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ORIGINAL INVESTIGATIONS
Electrophysiological, Electroanatomical,and Structural Remodeling of the Atria asConsequences of Sustained Obesity
Rajiv Mahajan, MD, PHD,* Dennis H. Lau, MBBS, PHD,* Anthony G. Brooks, PHD,* Nicholas J. Shipp, PHD,*Jim Manavis, PHD,y John P.M. Wood, D PHIL,z John W. Finnie, BVSC, PHD,x Chrishan S. Samuel, PHD,kSimon G. Royce, PHD,k Darragh J. Twomey, MBBS,* Shivshanker Thanigaimani, PHD,*Jonathan M. Kalman, MBBS, PHD,{ Prashanthan Sanders, MBBS, PHD*
ABSTRACT
Fro
Un
Ad
Pa
Ca
is s
BACKGROUND Obesity and atrial fibrillation (AF) are public health issues with significant consequences.
OBJECTIVES This study sought to delineate the development of global electrophysiological and structural substrate
for AF in sustained obesity.
METHODS Ten sheep fed ad libitum calorie-dense diet to induce obesity over 36 weeks were maintained in this state for
another 36 weeks; 10 lean sheep with carefully controlled weight served as controls. All sheep underwent electro-
physiological and electroanatomic mapping; hemodynamic and imaging assessment (echocardiography and dual-energy
x-ray absorptiometry); and histology and molecular evaluation. Evaluation included atrial voltage, conduction velocity
(CV), and refractoriness (7 sites, 2 cycle lengths), vulnerability for AF, fatty infiltration, atrial fibrosis, and atrial trans-
forming growth factor (TGF)-b1 expression.
RESULTS Compared with age-matched controls, chronically obese sheep demonstrated greater total body fat
(p < 0.001); LA volume (p < 0.001); LA pressure (p < 0.001), and PA pressures (p < 0.001); reduced atrial CV
(LA p < 0.001) with increased conduction heterogeneity (p < 0.001); increased fractionated electrograms (p < 0.001);
decreased posterior LA voltage (p < 0.001) and increased voltage heterogeneity (p < 0.001); no change in the effective
refractory period (ERP) (p > 0.8) or ERP heterogeneity (p > 0.3). Obesity was associated with more episodes (p ¼ 0.02),
prolongation (p ¼ 0.01), and greater cumulative duration (p ¼ 0.02) of AF. Epicardial fat infiltrated the posterior LA
in the obese group (p < 0.001), consistent with reduced endocardial voltage in this region. Atrial fibrosis (p ¼ 0.03)
and TGF-b1 protein (p ¼ 0.002) were increased in the obese group.
CONCLUSIONS Sustained obesity results in global biatrial endocardial remodeling characterized by LA enlargement,
m the *Centre for Heart Rhythm Disorders (CHRD), South Australian Health and Medical Research Institute (SAHMRI),
iversity of Adelaide and Royal Adelaide Hospital, Adelaide, Australia; ySchool of Medical Sciences, University of Adelaide,
elaide, Australia; zRoyal Adelaide Hospital and Department of Ophthalmology, University of Adelaide, Adelaide, Australia; xSAthology, Adelaide, Australia; kDepartment of Pharmacology, Monash University, Melbourne, Australia; and the {Department of
rdiology, Royal Melbourne Hospital and Department of Medicine, University of Melbourne, Melbourne, Australia. Dr. Mahajan
upported by the Leo J. Mahar Lectureship from the University of Adelaide. Dr. Lau is supported by a postdoctoral fellowship
Mahajan et al. J A C C V O L . 6 6 , N O . 1 , 2 0 1 5
Obesity and the Substrate for AF J U L Y 7 , 2 0 1 5 : 1 – 1 1
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A trial fibrillation (AF) is an importanthealth problem, with 2010 globalestimates suggesting that it affects
33.5 million individuals (1). This prevalenceis projected to increase 2.5-fold by 2050 (2).Emerging evidence suggests that aging alonedoes not account for the exponential rise inAF prevalence (2). It is in this setting thatnew risk factors, such as obesity, havebeen proposed as important contributors tothis epidemic (3). Obesity is a rampantepidemic, with more than one-third of thepopulation being overweight or obese. Anal-ysis of population-based studies suggeststhat obesity is associated with long-termincreased risk of AF, independent of otherrisk factors (4–6). In a meta-analysis byWanahita et al. (7), there was a graded dose-response relationship between obesity andAF in the general population.
SEE PAGE 12
The mechanisms by which obesity predisposes toAF are confounded by the coexistence of obstructivesleep apnea (OSA), hypertension, diabetes, and cor-onary artery disease, all well-established precursorsfor the development of AF. Using limited, open-chest,direct contact mapping, we have previously shownconduction slowing and atrial fibrosis with short-termweight gain in an ovine model (8). In the presentstudy, we investigate the global endocardial elec-trophysiological, electroanatomic, and structuralsubstrate with sustained obesity, a state more com-parable with humans.
METHODS
The animal research ethics committees of the Uni-versity of Adelaide and the South Australian Healthand Medical Research Institute, Adelaide, Australia,
ational Health and Medical Research Council of Australia (NHM
eart Foundation of Australia. Dr. Samuel is supported by an N
by the Leo J. Mahar Electrophysiology Scholarship from the Univ
etzel Electrophysiology Scholarship from the University of Ad
from the NHMRC; and has received research funding from St. Jud
r. Sanders is supported by a Practitioner Fellowship from the NH
edtronic, St. Jude Medical, Sanofi, and Merck, Sharpe and Dohm
ebster, Medtronic, St. Jude Medical, Boston Scientific, Merck,
search funding from Medtronic, St. Jude Medical, Boston Scie
at they have no relationships relevant to the contents of this pa
essions of the Heart Rhythm Society, May 2012, Boston, Massachu
). Drs. Lau and Sanders contributed equally to this paper.
his manuscript’s audio summary by JACC Editor-in-Chief Dr. Va
received February 3, 2015; revised manuscript received March 2
which adhere to the National Health and MedicalResearch Council of Australia Guidelines for the Careand Use of Animals for Research Purposes, approvedthe study.OBESE OVINE MODEL. A total of 10 sheep had obesityinduced through a previously described protocolusing an ad libitum regimen of hay and high-energypellets (9). At baseline, healthy sheep were com-menced on a high-calorie diet of energy-dense soy-bean oil (2.2%) and molasses–fortified grain andmaintenance hay with weekly weight measurement.Excess voluntary intake was predominantly of grassalfalfa silage and hay. For the obese sheep, pelletswere gradually introduced at 8% excess basalenergy requirements, and rationed to $70% oftotal dry-matter intake. Blood samples were periodi-cally collected to ensure electrolyte and acid-basehomeostasis. The sheep gradually gained weight,reaching maximal obesity at 36 weeks and weresubsequently maintained in this state for a further36 weeks.
CONTROL GROUP. Ten age-matched sheep weremaintained as controls at their baseline weight. To dothis, high-quality hay was provided ad libitum, whileenergy-dense pellets were rationed at 0.75% of bodyweight. The nutritional content of food and housingconditions were identical for both groups, with onlythe amount of food intake varying.
STUDY PREPARATION. Animals were pre-acclimatizedfor at least 1 week before any surgery. Shorn weightwas recorded immediately before surgery.
BODY COMPOSITION. Dual-energy x-ray absorpti-ometry scans were performed to determine total bodyfat in the animals.
TRANSTHORACIC ECHOCARDIOGRAPHY. An echo-cardiogram (Acuson Aspen, Siemens Healthcare,Malvern, Pennsylvania) was performed under generalanesthesia before the electrophysiology study. Theleft atrial (LA) dimensions were measured in the
RC). Drs. Brooks and Sanders are supported by the
HMRC Senior Research Fellowship. Dr. Twomey is
ersity of Adelaide. Dr. Thanigaimani is supported by
elaide. Dr. Kalman is supported by a Practitioner
e Medical, Biosense Webster, Medtronic, and Boston
MRC; has served on the advisory boards of Biosense
e; has received lecture and/or consulting fees from
Sharpe and Dohme, Biotronik, and Sanofi; and has
ntific, Biotronik, and Sorin. All other authors have
per to disclose. Previously presented at the Annual
setts, and published in abstract form (Heart Rhythm
TABLE 1 Structural and Hemodynamic Characteristics of the Control and
Obese Groups
Controls Obese p Value
Weight, kg 60 � 7 110 � 9 <0.001
Total body fat, kg 9 � 6 35 � 6 <0.001
Total body fat/total soft tissue (DEXA) 15.2 � 7.8 36.9 � 4.3 <0.001
LA major axis, mm 36.2 � 1.3 38.3 � 1.9 0.01
LA minor axis, mm 27.7 � 1.1 30.1 � 1.4 <0.001
LV posterior wall, mm 7.1 � 0.6 8.8 � 1.2 0.001
LVEF, % 67 � 5 70 � 6 0.8
LA pressure, mm Hg 3.7 � 1.4 8.1 � 1.6 <0.001
RA pressure, mm Hg 1.8 � 1.1 4.6 � 1.2 <0.001
PA pressure, mm Hg 9.8 � 2.6 15.0 � 0.9 <0.001
Systemic mean BP, mm Hg 71 � 12 86 � 13 0.02
Values are mean � SD.
BP ¼ blood pressure; DEXA ¼ dual-energy x-ray absorptiometry; LA ¼ left atrial; LV ¼ leftventricle/ventricular; LVEF ¼ left ventricular ejection fraction; PA ¼ pulmonary artery; RA ¼ rightatrium.
J A C C V O L . 6 6 , N O . 1 , 2 0 1 5 Mahajan et al.J U L Y 7 , 2 0 1 5 : 1 – 1 1 Obesity and the Substrate for AF
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apical 4-chamber view. The left ventricular (LV)dimensions were measured in M-mode in parasternallong-axis view at the level of mitral leaflet tips. TheLV dimensions were utilized for determination ofglobal LV function by the Teicholz formula.
HEMODYNAMIC ASSESSMENT. Invasive blood pres-sure (BP) monitoring was performed during theelectrophysiology study. LA, right atrial (RA), andpulmonary artery (PA) pressures were recorded.
ELECTROPHYSIOLOGICAL STUDY. Details of theelectrophysiological study are presented in theOnline Appendix and are based on previously pub-lished methodology (10). Briefly, venous access wasobtained through the right femoral and left internaljugular veins. A 10-pole catheter was advanced to thecoronary sinus (CS). A conventional trans-septalpuncture was performed using a BRK1 needle andSL0 sheath to access the left atrium. A 3.5-mm tipcatheter (Navistar, Biosense Webster, Diamond Bar,California) was used to create electroanatomic mapsof the RA and LA in sinus rhythm with the CARTO XPmapping system (Biosense Webster). The followingwere determined:
Effect ive refractory per iod . The effective re-fractory period (ERP) was measured from thefollowing 7 sites: 1) RA appendage; 2) RA lateral wall,upper; 3) RA free wall, lower; 4) proximal CS; 5) distalCS; 6) LA appendage (LAA); and 7) LA posterior wall.ERP heterogeneity was determined by the coefficientof variation (CoV) of ERP variation at each cyclelength (CL) (CoV ¼ SD/mean � 100%).
AF vulnerab i l i ty and durat ion . AF vulnerabilitywas assessed during ERP testing. AF was defined asrapid, irregular atrial activity lasting $2 s. AF lastingmore than 10 min was considered sustained; whenthis occurred, no further data were acquired.
Elect roanatomic mapping . Electroanatomic mapsof the LA/RA were created in sinus rhythm using theCARTO (Biosense Webster) mapping system. Detailsof the electroanatomic mapping and analysis arepresented in the Online Appendix and are based onpreviously published methodology (10). Each pointwas binned according to location (region), fraction-ation (presence or absence), scar (presence orabsence), and bipolar voltage amplitude to allowanalysis in a mixed-effects model. Regional atrialbipolar voltage and conduction velocity wereanalyzed off-line. The LA/RA maps were segmentedfor analysis, and the following parameters wereassessed as previously described (11):
1. Atrial conduction velocity: Conduction velocity foreach segment was determined by averaging the
conduction velocity between 3 to 5 pairs of points.An index of heterogeneity was determined bycalculating the CoV of the different regions in eachchamber.
2. Electrograms with a duration $50 ms and 3 or moredeflections crossing baseline were consideredcomplex fractionated electrograms; and doublepotentials were potentials separated by an isoelec-tric interval and with a total electrogram dura-tion $50 ms. For analysis, a fraction of the totalnumber of fractionated/double points was utilized.
3. Atrial voltage: Low-voltage areas were defined as3 contiguous points with a bipolar voltage <0.5mV. Electrically silent areas (scar) were defined as3 contiguous points with an absence of recordableactivity or bipolar voltage amplitude <0.05 mV.An index of heterogeneity was determined bycalculating the CoV of the different regions in eachchamber.
HISTOLOGICAL ASSESSMENT. Isolated atrial tissuesfrom the LA posterior wall and LAA were perfusion-fixed with 4% paraformaldehyde and immersed in10% buffered formalin. Sections were processed andembedded in paraffin. For each site, 5-mm serial sec-tions were then taken at 1-cm intervals from eachblock and stained with hematoxylin and eosin (HE)and Masson’s trichrome, respectively. Additionalsamples were frozen at �70�C.Fatty infi l t rat ion of atr ia l musc le by epicard ia lfat . Fat infiltration of the atrial muscle from theepicardial fat was evaluated in low-power (1.25�)magnification with hematoxylin and eosin stainingand confirmed with Oil Red O staining of frozen sec-tions. Fatty infiltration of the atrium by the overlying
TABLE 2 Electrophysiological and Structural Characteristics of the
ERP ¼ effective refractory period; LAA ¼ left atrial appendage; LAPW ¼ left atrial
posterior wall; RAA ¼ right atrial appendage; RAFW_U ¼ upper right atrial free wall;
RAFW_L ¼ lower right atrial free wall.
Mahajan et al. J A C C V O L . 6 6 , N O . 1 , 2 0 1 5
Obesity and the Substrate for AF J U L Y 7 , 2 0 1 5 : 1 – 1 1
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epicardial adipose tissue was graded 1 to 3 on thebasis of severity as follows:
1. None or focal infiltration of the adjacent outer thirdof the atrial muscle layer by the overlying epicar-dial adipose tissue.
2. Coalescent infiltration of the outer third and/orfocal infiltration up to the middle third of the atrialmuscle layer.
3. Coalescent infiltration extending from the epicar-dial adipose tissue to the middle or inner third ofthe atrial muscle layer.
F ibros i s assessment . Morphometric analysis ofMasson’s trichrome–stained sections to obtain aquantitative estimate of collagen within the tissue(described in the Online Appendix).Atr ia l TGF-b1 assessment . Western blotting wasused to assess changes in transforming growth factor(TGF)-b1 tissue expression in LA myocardium(described in the Online Appendix).
STATISTICAL ANALYSIS. Normally distributed con-tinuous data were expressed as mean � SD and testedwith unpaired Student t tests between groups.Skewed distributions were expressed as median andinterquartile range (IQR) and means tested usingMann-Whitney U tests. Mixed-effect models werefitted to the data in order to compare voltage, con-duction velocity, fractionation, and atrial refractoryperiod across regions, chambers, and groups (obeseand control). Fixed effects included combinations ofgroup (obese, control), region, and chamber (LA/RA)with a maximum of 2 fixed effects entered into thestatistical model at a time. Main effects and theirinteraction were tested. Random effects of sheepidentity were fitted to all models to account fordependence on observations from the same animal.To investigate LA regional patterns in both ap-proaches, region (posterior LA, anterior LA, septal LA,inferior LA, lateral LA, and LA roof) and group (obeseand control) were modeled as fixed effects with aninteraction term (region $ group). If a significantinteraction was present, mixed-effects post-hoc testp values were reported (with Sidak adjustment ofalpha level). In the case of skewed distribution (i.e.,for fibrosis assessment with Masson’s trichromestaining), data were log-transformed before furtheranalysis. Two-sided p values <0.05 were consideredstatistically significant. All analyses were performedusing SPSS/PASW version18 (SPSS, Chicago, Illinois).
RESULTS
GROUP CHARACTERISTICS. The obese sheep achievedpeak weight over a period of 36 weeks and remained
J A C C V O L . 6 6 , N O . 1 , 2 0 1 5 Mahajan et al.J U L Y 7 , 2 0 1 5 : 1 – 1 1 Obesity and the Substrate for AF
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in this state of sustained obesity for another36 weeks. The control sheep were maintained leanduring this period. The obese sheep were twice theweight of the control animals and had significantlygreater total body fat. Table 1 presents the charac-teristics of the 2 groups.
STRUCTURAL AND HEMODYNAMIC REMODELING. Table 1details the hemodynamic characteristics of the 2groups. The LA was enlarged (p ¼ 0.01) with increasesin LA pressure (p < 0.001) without a change in LV
FIGURE 2 Atrial Conduction Abnormalities With Chronic Obesity
Control
Control
Representative left atrial isochronal mapsA
Representative right atrial isochronal mapsB
(A and B) Representative LA and RA isochronal maps (5 ms), respective
the obese sheep are more crowded and greater time was required to ac
and RA, respectively. Conduction velocity was uniformly reduced across a
posterior; HI_SEP ¼ high septal; INF_LA ¼ inferior left atrium; LA ¼ left
ejection fraction (p ¼ 0.8) in the obese sheep, ascompared with the controls. In addition, there wasa significant increase in RA and PA pressures(p < 0.001) with obesity. Systemic BP was alsoelevated in the obese animals, as compared with thecontrols (p ¼ 0.02).
ELECTROPHYSIOLOGICAL AND ELECTROANATOMIC
REMODELING. Electroanatomic maps of the atriawere created in sinus rhythm. Table 2 summarizes theelectrophysiological findings. LA and RA volumes
2.00 P (group) <0.001
P (group) <0.001
Regional conduction velocity (mean)distribution in the left atriumObese
Obese
: LAO view C
: LAO view D Regional conduction velocity (mean)distribution in the right atrium
1.50
1.00
Mea
n Ve
loci
ty (m
/s)
Mea
n Ve
loci
ty (m
/s)
.50
.00
2.00
1.50
1.00
.50
.00
ANT_LA
ANTHI_L
AT
HI_POS
HI_SEP
LOW_LAT
LOW_POS
LOW_SEP
INF_LA
LAT_LA
POS_LA
ROO_LA
SEP_LA
Controls Obese
ly, demonstrating conduction in obese and control groups in sinus rhythm. The isochrones in
tivate the atria. (C and D) Regional distributions of conduction velocity (mean) of the LA
ll regions. ANT ¼ anterior; ANT_LA ¼ anterior left atrium; HI_LAT ¼ high lateral; HI_POS ¼ high
atrium; LAO ¼ left anterior oblique; LAT_LA ¼ lateral left atrium; LOW_LAT ¼ low lateral;
left atrium; RA ¼ right atrium; ROO_LA ¼ left atrial roof; SEP_LA ¼ septal left atrium.
FIGURE 3 Atrial V
A
B
(A and B) Represent
with the lower limit
in the obese animal.
voltage in the poste
Mahajan et al. J A C C V O L . 6 6 , N O . 1 , 2 0 1 5
Obesity and the Substrate for AF J U L Y 7 , 2 0 1 5 : 1 – 1 1
6
were increased in the obese sheep, as compared withthe controls (p < 0.001).
ATRIAL REFRACTORINESS. Atrial ERPs, at CLs of450 and 300 ms, from the 7 sites did not differsignificantly between the 2 groups (CL 300 ms,p ¼ 0.8; CL 450 ms, p ¼ 1.0) (Table 2, Figure 1). ERPheterogeneity at CLs of 450 and 300 ms also did not
oltage Abnormalities With Chronic Obesity
Control
Representative left atrial voltage maps: LAO view
Representative right atrial voltage maps: LAO view
Obese
Control Obese
ative LA and RA voltage (bipolar) maps, respectively, of obese and control g
set at 0.5 mV and the upper limit at 5.0 mV. Note the heterogeneity in the
There was absence of electrical scar. (C and D) Regional distribution of volt
rior LA was reduced (*p < 0.001) in the obese group. Abbreviations as in Fig
significantly differ between the obese and controlgroups (CL 300 ms, p ¼ 0.7; CL 450 ms, p ¼ 0.3).
CONDUCTION VELOCITY. The mean conductionvelocity was significantly reduced in the obese groupin both atrial chambers (LA 1.18 � 0.04 m/s and RA1.02 � 0.04 m/s), as compared with the controls (LA1.58 � 0.04 m/s and RA 1.43 � 0.04 m/s; p < 0.001)
D
C Regional mean voltage distributionin the left atrium
Regional mean voltage distributionin the right atrium
10.00 P (group) = 0.88
P (group) = 0.21
P (Interaction) <0.001
*
8.00
6.00
4.00
2.00
Mea
n Vo
ltage
(mV)
.00
ANT_LA
ANTHI_L
AT
HI_POS
HI_SEP
LOW_LAT
LOW_POS
LOW_SEP
INF_LA
LAT_LA
POS_LA
ROO_LA
SEP_LA
8.00
6.00
4.00
2.00
Mea
n Vo
ltage
(mV)
.00
Controls Obese
roups in sinus rhythm. The voltage range was selected manually
voltage and increased fractionated/double potentials (colored dots)
age (mean) of the LA and RA, respectively, in the 2 groups. The
ure 2.
FIGURE 4 Epicardial Fat: Novel Substrate for AF in
Chronic Obesity
PA view
ENDO EPI EPIENDO
Controls Obese
Gross specimenheart
Posterior leftatrial wall
Left atrialappendage
AP view
(Top) Gross specimen of sheep heart after removal of parietal
pericardium with pericardial fat. PA view: arrow points to
epicardial fat adjacent to the LAPW. AP view: arrow points to the
LAA, which has very little epicardial fat. (Middle and Bottom)
Representative H&E stained sections (1.25�) of the LAPW and
LAA, respectively, from control and obese groups. Arrow shows
infiltration of atrial musculature with fat cells. AF ¼ atrial
fibrillation; AP ¼ anterior–posterior; ENDO ¼ endocardial;
EPI ¼ epicardial; H&E ¼ hematoxylin and eosin; PA ¼ posterior–
anterior; other abbreviations as in Figure 1.
J A C C V O L . 6 6 , N O . 1 , 2 0 1 5 Mahajan et al.J U L Y 7 , 2 0 1 5 : 1 – 1 1 Obesity and the Substrate for AF
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(Figure 2). The reduction in conduction velocity wasconsistent across different LA segments (interactivep value [area � group] ¼ 0.6). In addition, conductionwas more heterogeneous in the obese as comparedwith the controls (CoV: 22.0 � 6.1% vs. 9.1 � 4.9%;p < 0.001) (Figure 1). After adjusting for changes in LApressure, the differences in conduction velocity(p < 0.001) and heterogeneity (p < 0.001) persisted inobese animals compared with controls.COMPLEX FRACTIONATION. The LA demonstratedgreater fractionation/double potentials, as comparedwith the RA in both the obese and control groups(p < 0.001). During sinus rhythm, 53.2 � 13.6% and36.0 � 12.3% of LA and RA signals, respectively, werefractionated/double potentials in the obese group. Bycontrast, only 10.8 � 4.4% and 8.2 � 2.8% of all LAand RA signals, respectively, were fractionated/double potentials in the control group (p < 0.001).
VOLTAGE. The mean global voltage did not differbetween obese (LA 4.5 � 1.7 mV; RA 4.1 � 1.6 mV) andcontrol groups (LA 4.4� 1.4 mV, p¼ 0.88; RA 3.6� 0.9,p¼0.21) (Figure 3). Therewere no areas of scar in eitherchamber in control or obese animals. However, the LAregional voltage patterns were different (interactionp < 0.001) in the obese and control groups, primarilydue to a significant reduction in the posterior LAvoltage (3.7 � 2.3 mV vs. 5.5 � 2.3 mV; p < 0.001) in theobese. Regional voltage heterogeneity was elevated inthe obese group, as compared with the controls (CoV:32.1 � 8.8% vs. 24.6 � 7.5%; p < 0.001).
VULNERABILITY FOR AF. The median number of AFepisodes was greater in the obese group, as comparedwith controls (4.5 [IQR: 2 to 7] vs. 1 [IQR: 0 to 2];p¼0.02). The total AF episode duration per animal wasincreased in the obese group as compared with thecontrols (46 [IQR: 10 to 112] s vs. 4.1 [IQR: 3 to 15] s;p¼0.05). Similarly, the average AF episode length waslonger in the obese as compared with control animals(7.9 [IQR: 5 to 17.7] s vs. 2.7 [IQR: 2 to 5] s; p ¼ 0.01).ATRIAL MUSCULATURE INFILTRATION BY EPICAR-
DIAL ADIPOSE TISSUE. Dist r ibut ion of ep icard ia lad ipose t i ssue . In relation to the atria, epicardialadipose tissue was distributed adjacent to the LAposterior wall and atrioventricular groove (Figure 4),with minimal epicardial adipose tissue adherent tothe appendage. By contrast, paracardial adipose tis-sue was more diffuse in distribution, with prominentdeposits between the appendage and great arteries.Fatty infi l t ra t ion by epicard ia l fat . In controlsheep, limited numbers of adipocytes were presentsubepicardially, interposed between cardiac myo-cytes, whereas in obese animals, adipocyte hyper-plasia resulted in abundant deposition of adipose
tissue in the epicardium and infiltration into the atrialmusculature. Moderate (Grade II) to severe (Grade III)LA posterior wall infiltration by overlying epicardialfat was seen significantly more in the obese sheep(mean grade: obese 2.5 � 0.7, controls 1.0 � 0.0;p < 0.001). There was minimal fatty infiltration in theLAA in both groups (p ¼ 0.18; mean grade: 1.2 � 0.4and 1 � 0 for obese and control groups, respectively).Grade III fatty infiltration was not seen in the LAA ineither group, where epicardial fat was minimal.Figure 4 depicts representative HE-stained sections(1.25�), demonstrating fatty infiltration of the atrialmusculature by epicardial adipose tissue in the obeseand control groups.
FIBROSIS. There was increased interstitial fibrosisin the obese animals, as compared with the con-trol animals (p ¼ 0.03; on log-transformed data).
FIGURE 5 Fibrosis and Profibrotic TGFb1 Expression With Chronic Obesity
ControlA
B
Obese
Control Obese
Control
Atria
l Fib
rosis
(%)
Atria
l TGF
-ββ1 P
rote
in
.0
4.00
3.00
2.00
1.00
.00
2.0
4.0
6.0
8.0
10.0
12.0
Obese
P=0.03
P=0.002
7 8 9 10
Control
Actin
TGFβ1
1 2 3 4 5 6
42kD
25kD
1 2 3 4 5 6 7 8 9 10
Obese
(A) Representative Masson’s trichrome-stained sections (5�) of the left atrial (LA) posterior wall. The bar graph demonstrates the degree of atrial fibrosis in the
control and obese groups. The fibrous tissue is stained blue and assessed morphometrically. (B) Western blots for TGF-b1 expression in LA tissue. The bar graph
demonstrates atrial TGF-b1 protein expression in control and obese groups. TGF ¼ transforming growth factor.
Mahajan et al. J A C C V O L . 6 6 , N O . 1 , 2 0 1 5
Obesity and the Substrate for AF J U L Y 7 , 2 0 1 5 : 1 – 1 1
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Morphometric analysis of the Masson’s trichrome–stained LA sections demonstrated 8.14 � 2.39%, and5.31 � 0.95% staining in the obese and control groups,respectively. Figure 5A shows representative Masson’strichrome–stained sections from the 2 groups.
ATRIAL TGF-b1 PROTEIN. Atrial TGF-b1 proteinexpression increased 5-fold in the obese group, ascompared with the control group (p ¼ 0.001 on log-transformed data) (Figure 5B, Table 2).
DISCUSSION
MAJOR FINDINGS. This study presents new informa-tion on the global endocardial electrophysiological,electroanatomic remodeling, and fatty infiltration ofthe atria as a result of sustained obesity. Animalsgained weight over 36 weeks to achieve stableobesity and maintained this for another 36 weeks toreplicate a state more comparable with chronic obesityin humans. The obese group accumulated 5-foldgreater total body fat (34.5 kg), as compared withcontrols (8.7 kg). The obese sheep model is unique,because it does not experience OSA, because ofthe typical habitus and sleeping posture, thereby
excluding the confounding effects of OSA. It demon-strates the following.
Structural and hemodynamic changes� Biatrial enlargement with diastolic dysfunction,
reflected by elevated LA pressure in the presenceof normal ventricular function
� Elevated right heart pressures and systemic BP� Increased expression of profibrotic TGF-b1 and
increased interstitial fibrosis� Fat infiltration of the atrial myocardium
Electrophysiological remodeling� Slowed and heterogeneous conduction� Increased complex fractionated electrograms� Increased voltage heterogeneity without signifi-
cant change in mean voltage or appearance of scar/low voltage
� No change in ERP and ERP heterogeneity
As a result of these hemodynamic, structural, andelectrophysiological changes, obese animals weremore vulnerable to AF. Importantly, this study pro-vides causative evidence that links obesity directlywith the development of the AF substrate.
CENTRAL ILLUSTRATION Obesity and the Substrate for AF
Mahajan, R. et al. J Am Coll Cardiol. 2015; 66(1):1–11.
Progressive weight gain has been demonstrated to result in atrial stretch and leads to the development of high-frequency triggers and the substrate for AF. With chronic
obesity, there is greater epicardial adipose tissue, activation of the cytokines, and the development of fibrosis. In addition, there is infiltration of the contiguous
atrial myocardium by fat cells. All of these result in the milieu of slowed and inhomogeneous conduction that forms the substrate for AF. AF ¼ atrial fibrillation;
LA ¼ left atrial; TGF ¼ transforming growth factor.
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ATRIAL SUBSTRATE PRE-DISPOSING TO AF. Overthe last decade, several studies have presented eval-uations of the atrial substrate in conditions known toresult in AF. Li et al. (12) were the first to distinguishthese abnormalities forming the “second factor” fromthe electrical remodeling associated with AF. Theyhighlighted the importance of conduction abnormal-ities and structural changes, particularly diffuse atrialfibrosis in an experimental heart failure model (12).These findings were subsequently confirmed to be theunifying feature of structural remodeling in otherconditions, in both pre-clinical studies (13–15) andclinical studies (10,11,16,17).
AF SUBSTRATE IN OBESITY. Although an epidemio-logical link has been established between obesity andAF, the underlying electrophysiological changes andmechanism still remain to be defined (4–6). OSA isclosely associated with obesity in humans and pre-disposes to AF by causing hypertension, diastolicdysfunction, LA stretch, and autonomic imbalanceduring sleep. Iwasaki et al. (18) demonstrated thatobesity facilitates AF inducibility in the presence ofacute OSA. However, despite the structural remodel-ing, AF inducibility was not enhanced in obese rats inthe absence of obstruction. The ovine model allowsevaluation of obesity in the absence of OSA and, in this
study, demonstrated diffuse conduction abnormal-ities and interstitialfibrosiswith chronic obesity alone.The Central Illustration summarizes the structuralchanges that result in electrical remodeling and pro-mote AF in obesity.
Global endocardial biatrial conduction slowing andincreased fractionation were demonstrated in chron-ically obese animals. However, the degree of slowingvaried in different regions, resulting in increasedconduction heterogeneity. This is consistent with thefinding with limited epicardial mapping with short-term weight gain in an ovine model (8). Mungeret al. (19) have also reported slowed longitudinalconduction velocities from the LA to the pulmonaryveins in obese patients with AF. However, this humanstudy did not observe any change in conduction ve-locity along the coronary sinus. The more pronouncedfindings in our animal model may result from extremeobesity and more detailed mapping. The obeseanimals did not demonstrate electrical scars or alter-ations in global voltage; however, there was reduc-tion in posterior LA voltage with increased voltageheterogeneity. Contiguous epicardial fat wasobserved to infiltrate the region demonstrating thevoltage reduction. Thus, we hypothesize that fattyinfiltration of the posterior LA by epicardial fat couldpotentially represent a unique substrate that could
Mahajan et al. J A C C V O L . 6 6 , N O . 1 , 2 0 1 5
Obesity and the Substrate for AF J U L Y 7 , 2 0 1 5 : 1 – 1 1
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predispose to AF in obesity. As with other studiesevaluating clinical substrate for AF, endocardial atrialrefractoriness was not altered with sustained obesity.This finding differs from that of Munger et al.,who reported shortened atrial refractoriness in obesepatients undergoing ablation for AF. However,they acknowledged that AF induced during ERPtesting could potentially affect atrial refractoriness.In addition, a marked increase in complex frac-tionated signals was observed with sustained obesityin our study. This could be a result of conductionslowing secondary to interstitial fibrosis or fatinfiltration.
Sustained obesity was associated with diffuseatrial interstitial fibrosis. This is consistent withchanges observed with heart failure (12,20) andchronic hypertension (14). Spach et al. (21) havedemonstrated elegantly that fibrosis can produceconduction abnormalities promoting re-entry and AF.The fibrosis observed in their study was only inter-stitial in nature, without the areas of replacementfibrosis usually seen with infarction. Moreover, therewas only a 50% increase in interstitial fibrous tissue,in comparison to the 16-fold increase observed withheart failure (12), suggesting a more subtle insultwith obesity.
TGF-b1 has been shown to be a crucial cytokine inthe signal transduction pathways responsible forfibrosis. It occupies a central position, downstream ofangiotensin and upstream of endothelin pathways,and acts in a paracrine–autocrine fashion. Verheuleet al. (22) have shown that overexpression of consti-tutional TGF-b1 in transgenic mice led to selectiveatrial fibrosis, conduction heterogeneity, and AF. Inour study, TGF-b1 expression was increased 5-foldwith sustained obesity and could explain the increasein interstitial fibrosis. We have previously reportedincreased endothelin receptor expression with short-term weight gain (8). There are similar reports ofTGF-b superfamily (23) and endothelin (24) signalingpathway overexpression in humans.
EPICARDIAL FAT AND AF. There is emerging evi-dence that localized epicardial fat depots may havea significant and independent role in developmentof AF (25–28). The development of the obese statehas been shown to be associated with hypoxia ofthe expanding adipose tissue, resulting in adiposetissue fibrosis and production of a myriad of adi-pocytokines, including those in the TGF-b super-family (29). The absence of fascial barriers betweenepicardial fat and the contiguous atrial musculature,and the common vascular supply may facilitateparacrine action. Venteclef et al. (30) elegantly
demonstrated paracrine action in an organ-culturemodel. They incubated rat atrial tissue in a secre-tome derived from human epicardial fat anddemonstrated atrial fibrosis mediated by membersof the TGF-b superfamily. We demonstrated several-fold increased expression of TGF-b1 in atrial tissue;however, the source was not evaluated. In addition,a new finding was observed with epicardial fatinfiltrating the underlying myocardium. Epicardialfat is predominantly deposited on the posterior LA.The reduction in posterior LA voltage noted onendocardial mapping was consistent with thisfinding. We hypothesize that fat infiltration sepa-rates myocytes and could result in conduction ab-normalities in a fashion similar to microfibrosis (21).Considering the infiltration was observed onlyadjacent to epicardial fat deposits, the distributionof epicardial fat could contribute to conductionheterogeneity.
STUDY LIMITATIONS. Although the observed elec-trical and structural abnormalities predispose to AF,the development of clinical AF is a complex process,with other factors, such as triggers and perpetuators,not addressed in the current study. This studyhas shown that epicardial fat cells infiltrate theposterior LA. However, a causal relationship be-tween fatty infiltration and AF vulnerability couldonly be studied to a limited extent in this model.Furthermore, the profibrotic signal transducingpathways responsible for AF in obesity were notfully elucidated.
CONCLUSIONS
Sustained chronic obesity results in chronic stretch,diffuse interstitial fibrosis, conduction abnormalities,and increased vulnerability to AF. A TGF-b signalingpathway may play an important role in mediatinginterstitial fibrosis in sustained obesity. Infiltration ofthe underlying atrial musculature by epicardial fatmay be a unique feature of the AF substrate inobesity. This study provides direct evidence for therole of obesity in development of a unique substratepredisposing to AF.
ACKNOWLEDGMENT The authors acknowledge thecontribution of Mr. Krupesh Patel for the morpho-metric analysis of inflammation and fibrosis.
REPRINT REQUESTS AND CORRESPONDENCE: Dr.Prashanthan Sanders, Centre for Heart Rhythm Disorders(CHRD), Department of Cardiology, Royal AdelaideHospital, Adelaide, SA 5000, Australia. E-mail: [email protected].
Sustained obesity, in the absence of sleep apnea, is
associated with diastolic ventricular dysfunction, eleva-
tion of atrial profibrotic TGF-b1, infiltration of atrial
musculature by contiguous epicardial fat, and atrial
fibrosis. These factors contribute to electrophysiological
remodeling and AF.
TRANSLATIONAL OUTLOOK: Further studies are
needed to determine whether therapies that inhibit fat
cell infiltration from the epicardial depot into the left
atrial wall of obese individuals could reduce their risk of
developing AF.
J A C C V O L . 6 6 , N O . 1 , 2 0 1 5 Mahajan et al.J U L Y 7 , 2 0 1 5 : 1 – 1 1 Obesity and the Substrate for AF
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KEY WORDS atrial fibrillation,epicardial fat, fibrosis, obesity, TGF-b1
APPENDIX For a supplemental Methodssection, please see the online version of thisarticle.
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