autologous dermal fibroblast injections slow atrioventricular conduction...
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DOI: 10.1161/CIRCEP.114.001536
1
Autologous Dermal Fibroblast Injections Slow Atrioventricular Conduction
and Ventricular Rate in Atrial Fibrillation in Swine
Running title: Tondato et al.; Fibroblasts Slow Ventricular Rate in AF
Fernando Tondato, MD, PhD1,2; Hong Zeng, MD1; Traci Goodchild, PhD1;
Fu Siong Ng, MD, PhD1; Nicolas Chronos, MD, FACC1; Nicholas S. Peters, MD2
1Myocardial Function Section, Imperial College & Imperial NHS Trust, London,
United Kingdom; 2Saint Joseph’s Translational Research Institute/ Saint Joseph’s
Hospital of Atlanta, Atlanta, GA
Correspondence:
Nicholas S. Peters, MD
ICTEM 4th Floor
Hammersmith Campus, Du Cane Rd
London, England, W12 0NN
United Kingdom
Tel: +44-207-594-1880
Fax: +44-207-594-1880
E-mail: [email protected]
Journal Subject Codes: [130] Animal models of human disease, [5] Arrhythmias, clinical electrophysiology, drugs, [106] Electrophysiology
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Abstract:
Background - Non-pharmacological ventricular rate control in atrial fibrillation (AF) without
producing atrioventricular block (AVB) remains a clinical challenge. We investigated the
hypothesis that autologous dermal fibroblast (ADF) injection into the AV nodal area (AVNA)
would reduce ventricular response during AF without causing AVB.
Methods and Results - Fourteen pigs underwent electrophysiology study before, immediately
and 28 days after ~200 million cultured ADFs (n=8) or saline (n=6) were injected under
electroanatomical guidance in the AVNA, with continuous 28-day ECG recording. In the ADF
group at 28 days post-injection there were prolongations of PR interval (after vs. before:
130±13msec vs. 113±14msec, P=0.04), of AH interval during both sinus rhythm (92±13msec vs.
76.8±8msec, P<0.01) and atrial pacing at 400ms 102±13msec vs. 91±9msec, P<0.01), and of AV
node Wenckebach cycle length (230±19mec vs. 213±24msec, P<0.01), with no changes in the
control group. The RR interval during induced AF 28 days after injections was 24% longer in
ADF-treated group compared to controls (488±120msec vs. 386±116msec, P<0.001).
Histological analysis revealed presence of ADF-labeled cells in the AVNA at 28 days. Transient
accelerated junctional rhythm during injections, and transient nocturnal Mobitz I AV conduction
occurred early post-injection in both groups.
Conclusions - Cells survived for 4 weeks and significantly slowed AV conduction and
ventricular rate in acutely induced AF. Critically, despite a large number of injections in the
AVNA and marked effects on AV conduction, AVB did not occur. Further studies are necessary
to determine the clinical feasibility and safety of this strategy for ventricular rate control in AF.
Key words: atrioventricular node, fibroblasts, cell transplantation
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Introduction
Drug treatment to maintain long-term sinus rhythm or achieve ventricular rate control in atrial
fibrillation (AF) is limited by lack of efficacy or intolerance of side effects1, 2. Non-
pharmacological approaches3, 4 such as pulmonary vein isolation are effective but this is not
practicable or appropriate for all patients. Several randomized controlled clinical trials5-7 have
shown that ventricular rate control in patients with atrial fibrillation can be an effective
therapeutic approach, with outcomes in some patient groups that are comparable to strategies for
maintenance of sinus rhythm. Rate control in AF therefore remains an appropriate and common
palliative strategy, and for symptomatic patients when all pharmacological and other non-
pharmacological approaches have failed, AV node ablation is a well-established and widely
practiced technique8, achieving control of symptoms in the majority of patients, but requires
permanent pacemaker implantation. Previously, various strategic approaches to attempt AV node
modification to control rather than abolish AV conduction have all been shown to have
prohibitive risk of complete AV block,9, 10 and are therefore rarely attempted in current clinical
practice.
Among the advances in the area of cell therapy11-14, multiple cell types have been used
for myocardial repair and regeneration15-18 including restoration of conduction in experimental
models19, 20. Cellular therapies have the potential to either enhance or block electrical conduction
of the heart, 21, 22 and although dermal fibroblasts injected in the atrioventricular node area have
been shown to modify conduction velocity,21 the clinical therapeutic goal in AF is not that of
slowing AV nodal conduction, but of controlling ventricular response rate. The aim of this study
was to address the hypothesis that injections of adult autologous dermal fibroblasts (ADF) in the
AV node area would inhibit AV node conduction, resulting in control of ventricular response
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during AF without inducing complete AV block or evidence of fibrotic changes in the lung.
Methods
Animal Preparation
The protocol was consistent with federal guidelines for the care and use of laboratory animals
and was approved by the Institutional Animal Care and Use Committee of the Saint Joseph’s
Translational Research Institute. Fourteen juvenile farm pigs weighing 48.7±10.1 Kg were
enrolled into the study. Anesthesia was induced by an intramuscular injection of a combination
of 2 to 4mg/Kg of telazol, 4mg/Kg of ketamine and 2mg/Kg of xylazine, followed by intubation
and general anesthesia induced and maintained using inhalant isoflurane (~1.5-2.5% in O2) .
Autologous Dermal Fibroblast Preparation
The groin regions were shaved, scrubbed and draped. A 6 x 2 cm sample of skin was harvested.
The tissue was minced and digested in 0.25% Trypsin-EDTA for 1h at 37oC. Dulbecco’s
modified eagles medium (DMEM) containing 15% fetal bovine serum (FBS) was added then
digest was filtered through 100 m strainer. Cells were washed twice with phosphate buffered
saline (PBS) and the final cell pellet was re-suspended in DMEM with 10% FBS with
Penicillin/Streptomycin (1%) and placed in culture at 37oC with 5% CO2. Fibroblasts were
grown to 80% confluence then passaged (22). Passage 2-4 was used for cell injections. Prior to
injection, fibroblasts were labeled with CM–DiI 2 l/ml (Invitrogen, Carlsbad, CA). Cell number
and viability was determined using the Trypan blue dye exclusion method before injection.
Electrophysiologic Study:
Standard quadripolar EP catheters (Cordis, 6F) were inserted through the right and/or left
femoral veins and advanced to the right atrial (RA), right ventricular apex (RVA), coronary sinus
and His bundle positions under fluoroscopic guidance. The electrodes were connected to an EP-3
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WorkMate® system (EP MedSystems, Inc., Mount Arlington, NJ, U.S.A.), and standard
electrophysiological study with programmed stimulation was performed to determine PR, AH
and HV intervals, AV node Wenckebach and RR intervals during AF induced by rapid RA
pacing. PR interval was determined as the time interval between the P wave onset and QRS onset
on the surface ECG. AH interval was determined as the time interval between the initial atrial
deflection to the initial H deflection recorded in the His bundle electrogram, and was measured
during sinus rhythm and atrial pacing at 400, 500, and 600msec drive cycle length by using
2.0msec square pulses at twice diastolic pacing threshold. HV interval was determined as the
time interval between the initial H deflection recorded in the His bundle electrogram and the
earliest deflection of the QRS complex on the surface ECG. The AV node Wenckebach cycle
length was determined by decremental pacing from RA (progressive reduction of pacing cycle
length, translating into faster pacing rates) at twice the diastolic pacing threshold until
anterograde type I second-degree AV block (Wenckebach) occurred. The above EP
measurements were performed before injections, immediately after injections and at post
injection time point of 28 days. AF was induced at follow-up (28 days) by RA pacing at a cycle
length of 100msec at 2.0msec square pulses and current 10.0mA, and sustained for a variable
period after cessation of pacing, during which ten consecutive RR intervals during AF were used
to calculate ventricular rate.
Autologous Dermal Fibroblast Injections:
Eight pigs received cell injections and six pigs received saline solution as a control.
An RA geometry was made with a 4mm tip eletroanatomical mapping catheter (Biosense
Webster, Diamond Bar, CA, U.S.A.) with a filling threshold of 15mm, marking the positions of
the His bundle and the ostium of coronary sinus (CARTOTM 4.0 system, Biosense, Diamond Bar,
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CA, U.S.A.). A Myostar catheter (Biosense Webster, Diamond Bar, CA, U.S.A.) - a deflectable
CARTO-compatible injection catheter, was then used for marking the locations of the multiple
injections on the RA geometry. The Myostar catheter has an adjustable needle depth.
Preliminary bench testing had determined the ideal cell concentration to maximize viability and
the optimal volume of each injection that could be retained in an ex-vivo myocardial preparation.
Based on these preliminary studies (data not shown), the yield from the fibroblast cell cultures
was diluted to a volume of 50ml for an injection volume of 0.8ml delivered by indeflator
pressurized to 20 atm, resulting in ~60 injections per case. The Myostar is deflectable and
designed to provide support for needle penetration on deployment, and preliminary in vivo
studies using radiopaque contrast in the injectate confirmed tissue penetration and contrast
retention, and 2mm as being an optimal needle-depth setting for the purposes of injection in the
region of the triangle of Koch in this study (data not shown).
Guided by the RA geometry, multiple 0.8ml injections of a total of 50ml of ADF or
saline were delivered along the slow and fast pathway regions around the AV node. Although
initial injections focused on the inputs to AV node, the large number of injections extended to
the region of confluence of slow and fast pathways, and therefore the region of the compact AV
node, which was not deliberately avoided. In each position, the needle was deployed and the
interval between each indeflator injection was approximately 60 seconds. The sites of injections
were tagged on the CARTO map (Figure 1). After injections, a trans-thoracic echocardiogram
(Acuson, Siemens, Mountain view, CA, U.S.A.) was performed to exclude complications and
evaluate atrial and ventricular form and function.
Preliminary bench testing had determined the ideal cell concentration to maximize
viability and the optimal volume of each injection that could be retained in an ex-vivo
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myocardial preparation. Based on these preliminary studies (data not shown), the yield from the
fibroblast cell cultures was diluted to a volume of 50ml for an injection volume of 0.8ml
delivered by indeflator pressurized to 20 atm, resulting in ~60 injections per case. The Myostar
is deflectable and designed to provide support for needle penetration on deployment, and
preliminary in vivo studies using radiopaque contrast in the injectate confirmed tissue
penetration and contrast retention, and 2mm as being an optimal needle-depth setting for the
purposes of injection in the region of the triangle of Koch in this study (data not shown).
Transmitter Implant and ECG Monitoring:
After completing injections and EP evaluation, animals underwent implantation of a telemetry
transmitter (TA10CTA-D70, DSI Systems™, Data Sciences International Inc, St. Paul, MN,
U.S.A.) in the subcutaneous space of the abdomen for continuous telemetry ECG monitoring and
recording over the ensuing 4 weeks. On full disclosure and examination of all ECG recording,
the PR interval at its longest and any changes indicative of AV block or other cardiac
arrhythmia during each period of 24 hours were reported.
Restudy and Histology:
At 28 days after injections, CARTO mapping and EP studies were repeated. Animals were
euthanized; the heart and the lungs were harvested and the injected area corresponding to Koch’s
triangle encompassing the AV node region was dissected for histological analysis (Figure 2).
Samples were divided into 3 pieces and each piece was further divided into 3 sections where one
was fixed in 10% buffered formalin, embedded in paraffin, 5 m sections cut and stained with
hematoxylin-eosin for assessment of overall cellularity and Verhoeff-Masson for collagen
deposition. The other two sections were processed for frozen sectioning to identify CM- DiI
positively labeled ADF. Frozen sections were cut and counterstained with 4', 6-diamidino-2-
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the subcutaneous space of the abdomen for continuous telemetry ECG monitorin
over the ensuing 4 weeks. On full disclosure and examination of all ECG recordi
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phenylindole (DAPI, Sigma, St. Louis, MO) to highlight cell nuclei. Images were acquired using
epifluorescence microscopy (Nikon Eclipse E400, Japan).
Data analysis
Data are expressed as mean ± standard deviation. Student’s t-tests were performed in order to
compare continuous variables with normal distribution. Paired t-tests were used to compare
repeated measurements in the same group. The non-parametric test of Mann-Whitney was used
for comparison between those samples without a normal distribution. A probability value of
For multiple repeated-measures comparisons, 2-way ANOVA
was used, using significant p value <0.05.
Results
Cell Culture and Injection
A total of 1.9 ± 0.6x108 cells were obtained from each culture (Figure 3). The average culture
time was 27±10 days. The cell viability after CM-DiI labeling and immediately before injection
was 96±5%.
Injections were performed without apparent adverse effect. The number of injections was
comparable between both groups (cell group (n=8): 66±20; control group (n=6): 59±17; p=0.52).
Electrophysiologic study
Brief episodes of junctional rhythm with normal QRS morphology during and immediately
following injections were common in both groups, and resolved with return to sinus rhythm in
the first few hours post-procedure. For this reason, PR and AH interval measurements could not
be consistently or reliably measured immediately after injections. PR and AH intervals in sinus
rhythm were comparable between groups at baseline. A summary of measured intervals is shown
in Table 1.
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During sinus rhythm at 28 days after injection there was significant prolongation of both
the PR (130±13msec vs. 113±14msec, p=0.04) and AH (92±13msec vs. 80±7msec, p=0.016)
intervals compared to baseline in the ADF-treated group, but not in the control group (Table 1).
The analysis of PR and AH intervals were limited to comparison between baseline and 28 days
follow-up (t-Student) due to frequent episodes of junctional rhythm immediately after injections.
During fixed atrial pacing at 600, 500 and 400 msec at 28 days, AH intervals were
significantly increased at all cycle lengths in the ADF group compared to baseline in the ADF-
treated group but not in the control group These findings translated into significant differences
in AH interval between ADF-treated and control groups at all paced cycle lengths (Table 2).
The ANOVA analysis showed that there were no significant variations in the measured HV
interval at baseline, immediately post injection and at follow-up (Table 1), neither in the cell
group (p=0.37), nor in the control group (p=0.47).
The AV node Wenckebach cycle length showed no difference between groups either at
baseline (210±32msec in control group, and 213±24msec in cell group, p=0.87), and prolonged
significantly at 28 days only in the ADF-treated group compared to baseline (230±19mec vs.
213±24msec, p=0.009).
Ventricular rate during AF
At 28 days after injection, the mean RR interval during acutely induced AF was ~100msec
longer in the ADF-treated group compared to controls (488±120msec vs. 386±116msec,
p<0.001).
Continuous ECG Telemetry
Real-time ECG monitoring during the 28 days of observation showed PR interval prolongation
during the first 5-7 days after injection in both groups, then progressively returning to baseline
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levels in the control group. The ADF group, however, presented a partial return of PR interval
but not back to baseline levels and remained steadily elevated for the ensuing period of
observation (see Figure 4). In one animal in ADF-treated group, two transient episodes of
isolated non-conducted P waves during nocturnal second-degree AV block occurred on the
second and third days of observation (Figure 5). Neither late AV block nor change in HV
intervals occurred in either group.
Necropsy and Histological Findings
No evidence of perforation, thrombosis or pericarditis was noted during macroscopic analysis.
Under light microscope, small isolated concentrations of scant mononuclear cells were found in
the perinodal myocardium in the ADF-treated group, probably representing mild localized
inflammation, (Figure 6) with no evidence of any major inflammatory response. Under
epifluorescent microscopy, only in the ADF group, CM-DiI-labeled cells were identified (Figure
7). The presence of CM-DiI at the membrane surface concordant with the DAPI counterstaining
in the nuclei confirms cell viability at the time of sacrifice. Extensive examination of the lungs
showed no macroscopic or microscopic abnormalities.
Discussion
The findings of this study are that ADFs can be safely injected into the AV nodal region,
resulting in slowing of AV nodal conduction and of the ventricular response rate in acutely
induced AF in pigs. Importantly, despite a deliberately large number of injections, no animal
developed complete AV block in the first 28 days, and only one animal had two brief episodes of
transient nocturnal second degree AV block (narrow QRS) during the first two days after
injection. No attempt was made to avoid the compact AV node, and that some of the injections
will have been directly in to this region was evident from the acute detectable effects on AV
ng g macroscopip c annnnalalalalyy
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dal mymymymyocococararara diddd umumumm in the ADF-treated grouuupp,p probably reprppp esssenenenenting mild localized
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nodal function (prolongation of AH and PR intervals) in the absence of AV block up to 28 days,
suggests a margin of safety that is unique among all previous attempted strategies for AV nodal
modification and a highly promising proof of concept.
Transient junctional rhythm and prolongation of the PR interval during injections were
both observed, but always settled with return to sinus rhythm and normalization of the PR
interval within a few hours after completion of the injections. The frequency, timing and
consistency of these transient changes in rhythm in both groups would implicate mechanical
trauma of the injection procedure and not the later biological response to ADF transplantation.
While the initial changes can be explained by a simple mechanical effect, the late
changes seen only in the ADF group - the prolongation of the AH interval, the Wenckebach
cycle length and, most importantly with respect to potential clinical applicability, prolongation of
the mean R-R interval in acutely induced AF, result from the late biological effect of the injected
ADF cells.
Dermal fibroblasts are mesenchymal cells that are readily isolated and cultured in the
laboratory and play an important role in tissue engineering and regeneration, including the
treatment of burns, chronic venous ulcers and several other clinical applications in dermatology
and plastic surgery23-27. Although previous studies have reported the effects of ADF on
myocardial conduction, including the AV node and focusing principally on conduction velocity
(20-27), no previous studies have addressed the critical safety concern of high-grade AV block
excluded by the extended and continuous rhythm monitoring in the present study. The putative
mechanisms for the effect of the transplanted ADFs in retarding AV conduction include
collagenous interruption of the myocardial syncytium and cell separation, and myocyte-
fibroblast gap-junctional coupling providing an alternative sink for charge transfer between
to ADF transpplantntntntatataa i
nical effeffffefef ctcttt, ththththee lalatetetete
en only in the ADF group - the prolongation of the AH interval, the Wenckebach
h t
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rmal fibroblasts are mesench mal cells that are readil isolated and c lt red in th
en ononononlylylyy iiiin n nn thttt eee AAADA F group - the prolongaaatitiitioon of the AHHH interererervav l, the Wenckebach
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cardiac myocytes28. We have shown previously that injection of autologous dermal fibroblasts in
chronic myocardial infarction can potentially lead to stabilization of arrhythmogenic burden in a
swine model, preventing development of both induced and spontaneous ventricular arrhythmias.
Kizana et al29 reported that fibroblasts can be genetically modified to produce excitable cells
capable of electrical coupling, with additional potential for treating cardiac conduction defects.
In the present study, histological examination showed that after 4 weeks there was no
significant inflammatory or major fibrotic response in either the perinodal myocardium or the
lungs, which will have received significant ADF overspill from injections, indicating that despite
a different tissue of origin, autologous cells for ventricular rate control in atrial fibrillation
remains a realistic proposition requiring further investigation.
Clinical application
Clinical trials continue to address the question of rate versus rhythm control in atrial fibrillation,
and some studies indicate that in certain populations of patients, a rate control strategy may be
preferable. Although the strategy of rhythm control has benefited from ablation and novel anti-
arrhythmic drugs, therapeutic options in rate control have remained largely unchanged for
decades and expose patients to drugs frequently with limited efficacy and side effects or the need
for permanent ventricular pacing after AV node ablation.
Specifically, attempts at AV nodal modification, such as with selective RF, cryo-, or
other destructive ablation with the intent to avoid complete abolition of AV nodal conduction
and retain normal ventricular activation sequence, have proven unsuccessful because of an
unacceptable incidence of AV block. Although of limited clinical utility as a measure of efficacy,
when assessing safety the PR and AH intervals were very important to measure at the time of
injection because this study was designed specifically to give a very large number of injections
ons, indicating g thhatatatat dd
l in aattrtrt iaiiiallll fifififibrbrbb ilililillalall titititionononon
r
p
als continue to address the question of rate versus rhythm control in atrial fibrilla
studies indicate that in certain populations of patients, a rate control strategy may
Altho gh the strateg of rh thm control has benefited from ablation and no el a
realalalisisistititit ccc prprprp opoo ososossitiii ion requiring further invvveese ttigation.
pppppliliil cation
als cocooontntntininininue ttto ddadddrddd ess thhhheeee qqquestiiiionononon oooffff rattte verrrsusususus rhrhrhrhyttthmhmhmhm controllll iiinnn n atriii llal ffffibibbib iirillllllla
studididid es iiindddicii atatateee e thhthhat iiin certt iaiin popp pupp lall tions fofff pppattiei nts, aa rate coccc tntrollll stratetetetegygygygy mmmmayyy
AlAlthth hgh tthhe ttr tat fof hh thth tnt ll hha bbe fifittedd ffr blbl tatiio dd ll
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in and around the AVN in an attempt to cause AV block – failure to achieve which, coupled with
the early modest changes in AH and PR, is reassuring of a much wider margin of safety of
critical importance to this proof of concept compared with previous interventions to modify
AVN, including RF and Cryo. Autologous fibroblast injection that can safely be delivered
transvenously to slow ventricular rate in AF as an alternative to long term drug treatment, is a
compelling treatment strategy of potential high impact requiring further investigation.
Before progressing to clinical use in humans, further studies will be necessary to
determine the ideal cell concentration, volume and total number of cells or injections per patient
in order to achieve maximum efficacy, without compromising the safety of the procedure.
Limitations
Despite the small numbers of animals, the lasting and beneficial effects on AV conduction
indicate a robust treatment effect for the 4 weeks of follow up. Further studies will be required
to investigate more long-term effects in progressing towards clinical application and to better
clarify the specific mechanism involved in the atrioventricular conduction delay induced by
ADF. The atrial fibrillation in this study was acutely induced episodes, and may differ from more
naturally occurring AF.
The present study did not evaluate the effect of isoproterenol challenge acutely after
injections because the expected effects of this intervention on AVN conduction, as proved to be
correct, took days to evolve, and procedural testing would not therefore have been informative.
In fact, acutely the main question was that of safety (AV block) rather than efficacy, and to have
given isoproterenol may potentially have masked this. In follow up, we considered the
telemetered ventricular rate monitoring that was performed in this study to be the most
informative measure.
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Conclusions
Transplantation of ADFs in the AV node area in pigs is feasible, safe and slows ventricular rate
in acutely induced AF. Despite a large number of injections, complete AV block did not occur.
These results encourage further investigation of a novel strategy for controlling ventricular rate
in AF.
Acknowledgment: The authors acknowledge British Heart Foundation Centre of Research
Excellence and Programme Grant RG/10/11/28457, NIHR Biomedical Research Centre, and the
great contribution of our colleague Dr. Keith Robinson, sadly deceased.
Funding Sources: This study was supported by a research grants from CardioPolymers, Inc.
(formerly Symphony Medical), Inc., and the British Heart Foundation (RG/10/11/28457 and the
ElectroCardioMaths Programme of Imperial BHF Centre), but all the contents of the manuscript
are solely the responsibility of the authors. The authors acknowledge NIHR Biomedical
Research Centre (UK) funding.
Conflict of Interest Disclosures: None
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SL, Atsma DE, van der Laarse A, Pijnappels DA, van Tuyn J, Fibbe WE, de Vrim
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erimimimenee tal coooondduuctionoon blooockckck in nn rararar ttt ccac rdddiiiomymyoocytytyty e cuccuc ltuuureeese . JJJ AmAmmm CCCColl CaCC rdrdiol.943-11119595952222.
en TTTTA,AA ddde BaBaBakkkkkkk er JJJM,MM van ddder HHHeyyydeddd n MAMAMA. MMeM senchyhyhyh mmmam l ll stststtem cellllls repeppepaiaiaiair rrn block. J J AmAmAmAm CCCCololololl ll CaCaCardrdrdrdioioioi l.l.l. 2222000000006;66;6;48484848:2:2:219191919-2-22-220202020. by guest on M
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Table 1: Basic interval measurements at baseline, post injection and at follow-up, expressed in milliseconds
ADF treated Control
Baseline Post injection 28-day p value Baseline Post injection 28-day p value
PR 113 ± 14 163 ± 17 130 ± 13 0.044* 113 ± 14 146 124 ± 12 0.161*
AH 80 ± 7 113 ± 18 92 ± 13 0.021* 75 ± 9 100 ± 25 76 ± 8 0.832*
HV 34 ± 3 33 ± 2 33 ± 1 0.372** 31 ± 4 32 ± 2 32 ± 4 0.471**
* Statistical comparison between 28-day and baseline** ANOVA analysis comparing baseline, immediately post injection and at follow-up
Table 2: AH interval measurements, expressed in milliseconds at pacing cycle length of 400, 500 and 600 ms
ADF treated Control
Baseline 28-day p value Baseline 28-day p value
400 ms 91 ± 9 102 ± 13 <0.01 87 ± 14 88 ± 12 0.47
500 ms 84 ± 9 96 ± 15 0.02 85 ± 15 82 ± 9 0.83
600 ms 82 ± 8 93 ± 16 0.04 81 ± 15 78 ± 10 0.61
32323232 ±±±± 2222
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Figure Legends:
Figure 1: Three- dimensional mapping of right atrium with CARTO 4.0 mapping system. His
bundle (orange dot) and CS ostium (pink dot) was marked on the map. Injections were
performed along the slow and fast pathways around peri-nodal region (red dots).
Figure 2: RA septum visualization during necropsy in one animal. The dotted lines represent the
histologic sections – A1, A2 and A3 were submitted to formalin fixation for 24 hours before
embedding in paraffin; sections A2, B2 and C2 were frozen after use of fixation and sections A3,
B3 and C3 were frozen without fixative. F. Ovalis = fossa ovalis; C.S. ostium = coronary sinus
ostium.
Figure 3: Pig autologous skin fibroblasts in culture Panel A: Pig ADF culture at 2 days, 100X
magnification. Panel B: Pig ADF culture at 28 days, 100X magnification.
Figure 4: PR interval on real-time ECG during 28 days of observation period
Figure 5: Presence of second degree AV block during real-time ECG monitoring in one animal
at 2 days after ADF injections
Figure 6: Right atrium histology. A, Site of injection stained with H&E. Arrows show sites of
oonn fofofofor r r r 24242424 hhhhouououoursrsrsrs bbbbefefefeforororor
f fi iii ddd iiig in paraffin; sections A2, B2 and C2 were frozen after use of fixation and section
were frozen without fixative. F. Ovalis = fossa ovalis; C.S. ostium = coronary si
Pi l ki fib bl i l P l A Pi ADF l 2 d 10
g in paraffin; sections A2, B2 and C2 were frozen after use of fixation and section
wewewewere frozen wiwiwiw thhhouououout t t t fififiixaxaxaxatitititivevevee. F.F.F. OOOvavavaliliis == ffosssssa a a ovvovvalaa isisiss; C.CC S.S.S. oosttiuiuiuiumm m === cococorororonanananaryryryry si
i l ki fib bl i l l A i A l 2 d 10
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injection (4X); B, Section shown in panel A under 10X magnification; C, Site of injection
stained with Masson Verhoeff. Arrows show the clusters of cells and D, shows the same area as
panel C under 10X magnification.
Figure 7: Identification of ADF labeled with CM-DiI (A) and nuclei counterstaining with DAPI
(B) 28 days after injection. The white arrows represent the edge of a site of the exact same field.
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PetersFernando Tondato, Hong Zeng, Traci Goodchild, Fu Siong Ng, Nicolas Chronos and Nicholas S.
Rate in Atrial Fibrillation in SwineAutologous Dermal Fibroblast Injections Slow Atrioventricular Conduction and Ventricular
Print ISSN: 1941-3149. Online ISSN: 1941-3084 Copyright © 2015 American Heart Association, Inc. All rights reserved.
Dallas, TX 75231is published by the American Heart Association, 7272 Greenville Avenue,Circulation: Arrhythmia and Electrophysiology
published online January 31, 2015;Circ Arrhythm Electrophysiol.
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