodes were surgically attached to the RA, RA appendage, LA, LA appendage, and right superior pulmonary vein. The AERP was measured at the above five epicardial sites by delivering a train of eight atrial paced beats S1 at a cycle length of 250 ms, followed by an extrastimulus (S2) introduced at coupling intervals. The S1 – S2 intervals were decreased from 180 ms to refractoriness initially by decrements of 10 ms. As the S1 –S2 intervals approached the AERP, decrements were reduced to 2 ms. Atrial effective refractory period dispersion was defined as the difference between the maximum and minimum AERP in the five sites. An S1S1 (120, 100, and 75 ms cycle length, respectively, lasting for 5 s each) programmed stimulus method was used to assess the inducibility and duration of AF. Atrial fibrillation was defined as irregular atrial rates faster than 500 b.p.m. associated with irregular atrioventricular conduction lasting .5 s. Atrial fibrillation times and AF duration separately refer to the average number of times and length of time that AF was induced in every dog within a group. Atrial fibrillation cycle length (AFCL) was calculated by measuring the interval at a timescale of 200 mm/s between the atrial excited signals in the whole recorded epicardial electrogram diagram of AF and averaged to obtain the mean AFCL. Measurements were repeated by two investigators.
Enzyme-linked immunosorbent assay Two millilitres of venous blood were collected in ethylenediaminetetraacetic acid vacutainers and centrifuged at 3000 gravities for 10 min at 48C at the baseline and endpoint of the protocol. The plasma was separately kept in microtubes and stored at 2808C until assay. The plasma levels of angiotensin II (Ang II; Ang II Elisa Kit, Enzo Life Sciences Inc.) and aldosterone (Aldosterone EIA Kit, Enzo Life Sciences Inc.) were examined using an enzyme-linked immunosorbent assay.
Morphological evaluation At the completion of the protocol, the animals were euthanized and the hearts were quickly excised. Segments from the atrium were fixed with 4% paraformaldehyde at 48C and then embedded in paraffin for morphological evaluation. Light microscopy was stained with Masson trichrome solution. The interstitial collagen volume fraction (CVF) in the atrium was determined via quantitative morphometry with an image analyzer [Image-Pro Plus 6.0 (IPP 6.0), Media Cybernetics]. For electron microscopy, segments of the LA and RA were immediately fixed with glutaraldehyde stationary liquid and stored at 48C. Ultrathin section (70 – 80 nm) was cut from each sample, counterstained with uranium acetate and lead citrate, and examined with a transmission electron microscope (Hitachi HT7700) by two blinded observers.
Immunohistochemistry Four micrometre sections were cut from paraffin blocks of the LA and RA. The sections were stained with connexin 43 (Cx43, monoclonal goat anti-Cx43, Santa Inc.; used at 1 : 500). Heterogeneity of Cx43 expression was quantified by transforming photomicrographs of subepicardium and mid-myocardium of Cx43. The mean density of these slides was used as a measure of Cx43 heterogeneity. We determined the
density by the computer-assisted IPP 6.0 software. Each slide was examined under a microscope with ×40 objectives to select three fields with the highest density. The density was the positive area divided by the total area examined. The mean density in these three selected fields was used to represent the density of that slide.
Statistical analysis Data are expressed as mean + standard deviation (SD). Analyses were performed using SPSS version 17. Paired t-tests were used to compare the means of the baseline with that of the endpoint in the same group. Differences between groups were tested with one-way analysis of variance (ANOVA) ( post hoc Student–Newman–Keuls); cases of significant difference were further analysed with the Tukey–Kramer test. When data were not normally distributed, a Kruskal–Wallis one-way ANOVA on ranks was performed ( post hoc Dunn method). All statistical tests were two-sided, and statistical significance was established at P , 0.05.
Results Effect of renal sympathetic denervation on atrial structure and function The averaged echocardiographic data for LAD, LVEDD, LAV, and LAEF are shown in Table 1. At the endpoint of the protocol compared with sham-operated dogs, LAD, LAVmax, and LAVmin increased significantly in the control and RSD groups, while LAEF decreased after termination of long-term intermittent atrial pacing. Compared with the control group, the LAD, LAVmax, and LAVmin decreased, whereas the LAEF increased in the RSD group after RAP for 8 h a day and a total of 12 weeks (P , 0.05). There was no significant difference in LVEDD among the three groups (P . 0.05).
Haemodynamic changes Table 2 summarizes the average RA pressure, MPAP, PCWP, systolic BP, and diastolic BP responses in all dogs. Compared with the control group, RSD decreased systolic and diastolic BP (P , 0.05). Similarly, RSD prevented the increase of RA pressure, MPAP, and PAWP produced by long-term intermittent atrial pacing (P , 0.05).
Effect of renal sympathetic denervation on atrial vulnerability As Table 3 showed the baseline characteristics of the frequency and duration of AF among the three groups were similar (P . 0.05). At the endpoint of the study compared with the sham-operated group, the control group dogs had significantly more episodes of AF (P , 0.01), and longer durations of AF (P , 0.05) after long-term intermittent atrial pacing, while the RSD group dogs had similar episodes of AF (P . 0.05), and durations of AF (P . 0.05). Compared with the control group, RSD group dogs subjected to long-term intermittent atrial pacing had significantly fewer episodes of AF (P , 0.01) and shorter durations of AF (P , 0.05).
Effect of renal sympathetic denervation on atrial electrophysiology The changes in the electrophysiological properties of three groups were shown in Table 3. The results indicated that AERP in both control group and RSD group beagles were significantly shortened, while P-wave duration and dispersion were significantly prolonged
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Table 1 Changes in echocardiographic indices before and after rapid atrial pacing LAD (mm)
LVEDD (mm)
19.0 + 1.5 19.3 + 0.8
27.5 + 1.5 27.8 + 1.2
18.7 + 1.2 23.0 + 1.1**,††
28.0 + 1.9 29.3 + 1.0
18.8 + 1.7 21.4 + 1.0**,††,‡
27.9 + 2.0 28.7 + 1.4
LAVmax (cm3)
LAVmin (cm3)
LAEF (%)
2.0 + 0.6 2.3 + 0.5
71.7 + 7.2 68.9 + 4.5
2.2 + 0.8 5.8 + 1.2**,††
68.8 + 9.0 52.2 + 5.8*,††
2.1 + 0.7 3.4 + 0.5**,†,‡‡
70.4 + 7.4 61.7 + 5.7**,††,‡
............................................................................................................................................................................... Sham-operated group Baseline Endpoint
7.0 + 0.9 7.5 + 1.1
Control group Baseline Endpoint
6.8 + 0.8 12.2 + 1.6**,††
RSD group Baseline Endpoint
7.1 + 0.9 9.0 + 1.2**,†,‡‡
LAD, left atrial diameter; LVEDD, left ventricular end-diastolic dimension; LAVmax, left atrial maximal volume; LAVmin, left atrial minimal volume; LAEF, left atrial ejection fraction. *P , 0.05, **P , 0.01, compared with baseline in the same group. † P , 0.05, ††P , 0.01, compared with the sham-operated group at the same point. ‡ P , 0.05, ‡‡P , 0.01, compared with the control group at the same point.
Table 2 Haemodynamic parameters in the sham-operated, control, and RSD groups (mmHg) Systolic BP
Diastolic BP
RA pressure
MPAP
PCWP
............................................................................................................................................................................... Sham-operated group Baseline Endpoint
137 + 13
84 + 9
7+2
21 + 4
8+3
134 + 17
86 + 10
8+1
22 + 5
9+3
8+2
22 + 4
Control group Baseline
140 + 16
88 + 11
Endpoint
137 + 14
86 + 9
RSD group Baseline
135 + 18
85 + 10
8+1
21 + 3
8+2
127 + 12‡
78 + 9†‡
9 + 3‡‡
24 + 5‡
10 + 3‡
Endpoint
16 + 3**,††
32 + 6*,††
9+2 18 + 4**,††
BP, blood pressure; MPAP, mean pulmonary artery pressure; PCWP, pulmonary capillary wedge pressure. *P , 0.05, **P , 0.01, compared with baseline in the same group. † P , 0.05, ††P , 0.01, compared with the sham-operated group at the same point. ‡ P , 0.05, ‡‡P , 0.01, compared with the control group at the same point.
after long-term intermittent atrial pacing (P , 0.05). At the endpoint of the study compared with the sham-operated group, AERP dispersion, P-wave duration and dispersion in control beagles were significantly lengthened; however, the increasing trends of AERP dispersion and P-wave duration in the RSD group were not significantly different (P . 0.05). Compared with the control group, RSD group dogs had significantly longer AERP and shorter AERP dispersion, P-wave duration and dispersion after RAP for 8 h a day and a total of 12 weeks (P , 0.05). There were six dogs induced AF in the control group of six dogs while four of seven dogs in the RSD group in the RA after long-term intermittent atrial pacing. Moreover, the AFCL were longer in the RSD group than that of the control group (122.5 + 6.6 vs. 102.3 + 8.7 ms, P , 0.01).
Effect of renal sympathetic denervation on the plasma levels of angiotensin II and aldosterone Compared with the baseline, control group and RSD group dogs had a significant increase in the plasma levels of Ang II and aldosterone
after long-term intermittent atrial pacing (P , 0.01). At the endpoint of the protocol, compared with the sham-operated group, both control group and RSD group beagles showed a statistically significant increase in the plasma levels of Ang II (control group, 2877.9 + 376.9 vs. 676.3 + 166.3 pg/mL, P , 0.01; RSD group, 1174.9 + 181.9 vs. 676.3 + 166.3 pg/mL, P , 0.01) and aldosterone (control group, 377.9 + 63.7 vs. 62.5 + 18.8 pg/mL, P , 0.01; RSD group, 161.4 + 38.3 vs. 62.5 + 18.8 pg/mL, P , 0.01); rather, the plasma levels of Ang II and aldosterone were significantly reduced after RSD compared with the values for the control group (Ang II, 1174.9 + 181.9 vs. 2877.9 + 376.9 pg/mL, P , 0.01; aldosterone, 161.4 + 38.3 vs. 377.9 + 63.7 pg/mL, P , 0.01).
Effect of renal sympathetic denervation on atrial fibrosis Representative histological sections of atrial myocardium are shown in Figure 2. Atrial Masson staining for collagen showed a normal intercellular space in the samples from sham-operated dogs with CVF values of 3%, which was significantly increased in both LA and
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Effect of RSD on progression of paroxysmal AF in canines
Table 3 Changes in the electrophysiological properties among the three groups before and after rapid atrial pacing AF times
AF duration (s)
AERP (ms)
AERP dispersion (ms)
P-wave duration (ms)
P-wave dispersion (ms)
55.5 + 3.1
12.3 + 2.6
5.0 + 2.1
60.0 + 3.4
12.7 + 3.0
56.7 + 6.0
13.0 + 2.7
76.5 + 9.2**,††
27.7 + 3.3**,††
55.0 + 5.0
13.9 + 3.5
66.1 + 5.5**,‡
19.7 + 2.8**,††,‡‡
............................................................................................................................................................................... Sham-operated group Baseline
0.0 + 0.0
0.0 + 0.0
126.0 + 1.8
Endpoint Control group
0.2 + 0.4
1.2 + 2.9
122.7 + 2.4
0.8 + 2.0
126.7 + 4.3
Baseline Endpoint RSD group
0.2 + 0.4 2.8 + 0.4**,††
40.7 + 24.2*†
108.3 + 5.6**,††
Baseline
0.0 + 0.0
0.0 + 0.0
126.6 + 1.9
Endpoint
0.7 + 0.8*,‡‡
4.6 + 5.7‡
117.4 + 2.2**,†,‡‡
24.3 + 5.9††
9.1 + 4.3‡‡
AF, atrial fibrillation; AERP, atrial effective refractory period. AF times and AF duration separately refer to the average number of times and length of time that AF was induced in every dog within a group. *P , 0.05, **P , 0.01, compared with baseline in the same group. † P , 0.05, ††P , 0.01, compared with the sham-operated group at the same point. ‡ P , 0.05, ‡‡P , 0.01, compared with the control group at the same point.
Figure 2 Effect of RSD on fibrosis in the atria detected with Masson’s trichrome staining. Red areas represent myocytes, and blue areas represent collagen. (A) Examples of sham-operated LA; (B) examples of control LA; (C) examples of RSD LA; (A′ ) examples of sham-operated RA; (B′ ) examples of control RA; (C′ ) examples of RSD RA (original magnification: ×400).
RA of the control group (LA, 14.2 + 4.5 vs. 3.3 + 1.0%, P , 0.01; RA, 15.8 + 5.2 vs. 2.8 + 0.9%, P , 0.01). In comparison, RSD clearly decreased the volume fraction of collagen compared with that of the control group (LA, 5.2 + 3.1 vs. 14.2 + 4.5%, P , 0.01; RA, 9.7 + 3.4 vs. 15.8 + 5.2%, P , 0.05).
Ultrastructural changes Atrial myocardial ultrastructure was documented by electron microscopy, as shown in Figure 3. Atrial myocytes from the sham-operated dogs showed a highly organized sarcomeric structure
throughout the cytoplasm, with rows of uniformly sized mitochondria and nuclei with clustered heterochromatin. A marked increase in the number and size of the mitochondria, loss of the banding pattern, and integrity of contractile elements were observed after RAP for 8 h a day and a total of 12 weeks, which were the characteristics of ultrastructural abnormalities. In contrast, the long-term intermittent atrial tachypacing-induced ultrastructural changes were dramatically suppressed by RSD. Ultrastructural abnormalities in the LA were similar to that corresponded to the RA in the control and RSD groups.
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Figure 3 Typical ultrastructural changes by transmission electron microscopy separately in LA and RA tissue. Exemplary pictures of electron microscopy in the sham-operated group (A and A′ ), the control group (B and B′ ), and the RSD group (C and C′ ). (A– C) Representative of LA; (A′ – C′ ) representative of RA. LA, left atrium; RA, right atrium (magnification: ×5.0 k at 1.0 mm).
Effects of renal sympathetic denervation on heterogeneity of connexin 43 expression in the atria Figure 4 shows the immunostaining results of Cx43 expression in the atrium among all groups. The heterogeneity of Cx43 expression, expressed as mean of the SD of different parts, was 3162.5 + 780.7, 5542.40 + 506.3, 5396.8 + 714.7, 3734.1 + 729.6, 16 322.6 + 2162.4, and 7723.4 + 1499.3 mm2/mm2 for sham-operated subepicardial, control subepicardial, RSD subepicardial, sham-operated midmyocardial, control mid-myocardial, and RSD mid-myocardial, respectively. Analysis of variance revealed that Cx43 heterogeneity in sham-operated subepicardial and sham-operated mid-myocardial was not statistically different (P . 0.05), so was that in RSD subepicardial and RSD mid-myocardial. Compared with the RSD group, Cx43 distribution in control subepicardial and control midmyocardial was significantly more heterogeneous (P , 0.05).
Discussion Major finding This study shows that the induced AF produced by long-term intermittent atrial pacing could be reduced by catheter-based RSD. In addition to increased AERP and AFCL, AERP dispersion, P-wave duration and dispersion were significantly decreased in the RSD group compared with the control group. In connection with decreased plasma
levels of Ang II and aldosterone, atrial structural remodelling including LA dilatation, atrial morphology together with the heterogeneity of Cx43 were markedly attenuated in the RSD dogs compared with controls.
Effect of renal sympathetic denervation on haemodynamics and paroxysmal atrial fibrillation induced by long-term intermittent atrial pacing Renal sympathetic denervation selectively reduces both renal sympathetic efferent and afferent nerve activities and is originally found to treat resistant hypertension, which is indicated by a reduction in plasma renin activity.12 Nowadays, the beneficial effect of this procedure is extended to other syndromes in which sympathetic hyperactivity is a component of the underlying disease process. Our previous studies have demonstrated that renal denervation could effectively suppress incidences of AF and atrial remodelling in canines with atrial pacing, which might be related to the inhibition of RAAS.10,11 It is well known that haemodynamic changes, resulting in atrial stretch and alterations in neurohormonal activation, may result in atrial remodelling which may favour an environment vulnerable to the development of atrial arrhythmia. In the present study, we found the same effect of RSD on BP and induced AF in beagles with RAP for 8 h a day and a total of 12 weeks, which simulated the model of paroxysmal AF. Furthermore, we also found that the increase of RA pressure, MPAP, and PAWP induced by long-term
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Effect of RSD on progression of paroxysmal AF in canines
Figure 4 Heterogeneity of Cx43 expression in the LA among all groups. (A) Sham-operated subepicardial; (B) control subepicardial; (C) RSD subepicardial; (A′ ) sham-operated mid-myocardial; (B′ ) control mid-myocardial; (C′ ) RSD mid-myocardial (original magnification: ×400).
intermittent atrial pacing was prevented by RSD. The results of plasma levels of Ang II and aldosterone indicated that the inhibition of RAAS still may be a mechanism underlying these results.
Effect of renal sympathetic denervation on atrial electrical and structural heterogeneity Atrial fibrillation causes atrial electrical and structural remodelling, which have an important role in the perpetuation and progression of the arrhythmia. Our prior studies of AERP in canines during atrial pacing have demonstrated that the shortening in AERP induced by RAP was reversible after RSD.10,11 In the present study, the same phenomenon that RSD attenuated the shortening in AERP produced by RAP for 8 h a day and a total of 12 weeks can be observed. In addition, we found that AERP dispersion was completely increased after long-term intermittent atrial pacing while RSD prevented RAP-induced increasing of AERP dispersion. The statistical importance of AERP dispersion supports the importance of RAP-induced electrical heterogeneity in the substrate for AF. Data from the present study pointed out RSD could inhibit the increase in regional heterogeneity of refractoriness in experimental paroxysmal AF, which contributes to the maintenance of AF. There are large number of clinical studies confirming the role of P-wave duration and dispersion as non-invasive useful markers associated with paroxysmal AF.13,14 Prolonged P-wave duration and dispersion were reported to be commonly found in patients with a history of paroxysmal AF.13,15 These simple, non-invasive tests are still applied successfully by many investigators.16 – 22 The results of our study showed that the occurrence of paroxysmal AF caused a significant increase in P-wave duration and dispersion, and RSD attenuated the increase of P-wave duration and dispersion induced by long-term intermittent atrial pacing. This finding agrees with
published observations supporting a role for atrial electrical heterogeneity in canines with paroxysmal AF. Furthermore, it seems that RSD decreases sympathetic renal afferent nerve activity and central sympathetic drive, leading to decreased atrial electrical heterogeneity. Furthermore, AFCL was also observed as atrial electrical heterogeneity in the present study. Previous studies of AFCL in animals and humans have shown a good correlation between AFCL and AF.23 – 26 In these studies, a shorter AFCL was observed in chronic AF compared with acute AF23 and the surface ECG AFCL independently predicted procedural termination of persistent AF.24 Atrial fibrillation cycle length was longer in the RSD group compared with the control group, further suggesting that RSD might reduce the heterogeneity in atrial electrophysiology and facilitate AF termination. Our previous study showed that RSD could suppress the atrial substrate remodelling in ambulatory canines during prolonged RAP, including the significantly increased atrial fibrosis, inflammation, and apoptosis, together with atrial gap junctional remodelling and neural remodelling.11 Left atrial enlargement and fibrosis have been proposed as two of the core processes involved in atrial remodelling.27 – 30 Moreover, LA size and fibrosis19 could also play a role in determining P-wave duration, that was an important role for atrial electrical heterogeneity. The results of echocardiography showed that the LA dilation and hypofunction in experimental paroxysmal AF were markedly improved by RSD. Fibrosis in both LA and RA caused by long-term intermittent atrial pacing were attenuated after RSD. The results under electron microscopy suggested that the long-term intermittent tachypacing-induced atrial ultrastructural changes were dramatically attenuated by RSD. These findings implied that RSD might inhibit atrial structural remodelling in canines with paroxysmal AF. Immunohistochemistry results showed that the heterogeneity of Cx43 distribution produced by long-term intermittent atrial pacing was significantly reduced after RSD. All these suggested
654 renal denervation suppress the atrial structural heterogeneity after long-term intermittent RAP, especially for Cx43 heterogeneous distribution.
Conclusion Our previous studies showed that the ablation of renal artery could be applied to AF therapy in canines during short-term or sustained RAP.10,11 In the present study, we further demonstrated that RSD could reduce the incidences of AF and suppress the atrial electrophysiology and structural heterogeneity after long-term intermittent RAP. All these indicate that renal denervation might provide a new strategy useful for preventing the progression of paroxysmal AF. Conflict of interest: none declared.
Funding This study was supported by the National Science and Technology Pillar Program of China (2011BAI11B12) and National Key Basic Research Development Program of China (The ‘973’ Program, 2012CB518604).
References 1. Krum H, Schlaich M, Whitbourn R, Sobotka PA, Sadowski J, Bartus K et al. Catheterbased renal sympathetic denervation for resistant hypertension: a multicentre safety and proof-of-principle cohort study. Lancet 2009;373:1275 –81. 2. Symplicity HTN-2 Investigators, Esler MD, Krum H, Sobotka PA, Schlaich MP, Schmieder RE et al. Renal sympathetic denervation in patients with treatmentresistant hypertension (The Symplicity HTN-2 Trial): a randomised controlled trial. Lancet 2010;376:1903 –9. 3. Bhatt DL, Kandzari DE, O’Neill WW, D’Agostino R, Flack JM, Katzen BT et al. A controlled trial of renal denervation for resistant hypertension. N Engl J Med 2014;370:1393 – 401. 4. Davies JE, Manisty CH, Petraco R, Barron AJ, Unsworth B, Mayet J et al. First-in-man safety evaluation of renal denervation for chronic systolic heart failure: primary outcome from REACH-Pilot study. Int J Cardiol 2013;162:189 –92. 5. Brandt MC, Mahfoud F, Reda S, Schirmer SH, Erdmann E, Bo¨hm M et al. Renal sympathetic denervation reduces left ventricular hypertrophy and improves cardiac function in patients with resistant hypertension. J Am Coll Cardiol 2012;59:901 –9. 6. Linz D, Mahfoud F, Schotten U, Ukena C, Neuberger HR, Wirth K et al. Renal sympathetic denervation suppresses postapneic blood pressure rises and atrial fibrillation in a model for sleep apnea. Hypertension 2012;60:172–8. 7. Pokushalov E, Romanov A, Corbucci G, Artyomenko S, Baranova V, Turov A et al. A randomized comparison of pulmonary vein isolation with versus without concomitant renal artery denervation in patients with refractory symptomatic atrial fibrillation and resistant hypertension. J Am Coll Cardiol 2012;60:1163 –70. 8. Linz D, Mahfoud F, Schotten U, Ukena C, Hohl M, Neuberger HR et al. Renal sympathetic denervation provides ventricular rate control but does not prevent atrial electrical remodeling during atrial fibrillation. Hypertension 2013;61:225–31. 9. Hu J, Ji M, Niu C, Aini A, Zhou Q, Zhang L et al. Effects of renal sympathetic denervation on post-myocardial infarction cardiac remodeling in rats. PLoS ONE 2012;7:e45986. doi:10.1371/journal.pone.0045986.
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10. Zhao Q, Yu S, Zou M, Dai Z, Wang X, Xiao J et al. Effect of renal sympathetic denervation on the inducibility of atrial fibrillation during rapid atrial pacing. J Interv Card Electrophysiol 2012;35:119 –25. 11. Wang X, Zhao Q, Huang H, Tang Y, Xiao J, Dai Z et al. Effect of renal sympathetic denervation on atrial substrate remodeling in ambulatory canines with prolonged atrial pacing. PLoS ONE 2013;8:e64611. doi:10.1371/journal.pone.0064611. 12. Schlaich MP, Sobotka PA, Krum H, Lambert E, Esler MD. Renal sympathetic-nerve ablation for uncontrolled hypertension. N Engl J Med 2009;361:932 –4. 13. Dilaveris PE, Gialafos EJ, Sideris SK, Theopistou AM, Andrikopoulos GK, Kyriakidis M et al. Simple electrocardiographic markers for the prediction of paroxysmal idiopathic atrial fibrillation. Am Heart J 1998;135:733 – 8. 14. Dilaveris PE, Gialafos EJ, Andrikopoulos GK, Richter DJ, Papanikolaou V, Poralis K et al. Clinical and electrocardiographic predictors of recurrent atrial fibrillation. Pacing Clin Electrophysiol 2000;23:352 –8. 15. Yamada T, Fukunami M, Shimonagata T, Kumagai K, Sanada S, Ogita H et al. Dispersion of signal-averaged P wave duration on precordial body surface in patients with paroxysmal atrial fibrillation. Eur Heart J 1999;20:211–20. 16. Koide Y, Yotsukura M, Ando H, Aoki S, Suzuki T, Sakata K et al. Usefulness of P-wave dispersion in standard twelve-lead electrocardiography to predict transition from paroxysmal to persistent atrial fibrillation. Am J Cardiol 2008;102:573 –7. 17. Bissinger A, Grycewicz T, Grabowicz W, Lubinski A. The effect of diabetic autonomic neuropathy on P-wave duration, dispersion and atrial fibrillation. Arch Med Sci 2011;7:806 –12. 18. Hashemi Jazi M, Amirpour A, Zavvar R, Behjati M, Gharipour M. Predictive value of P-wave duration and dispersion in post coronary artery bypass surgery atrial fibrillation. ARYA Atheroscler J 2012;8:59 –62. 19. Ehrlich JR, Schadow K, Steul K, Zhang GQ, Israel CW, Hohnloser SH. Prediction of early recurrence of atrial fibrillation after external cardioversion by means of P wave signal-averaged electrocardiogram. Z Kardiol 2003;92:540–6. 20. Buber J, Luria D, Sternik L, Kuperstein R, Grupper A, Goldenberg I et al. Morphological features of the P-waves at surface electrocardiogram as surrogate to mechanical function of the left atrium following a successful modified maze procedure. Europace 2014;16:578 –86. 21. Blanche C, Tran N, Rigamonti F, Burri H, Zimmermann M. Value of P-wave signal averaging to predict atrial fibrillation recurrences after pulmonary vein isolation. Europace 2013;15:198 –204. 22. Masuda M, Inoue K, Iwakura K, Okamura A, Toyoshima Y, Doi A et al. Impact of pulmonary vein isolation on atrial late potentials: association with the recurrence of atrial fibrillation. Europace 2013;15:501 –7. 23. Sih HJ, Zipes DP, Berbari EJ, Adams DE, Olgin JE. Differences in organization between acute and chronic atrial fibrillation in dogs. J Am Coll Cardiol 2000;36: 924 –31. 24. Matsuo S, Lellouche N, Wright M, Bevilacqua M, Knecht S, Nault I et al. Clinical predictors of termination and clinical outcome of catheter ablation for persistent atrial fibrillation. J Am Coll Cardiol 2009;54:788 –95. 25. Gaspo R, Bosch RF, Talajic M, Nattel S. Functional mechanisms underlying tachycardia-induced sustained atrial fibrillation in a chronic dog model. Circulation 1997;96:4027 –35. 26. Limantoro I, de Vos CB, Delhaas T, Weijs B, Blaauw Y, Schotten U et al. Clinical correlates of echocardiographic tissue velocity imaging abnormalities of the left atrial wall during atrial fibrillation. Europace 2014;16:1546 – 53. 27. Allessie M, Ausma J, Schotten U. Electrical, contractile and structural remodeling during atrial fibrillation. Cardiovasc Res 2002;54:230–46. 28. Burstein B, Nattel S. Atrial fibrosis: mechanisms and clinical relevance in atrial fibrillation. J Am Coll Cardiol 2008;51:802 –9. 29. Schoonderwoerd BA, Van Gelder IC, Van Veldhuisen DJ, Van den Berg MP, Crijns HJ. Electrical and structural remodeling: role in the genesis and maintenance of atrial fibrillation. Prog Cardiovasc Dis 2005;48:153 –68. 30. Goudis CA, Kallergis EM, Vardas PE. Extracellular matrix alterations in the atria: insights into the mechanisms and perpetuation of atrial fibrillation. Europace 2012; 14:623 – 30.
Europace (2015) 17, 647–654 doi:10.1093/europace/euu212
Effect of renal sympathetic denervation on the progression of paroxysmal atrial fibrillation in canines with long-term intermittent atrial pacing Xule Wang 1,2, Congxin Huang 1,2*, Qingyan Zhao1,2*, He Huang 1,2, Yanhong Tang 1,2, Zixuan Dai 1,2, Xiaozhan Wang 1,2, Zongwen Guo 1,2, and Jinping Xiao 1,2 1 Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan City 430060, People’s Republic of China; and 2Cardiovascular Research Institute of Wuhan University, Wuhan City 430060, People’s Republic of China
Received 25 March 2014; accepted after revision 3 July 2014; online publish-ahead-of-print 27 October 2014
Aims
The aim of the present study was to explore the effect of renal sympathetic denervation (RSD) on the progression of paroxysmal atrial fibrillation (AF) in canines with long-term intermittent atrial pacing. ..................................................................................................................................................................................... Methods Nineteen beagles were randomly divided into sham-operated group (six dogs), control group (six dogs), and RSD group and results (seven dogs). Sham-operated group were implanted with pacemakers without pacing; control group were implanted with pacemakers with long-term intermittent atrial pacing; and RSD group underwent catheter-based RSD bilaterally and were simultaneously implanted with pacemakers. Atrial pacing was maintained for 8 h a day and a total of 12 weeks in the control group and RSD group. Echocardiography showed that the left atrial structure and function were significantly improved in the RSD group compared with the control group (P , 0.05). Compared with the control group, the RSD group had fewer incidences of AF and a shorter duration of AF (P , 0.05) after long-term intermittent atrial pacing. In addition to increased atrial effective refractory period (AERP) and AF cycle length, AERP dispersion and P-wave duration and dispersion were significantly decreased in the RSD group compared with the control group (P , 0.05). Atrial morphological evaluation suggested that fibrosis and ultrastructural changes induced by long-term intermittent atrial pacing were markedly suppressed in the RSD dogs compared with controls (P , 0.05). Immunohistochemistry results showed that connexin 43 distribution in RSD mid-myocardial was significantly fewer heterogeneous than that in control midmyocardial (P , 0.05). ..................................................................................................................................................................................... Conclusion Renal denervation inhibits the progression of paroxysmal AF, which might be related to the suppression of atrial electrophysiology and structural heterogeneity.
----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords
Renal sympathetic nerve † Ablation † Atrial fibrillation † Atrial electrophysiology † Heterogeneity
Introduction In the recent few years, renal sympathetic denervation (RSD) applied to the treatment of cardiovascular disease has become a hot spot in the clinical trials and animal experiments. The SYMPLICITY HTN-11 and HTN-2 studies2 have suggested that RSD has achieved a breakthrough in the treatment of resistant hypertension, which might have related to the inhibition of chronic sympathetic nervous overactivation. Although optimism has recently been tempered with the broadcast alert from Medtronic that the SYMPLICITY HTN-3 did
not meet its primary efficacy endpoint,3 RSD has great potential in the treatment of other cardiovascular diseases, such as heart failure, left ventricular hypertrophy, refractory atrial fibrillation (AF), and post-myocardial infarction cardiac remodelling.4 – 9 In recent reports from our laboratory,10,11 we demonstrated that RSD not only could suppress AF in canines with 7 h rapid right atrial pacing (RAP), but also could effectively suppress the incidences of AF and atrial remodelling in canines during sustained RAP for 5 weeks, which might be related to the inhibition of renin–angiotensin –aldosterone system (RAAS). Whether renal denervation can effectively
* Corresponding author. No. 238 Jiefang Road, Wuchang District, Wuhan City, Hubei Province, China. Tel: +86 13871329139; fax: +8627 88042292. E-mail address: huangcongxin@vip. 163.com (C.H.) or [email protected] (Q.Z.) Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2014. For permissions please email: [email protected].
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What’s new? † This study showed that the induced atrial fibrillation produced by long-term intermittent atrial pacing could be reduced by catheter-based renal sympathetic denervation (RSD) in ambulatory canines, suggesting that renal denervation inhibits the progression of paroxysmal atrial fibrillation. † Furthermore, we found that the increase of right atrial pressure, mean pulmonary artery pressure, and pulmonary capillary wedge pressure produced by long-term intermittent atrial pacing was prevented by RSD. † In addition to increased atrial effective refractory period (AERP) and atrial fibrillation cycle length, AERP dispersion, P-wave duration and dispersion were significantly decreased in the RSD group compared with the control group, suggesting that RSD suppresses the heterogeneity of atrial electrophysiology after long-term intermittent right atrial pacing. † We also found that atrial structural remodelling including left atrial dilatation, atrial fibrosis, and ultrastructural changes together with the heterogeneity of connexin 43 were markedly suppressed in the RSD dogs compared with controls.
suppress the progression of paroxysmal AF is unknown. The aim of the present study was to explore the effect of RSD on the progression of paroxysmal AF in canines with long-term intermittent atrial pacing, and further observe the effect of RSD on functional and morphological changes induced by pacing in the canine model.
Methods Experimental animals and study protocol The study protocol was approved by the Ethical Committee of the Wuhan University School of Medicine, and all animal handling was performed in accordance with the Wuhan Directive for Animal Research and the current Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 85 – 23, revised 1996). We studied 19 healthy adult beagles (weight, 11.2 – 14.5 kg) assigned randomly to three groups. The sham-operated group consisted of six dogs that were implanted with pacemakers without pacing; the control group consisted of six dogs that were implanted with pacemakers with long-term intermittent atrial pacing; and the RSD group consisted of seven dogs that underwent catheterbased RSD and simultaneously were implanted with pacemakers. Anaesthesia was induced by administering 30 mg kg21 pentobarbital sodium intravenously and was maintained by isoflurane. All of the dogs were intubated and ventilated with room air supplemented with oxygen from a respirator (MAO01746, Harvard Apparatus). Normal saline at 50 – 100 mL h21 was infused to replace spontaneous fluid losses. Standard surface 12-lead electrocardiograms (ECGs) (MAC1200, GE) were monitored continuously throughout the procedure. Under fluoroscopy, an atrial endocardial pacing electrode (1642T, St Jude Medical, Inc.) was delivered to the RA appendage via the right external jugular vein and connected to a high-rate cardiac pacemaker (Fudan University, Shanghai, China), which was implanted in a subcutaneous pocket of the neck. After 3 days for recovery, RSD group and control group dogs were paced at 400 b.p.m. for 8 h a day and a total of 12 weeks (Figure 1).
Beagles (n = 19)
Control group (n = 6)
Sham-operated group (n = 6)
RSD group (n = 7)
Echocardiography, haemodynamic study, electrophysiological measurements, venous blood collected
Pacemakers without pacing, with feed for 12 weeks
Atrial pacing in 400 b.p.m. for 8 h a day and 12 weeks
After RSD, atrial pacing in 400 b.p.m. for 8 h a day and 12 weeks
Echocardiography, haemodynamic study, electrophysiological measurements, venous blood and atrial tissue collected
Figure 1 Flowchart of the study.
In the RSD group dogs, a tailor-made quadrupole radiofrequency ablation catheter was inserted into each renal artery via femoral artery under fluoroscopy. (When the quadrupole ablation catheter was introduced into each renal artery, its four tips were expanded automatically and applied to the wall of renal artery.) We applied radiofrequency ablations of 6 W or less and lasting up to 90 s within each renal artery. The catheter system monitored tip temperature and impedance, altering radiofrequency energy delivery in response to a predetermined algorithm. In sham-operated group and control group dogs, the ablation catheter was inserted into each renal artery without ablation.
Echocardiography The structure of left atrium (LA) and left ventricle were assessed via transthoracic echocardiographic examinations (IE33, S5-1, Philips) at both the baseline and endpoint of the study in all dogs. The LA diameter (LAD) and left ventricle end-diastolic dimension (LVEDD) were measured as the maximum dimension along the parasternal long-axis view using twodimensionally guided M-mode tracings. The LA volume (LAV) was measured using a single-plane Simpson’s method in the apical view showing the largest and smallest LA area. All of the volumes were measured in triplicate, and the averages were reported. Left atrial ejection fraction (LAEF) was calculated as (LAVmax 2 LAVmin)/LAVmax. An independent echocardiography expert reviewed the images and the parameters.
Haemodynamic study A 6F haemostatic sheath was inserted into the femoral artery, and femoral systolic and diastolic blood pressure (BP) was measured at the baseline and at the endpoint of the protocol in each dog. Furthermore, RA pressure, mean pulmonary artery pressure (MPAP), and pulmonary capillary wedge pressure (PCWP) were monitored by a balloon wedge pressure catheter (REF AI-07126, Arrow International, Inc.) through a 6F sheath placed in the femoral vein.
Electrophysiological measurements P-wave duration was measured from the surface ECG at baseline and after pacing. Twelve-lead ECG was recorded at the speed of 200 mm/s in supine position. P-wave duration was measured from the onset to the offset of the P-wave in the lead with the longest P-wave. The onset
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and offset were defined as the junction between the P-wave pattern and the isoelectric line. P-wave dispersion was defined as the difference between the maximum and minimum P-wave duration in 12 leads. A multipolar electrode catheter was introduced into the femoral veins through a 6F haemostatic sheath and placed in the high RA. Atrial vulnerability and atrial effective refractory period (AERP) were determined at the RA at the baseline and after pacing. Once intermittent atrial pacing up to 12 weeks, the chest was opened by means of a median sternotomy and the heart was exposed and suspended in a pericardial sling. Four quadripolar plaque electrodes were surgically attached to the RA, RA appendage, LA, LA appendage, and right superior pulmonary vein. The AERP was measured at the above five epicardial sites by delivering a train of eight atrial paced beats S1 at a cycle length of 250 ms, followed by an extrastimulus (S2) introduced at coupling intervals. The S1 – S2 intervals were decreased from 180 ms to refractoriness initially by decrements of 10 ms. As the S1 –S2 intervals approached the AERP, decrements were reduced to 2 ms. Atrial effective refractory period dispersion was defined as the difference between the maximum and minimum AERP in the five sites. An S1S1 (120, 100, and 75 ms cycle length, respectively, lasting for 5 s each) programmed stimulus method was used to assess the inducibility and duration of AF. Atrial fibrillation was defined as irregular atrial rates faster than 500 b.p.m. associated with irregular atrioventricular conduction lasting .5 s. Atrial fibrillation times and AF duration separately refer to the average number of times and length of time that AF was induced in every dog within a group. Atrial fibrillation cycle length (AFCL) was calculated by measuring the interval at a timescale of 200 mm/s between the atrial excited signals in the whole recorded epicardial electrogram diagram of AF and averaged to obtain the mean AFCL. Measurements were repeated by two investigators.
Enzyme-linked immunosorbent assay Two millilitres of venous blood were collected in ethylenediaminetetraacetic acid vacutainers and centrifuged at 3000 gravities for 10 min at 48C at the baseline and endpoint of the protocol. The plasma was separately kept in microtubes and stored at 2808C until assay. The plasma levels of angiotensin II (Ang II; Ang II Elisa Kit, Enzo Life Sciences Inc.) and aldosterone (Aldosterone EIA Kit, Enzo Life Sciences Inc.) were examined using an enzyme-linked immunosorbent assay.
Morphological evaluation At the completion of the protocol, the animals were euthanized and the hearts were quickly excised. Segments from the atrium were fixed with 4% paraformaldehyde at 48C and then embedded in paraffin for morphological evaluation. Light microscopy was stained with Masson trichrome solution. The interstitial collagen volume fraction (CVF) in the atrium was determined via quantitative morphometry with an image analyzer [Image-Pro Plus 6.0 (IPP 6.0), Media Cybernetics]. For electron microscopy, segments of the LA and RA were immediately fixed with glutaraldehyde stationary liquid and stored at 48C. Ultrathin section (70 – 80 nm) was cut from each sample, counterstained with uranium acetate and lead citrate, and examined with a transmission electron microscope (Hitachi HT7700) by two blinded observers.
Immunohistochemistry Four micrometre sections were cut from paraffin blocks of the LA and RA. The sections were stained with connexin 43 (Cx43, monoclonal goat anti-Cx43, Santa Inc.; used at 1 : 500). Heterogeneity of Cx43 expression was quantified by transforming photomicrographs of subepicardium and mid-myocardium of Cx43. The mean density of these slides was used as a measure of Cx43 heterogeneity. We determined the
density by the computer-assisted IPP 6.0 software. Each slide was examined under a microscope with ×40 objectives to select three fields with the highest density. The density was the positive area divided by the total area examined. The mean density in these three selected fields was used to represent the density of that slide.
Statistical analysis Data are expressed as mean + standard deviation (SD). Analyses were performed using SPSS version 17. Paired t-tests were used to compare the means of the baseline with that of the endpoint in the same group. Differences between groups were tested with one-way analysis of variance (ANOVA) ( post hoc Student–Newman–Keuls); cases of significant difference were further analysed with the Tukey–Kramer test. When data were not normally distributed, a Kruskal–Wallis one-way ANOVA on ranks was performed ( post hoc Dunn method). All statistical tests were two-sided, and statistical significance was established at P , 0.05.
Results Effect of renal sympathetic denervation on atrial structure and function The averaged echocardiographic data for LAD, LVEDD, LAV, and LAEF are shown in Table 1. At the endpoint of the protocol compared with sham-operated dogs, LAD, LAVmax, and LAVmin increased significantly in the control and RSD groups, while LAEF decreased after termination of long-term intermittent atrial pacing. Compared with the control group, the LAD, LAVmax, and LAVmin decreased, whereas the LAEF increased in the RSD group after RAP for 8 h a day and a total of 12 weeks (P , 0.05). There was no significant difference in LVEDD among the three groups (P . 0.05).
Haemodynamic changes Table 2 summarizes the average RA pressure, MPAP, PCWP, systolic BP, and diastolic BP responses in all dogs. Compared with the control group, RSD decreased systolic and diastolic BP (P , 0.05). Similarly, RSD prevented the increase of RA pressure, MPAP, and PAWP produced by long-term intermittent atrial pacing (P , 0.05).
Effect of renal sympathetic denervation on atrial vulnerability As Table 3 showed the baseline characteristics of the frequency and duration of AF among the three groups were similar (P . 0.05). At the endpoint of the study compared with the sham-operated group, the control group dogs had significantly more episodes of AF (P , 0.01), and longer durations of AF (P , 0.05) after long-term intermittent atrial pacing, while the RSD group dogs had similar episodes of AF (P . 0.05), and durations of AF (P . 0.05). Compared with the control group, RSD group dogs subjected to long-term intermittent atrial pacing had significantly fewer episodes of AF (P , 0.01) and shorter durations of AF (P , 0.05).
Effect of renal sympathetic denervation on atrial electrophysiology The changes in the electrophysiological properties of three groups were shown in Table 3. The results indicated that AERP in both control group and RSD group beagles were significantly shortened, while P-wave duration and dispersion were significantly prolonged
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Table 1 Changes in echocardiographic indices before and after rapid atrial pacing LAD (mm)
LVEDD (mm)
19.0 + 1.5 19.3 + 0.8
27.5 + 1.5 27.8 + 1.2
18.7 + 1.2 23.0 + 1.1**,††
28.0 + 1.9 29.3 + 1.0
18.8 + 1.7 21.4 + 1.0**,††,‡
27.9 + 2.0 28.7 + 1.4
LAVmax (cm3)
LAVmin (cm3)
LAEF (%)
2.0 + 0.6 2.3 + 0.5
71.7 + 7.2 68.9 + 4.5
2.2 + 0.8 5.8 + 1.2**,††
68.8 + 9.0 52.2 + 5.8*,††
2.1 + 0.7 3.4 + 0.5**,†,‡‡
70.4 + 7.4 61.7 + 5.7**,††,‡
............................................................................................................................................................................... Sham-operated group Baseline Endpoint
7.0 + 0.9 7.5 + 1.1
Control group Baseline Endpoint
6.8 + 0.8 12.2 + 1.6**,††
RSD group Baseline Endpoint
7.1 + 0.9 9.0 + 1.2**,†,‡‡
LAD, left atrial diameter; LVEDD, left ventricular end-diastolic dimension; LAVmax, left atrial maximal volume; LAVmin, left atrial minimal volume; LAEF, left atrial ejection fraction. *P , 0.05, **P , 0.01, compared with baseline in the same group. † P , 0.05, ††P , 0.01, compared with the sham-operated group at the same point. ‡ P , 0.05, ‡‡P , 0.01, compared with the control group at the same point.
Table 2 Haemodynamic parameters in the sham-operated, control, and RSD groups (mmHg) Systolic BP
Diastolic BP
RA pressure
MPAP
PCWP
............................................................................................................................................................................... Sham-operated group Baseline Endpoint
137 + 13
84 + 9
7+2
21 + 4
8+3
134 + 17
86 + 10
8+1
22 + 5
9+3
8+2
22 + 4
Control group Baseline
140 + 16
88 + 11
Endpoint
137 + 14
86 + 9
RSD group Baseline
135 + 18
85 + 10
8+1
21 + 3
8+2
127 + 12‡
78 + 9†‡
9 + 3‡‡
24 + 5‡
10 + 3‡
Endpoint
16 + 3**,††
32 + 6*,††
9+2 18 + 4**,††
BP, blood pressure; MPAP, mean pulmonary artery pressure; PCWP, pulmonary capillary wedge pressure. *P , 0.05, **P , 0.01, compared with baseline in the same group. † P , 0.05, ††P , 0.01, compared with the sham-operated group at the same point. ‡ P , 0.05, ‡‡P , 0.01, compared with the control group at the same point.
after long-term intermittent atrial pacing (P , 0.05). At the endpoint of the study compared with the sham-operated group, AERP dispersion, P-wave duration and dispersion in control beagles were significantly lengthened; however, the increasing trends of AERP dispersion and P-wave duration in the RSD group were not significantly different (P . 0.05). Compared with the control group, RSD group dogs had significantly longer AERP and shorter AERP dispersion, P-wave duration and dispersion after RAP for 8 h a day and a total of 12 weeks (P , 0.05). There were six dogs induced AF in the control group of six dogs while four of seven dogs in the RSD group in the RA after long-term intermittent atrial pacing. Moreover, the AFCL were longer in the RSD group than that of the control group (122.5 + 6.6 vs. 102.3 + 8.7 ms, P , 0.01).
Effect of renal sympathetic denervation on the plasma levels of angiotensin II and aldosterone Compared with the baseline, control group and RSD group dogs had a significant increase in the plasma levels of Ang II and aldosterone
after long-term intermittent atrial pacing (P , 0.01). At the endpoint of the protocol, compared with the sham-operated group, both control group and RSD group beagles showed a statistically significant increase in the plasma levels of Ang II (control group, 2877.9 + 376.9 vs. 676.3 + 166.3 pg/mL, P , 0.01; RSD group, 1174.9 + 181.9 vs. 676.3 + 166.3 pg/mL, P , 0.01) and aldosterone (control group, 377.9 + 63.7 vs. 62.5 + 18.8 pg/mL, P , 0.01; RSD group, 161.4 + 38.3 vs. 62.5 + 18.8 pg/mL, P , 0.01); rather, the plasma levels of Ang II and aldosterone were significantly reduced after RSD compared with the values for the control group (Ang II, 1174.9 + 181.9 vs. 2877.9 + 376.9 pg/mL, P , 0.01; aldosterone, 161.4 + 38.3 vs. 377.9 + 63.7 pg/mL, P , 0.01).
Effect of renal sympathetic denervation on atrial fibrosis Representative histological sections of atrial myocardium are shown in Figure 2. Atrial Masson staining for collagen showed a normal intercellular space in the samples from sham-operated dogs with CVF values of 3%, which was significantly increased in both LA and
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Table 3 Changes in the electrophysiological properties among the three groups before and after rapid atrial pacing AF times
AF duration (s)
AERP (ms)
AERP dispersion (ms)
P-wave duration (ms)
P-wave dispersion (ms)
55.5 + 3.1
12.3 + 2.6
5.0 + 2.1
60.0 + 3.4
12.7 + 3.0
56.7 + 6.0
13.0 + 2.7
76.5 + 9.2**,††
27.7 + 3.3**,††
55.0 + 5.0
13.9 + 3.5
66.1 + 5.5**,‡
19.7 + 2.8**,††,‡‡
............................................................................................................................................................................... Sham-operated group Baseline
0.0 + 0.0
0.0 + 0.0
126.0 + 1.8
Endpoint Control group
0.2 + 0.4
1.2 + 2.9
122.7 + 2.4
0.8 + 2.0
126.7 + 4.3
Baseline Endpoint RSD group
0.2 + 0.4 2.8 + 0.4**,††
40.7 + 24.2*†
108.3 + 5.6**,††
Baseline
0.0 + 0.0
0.0 + 0.0
126.6 + 1.9
Endpoint
0.7 + 0.8*,‡‡
4.6 + 5.7‡
117.4 + 2.2**,†,‡‡
24.3 + 5.9††
9.1 + 4.3‡‡
AF, atrial fibrillation; AERP, atrial effective refractory period. AF times and AF duration separately refer to the average number of times and length of time that AF was induced in every dog within a group. *P , 0.05, **P , 0.01, compared with baseline in the same group. † P , 0.05, ††P , 0.01, compared with the sham-operated group at the same point. ‡ P , 0.05, ‡‡P , 0.01, compared with the control group at the same point.
Figure 2 Effect of RSD on fibrosis in the atria detected with Masson’s trichrome staining. Red areas represent myocytes, and blue areas represent collagen. (A) Examples of sham-operated LA; (B) examples of control LA; (C) examples of RSD LA; (A′ ) examples of sham-operated RA; (B′ ) examples of control RA; (C′ ) examples of RSD RA (original magnification: ×400).
RA of the control group (LA, 14.2 + 4.5 vs. 3.3 + 1.0%, P , 0.01; RA, 15.8 + 5.2 vs. 2.8 + 0.9%, P , 0.01). In comparison, RSD clearly decreased the volume fraction of collagen compared with that of the control group (LA, 5.2 + 3.1 vs. 14.2 + 4.5%, P , 0.01; RA, 9.7 + 3.4 vs. 15.8 + 5.2%, P , 0.05).
Ultrastructural changes Atrial myocardial ultrastructure was documented by electron microscopy, as shown in Figure 3. Atrial myocytes from the sham-operated dogs showed a highly organized sarcomeric structure
throughout the cytoplasm, with rows of uniformly sized mitochondria and nuclei with clustered heterochromatin. A marked increase in the number and size of the mitochondria, loss of the banding pattern, and integrity of contractile elements were observed after RAP for 8 h a day and a total of 12 weeks, which were the characteristics of ultrastructural abnormalities. In contrast, the long-term intermittent atrial tachypacing-induced ultrastructural changes were dramatically suppressed by RSD. Ultrastructural abnormalities in the LA were similar to that corresponded to the RA in the control and RSD groups.
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Figure 3 Typical ultrastructural changes by transmission electron microscopy separately in LA and RA tissue. Exemplary pictures of electron microscopy in the sham-operated group (A and A′ ), the control group (B and B′ ), and the RSD group (C and C′ ). (A– C) Representative of LA; (A′ – C′ ) representative of RA. LA, left atrium; RA, right atrium (magnification: ×5.0 k at 1.0 mm).
Effects of renal sympathetic denervation on heterogeneity of connexin 43 expression in the atria Figure 4 shows the immunostaining results of Cx43 expression in the atrium among all groups. The heterogeneity of Cx43 expression, expressed as mean of the SD of different parts, was 3162.5 + 780.7, 5542.40 + 506.3, 5396.8 + 714.7, 3734.1 + 729.6, 16 322.6 + 2162.4, and 7723.4 + 1499.3 mm2/mm2 for sham-operated subepicardial, control subepicardial, RSD subepicardial, sham-operated midmyocardial, control mid-myocardial, and RSD mid-myocardial, respectively. Analysis of variance revealed that Cx43 heterogeneity in sham-operated subepicardial and sham-operated mid-myocardial was not statistically different (P . 0.05), so was that in RSD subepicardial and RSD mid-myocardial. Compared with the RSD group, Cx43 distribution in control subepicardial and control midmyocardial was significantly more heterogeneous (P , 0.05).
Discussion Major finding This study shows that the induced AF produced by long-term intermittent atrial pacing could be reduced by catheter-based RSD. In addition to increased AERP and AFCL, AERP dispersion, P-wave duration and dispersion were significantly decreased in the RSD group compared with the control group. In connection with decreased plasma
levels of Ang II and aldosterone, atrial structural remodelling including LA dilatation, atrial morphology together with the heterogeneity of Cx43 were markedly attenuated in the RSD dogs compared with controls.
Effect of renal sympathetic denervation on haemodynamics and paroxysmal atrial fibrillation induced by long-term intermittent atrial pacing Renal sympathetic denervation selectively reduces both renal sympathetic efferent and afferent nerve activities and is originally found to treat resistant hypertension, which is indicated by a reduction in plasma renin activity.12 Nowadays, the beneficial effect of this procedure is extended to other syndromes in which sympathetic hyperactivity is a component of the underlying disease process. Our previous studies have demonstrated that renal denervation could effectively suppress incidences of AF and atrial remodelling in canines with atrial pacing, which might be related to the inhibition of RAAS.10,11 It is well known that haemodynamic changes, resulting in atrial stretch and alterations in neurohormonal activation, may result in atrial remodelling which may favour an environment vulnerable to the development of atrial arrhythmia. In the present study, we found the same effect of RSD on BP and induced AF in beagles with RAP for 8 h a day and a total of 12 weeks, which simulated the model of paroxysmal AF. Furthermore, we also found that the increase of RA pressure, MPAP, and PAWP induced by long-term
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Effect of RSD on progression of paroxysmal AF in canines
Figure 4 Heterogeneity of Cx43 expression in the LA among all groups. (A) Sham-operated subepicardial; (B) control subepicardial; (C) RSD subepicardial; (A′ ) sham-operated mid-myocardial; (B′ ) control mid-myocardial; (C′ ) RSD mid-myocardial (original magnification: ×400).
intermittent atrial pacing was prevented by RSD. The results of plasma levels of Ang II and aldosterone indicated that the inhibition of RAAS still may be a mechanism underlying these results.
Effect of renal sympathetic denervation on atrial electrical and structural heterogeneity Atrial fibrillation causes atrial electrical and structural remodelling, which have an important role in the perpetuation and progression of the arrhythmia. Our prior studies of AERP in canines during atrial pacing have demonstrated that the shortening in AERP induced by RAP was reversible after RSD.10,11 In the present study, the same phenomenon that RSD attenuated the shortening in AERP produced by RAP for 8 h a day and a total of 12 weeks can be observed. In addition, we found that AERP dispersion was completely increased after long-term intermittent atrial pacing while RSD prevented RAP-induced increasing of AERP dispersion. The statistical importance of AERP dispersion supports the importance of RAP-induced electrical heterogeneity in the substrate for AF. Data from the present study pointed out RSD could inhibit the increase in regional heterogeneity of refractoriness in experimental paroxysmal AF, which contributes to the maintenance of AF. There are large number of clinical studies confirming the role of P-wave duration and dispersion as non-invasive useful markers associated with paroxysmal AF.13,14 Prolonged P-wave duration and dispersion were reported to be commonly found in patients with a history of paroxysmal AF.13,15 These simple, non-invasive tests are still applied successfully by many investigators.16 – 22 The results of our study showed that the occurrence of paroxysmal AF caused a significant increase in P-wave duration and dispersion, and RSD attenuated the increase of P-wave duration and dispersion induced by long-term intermittent atrial pacing. This finding agrees with
published observations supporting a role for atrial electrical heterogeneity in canines with paroxysmal AF. Furthermore, it seems that RSD decreases sympathetic renal afferent nerve activity and central sympathetic drive, leading to decreased atrial electrical heterogeneity. Furthermore, AFCL was also observed as atrial electrical heterogeneity in the present study. Previous studies of AFCL in animals and humans have shown a good correlation between AFCL and AF.23 – 26 In these studies, a shorter AFCL was observed in chronic AF compared with acute AF23 and the surface ECG AFCL independently predicted procedural termination of persistent AF.24 Atrial fibrillation cycle length was longer in the RSD group compared with the control group, further suggesting that RSD might reduce the heterogeneity in atrial electrophysiology and facilitate AF termination. Our previous study showed that RSD could suppress the atrial substrate remodelling in ambulatory canines during prolonged RAP, including the significantly increased atrial fibrosis, inflammation, and apoptosis, together with atrial gap junctional remodelling and neural remodelling.11 Left atrial enlargement and fibrosis have been proposed as two of the core processes involved in atrial remodelling.27 – 30 Moreover, LA size and fibrosis19 could also play a role in determining P-wave duration, that was an important role for atrial electrical heterogeneity. The results of echocardiography showed that the LA dilation and hypofunction in experimental paroxysmal AF were markedly improved by RSD. Fibrosis in both LA and RA caused by long-term intermittent atrial pacing were attenuated after RSD. The results under electron microscopy suggested that the long-term intermittent tachypacing-induced atrial ultrastructural changes were dramatically attenuated by RSD. These findings implied that RSD might inhibit atrial structural remodelling in canines with paroxysmal AF. Immunohistochemistry results showed that the heterogeneity of Cx43 distribution produced by long-term intermittent atrial pacing was significantly reduced after RSD. All these suggested
654 renal denervation suppress the atrial structural heterogeneity after long-term intermittent RAP, especially for Cx43 heterogeneous distribution.
Conclusion Our previous studies showed that the ablation of renal artery could be applied to AF therapy in canines during short-term or sustained RAP.10,11 In the present study, we further demonstrated that RSD could reduce the incidences of AF and suppress the atrial electrophysiology and structural heterogeneity after long-term intermittent RAP. All these indicate that renal denervation might provide a new strategy useful for preventing the progression of paroxysmal AF. Conflict of interest: none declared.
Funding This study was supported by the National Science and Technology Pillar Program of China (2011BAI11B12) and National Key Basic Research Development Program of China (The ‘973’ Program, 2012CB518604).
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