autologous dermal fibroblast injections slow atrioventricular conduction...

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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DOI: 10.1161/CIRCEP.114.001536

2

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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DOI: 10.1161/CIRCEP.114.001536

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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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DOI: 10.1161/CIRCEP.114.001536

4

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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DOI: 10.1161/CIRCEP.114.001536

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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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7

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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8

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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10

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

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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

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en only in the ADF group - the prolongation of the AH interval, the Wenckebach

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rmal fibroblasts are mesench mal cells that are readil isolated and c lt red in th

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12

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

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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

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13

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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14

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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a MGGGG, , ArAAArnonoldldldld JJJJM,M,MM BBBBuuhnloseeeerrrr SHSHSHSH LaLaLaLambmbmbmbererert

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MMMMedededed. 2008;358:22667-7-7-267777777.

der ICICICIC, HaHaHaH gegens VVVVE,E,E BBBBoskekekekerrr HHHHA, KiKiKiKingngngma JJJHHH,H KKKKamamampppp O,OOO KKKKinii gma TT,T,T SSSSaid ddd SASASAA, a JI, TiTiTiT mmmmmmererermamamamansnsnsn AAAAJ,J,J, TTTTijijjjssssssseene JJJJG,G,G,G CCCCrriririjjnj sss HJHJHJ. A A AA cocococ mpmpmpmpararararisissisononon ooof f f f rarararatetete ccccononontrtrtrtrolololl aaandndndd rhpppatients withhh rrreeece urrent ppper isiistent atriiiial ffffibibib iirilllllllatioii n. NN N EnEnEnEnglglgl JJJ MMMMedddd. 22220000000 2;22 34343447:7:7:7 11181 34



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sts foror mmyoyocaca ddrdrdiaiallll

SL, Atsma DE, van der Laarse A, Pijnappels DA, van Tuyn J, Fibbe WE, de Vrim

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SSSSL,L,L,L Atsmaaaa DDDE,EE,E vananan dddereere LLaaaaaaaarsrrsr e ee A,AA PPPijijijnann pppeels DADADADA, vaaaannnn TuTuTuynynynn J, FiiiFibbbbbbbbeee WEWEEWE, , dededee VVrDDDDLLL,L van der WWaall EEEEEE, ScScScchaliiiijjj MJMMJ. Huummmann aadulululu t t bbbob nne mmmarrooww memm sennnchhchymymal sssteem

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

nd baselinemnd babababaseseseselilililinenenenemmemememediatellly yyy popopopostss iinjnjnjnjececece tionononon aaaandndnd at t t fofofof llowowow-up p p p



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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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20

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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