Persistent lone atrial fibrillation: Clinicopathologic studyof 19 casesDomenico Corradi, MD,* Sergio Callegari, MD,† Laura Manotti, MD,*

David Ferrara, MD,‡ Matteo Goldoni, PhD,§ Rossella Alinovi, BsSc,§ Silvana Pinelli, BsSc,§

Paola Mozzoni, BsSc, PhD,§║ Roberta Andreoli, PhD,§║ Angeliki Asimaki, PhD,¶

Alberto Pozzoli, MD,‡ Gabriella Becchi, PhD,* Antonio Mutti, MD,§

Stefano Benussi, MD, PhD,‡ Jeffrey E. Saffitz, MD, PhD,¶ Ottavio Alfieri, MD‡

From the *Department of Biomedical, Biotechnological, and Translational Sciences (S.Bi.Bi.T.), Unit ofPathology, University of Parma, Parma, Italy, †Division of Cardiology, Vaio Hospital, Fidenza, Italy,‡Cardiothoracic Surgery Unit, Department of Cardiology, San Raffaele University Hospital, Milan, Italy,§Department of Clinical and Experimental Medicine, Laboratory of Industrial Toxicology, University ofParma, Parma, Italy, ║Italian Workers’ Compensation Authority (INAIL) Research Center at the University ofParma, Parma, Italy, and ¶Department of Pathology, Beth Israel Deaconess Medical Center and HarvardMedical School, Boston, Massachusetts.

BACKGROUND The extent to which atrial myocardium is remod-eled in patients with persistent lone atrial fibrillation (LAF) islargely unknown.

OBJECTIVE The purpose of this study was to perform a clinicopa-thologic investigation in patients with persistent LAF.

METHODS We characterized structural and molecular remodeling inatrial biopsies from 19 patients (17 males, mean age 49 years) withpersistent (47 days; n ¼ 8) or long-lasting persistent (41 year;n ¼ 11) LAF who underwent surgical ablation. Atrial tissue from15 autopsy samples without clinicopathologic evidence of heartdisease served as controls.

RESULTS Morphometric analysis showed cardiomyocyte hypertro-phy and greater amounts of myolytic damage and interstitialfibrosis in persistent LAF patients compared to controls(P o.0001). Atrial tissue levels of heme oxygenase-1 and 3-nitrotyrosine were increased in persistent LAF patients (P o.001),consistent with oxidative stress. Levels of superoxide dismutase-2,interleukin-8, interleukin-10, tumor necrosis factor-α, and thio-barbituric acid reactive substance were greater in controls than inpersistent LAF patients. Immunoreactive signal for connexin43 wasreduced more frequently in persistent LAF patients than controls.There was no correlation between features of structural or molecular

Drs. Saffitz and Alfieri are co-senior authors. Address reprint requestsand correspondence: Dr. Domenico Corradi, Department of Biomedical,Biotechnological, and Translational Sciences (S.Bi.Bi.T.), Unit of Pathol-ogy, University of Parma, Via Gramsci 14, 43126 Parma, Italy. E-mailaddress: domenico.corradi@unipr.it.

1547-5271/$-see front matter B 2014 Heart Rhythm Society. All rights reserved.

remodeling and clinical parameters, including persistent LAFduration.

CONCLUSION In persistent LAF patients, the atria are modified bystructural remodeling and molecular changes of oxidative stress.Tissue changes in persistent LAF appear to occur early after itsonset and are qualitatively no different than those observed inpatients with atrial fibrillation related to conventional risk factors.These findings suggest that different types of atrial fibrillation areassociated with the same spectrum of tissue lesions. Early inter-vention to restore sinus rhythm in persistent LAF patients mayprevent irreversible tissue change, especially interstitial fibrosis.

KEYWORDS Lone atrial fibrillation; Remodeling; Structure; Atrialcardiomyocyte; Pathology

ABBREVIATIONS AF ¼ atrial fibrillation; Cx43 ¼ connexin43;HO-1 ¼ heme oxygenase-1; IL ¼ interleukin; LAF ¼ lone atrialfibrillation; NYHA ¼ New York Heart Association; PAS ¼ periodicacid Schiff; SOD-2 ¼ superoxide dismutase-2; TBARS ¼thiobarbituric acid reactive substance; TNF-α ¼ tumor necrosisfactor-α

(Heart Rhythm 2014;0:1–9) I 2014 Heart Rhythm Society. Allrights reserved.

Introduction“Lone” atrial fibrillation (LAF), also known as “idiopathic”atrial fibrillation (AF), is an ill-defined clinical entity

characterized mainly by the absence of any apparent explan-ation or underlying etiology, especially associated cardiopul-monary disorders including hypertension.1 Over the last fewyears, the number of patients with true LAF has decreased asnew epidemiologic associations with AF have emerged (e.g.,sleep apnea syndrome, obesity, high coffee or alcohol con-sumption, endurance sports activities, gene mutations).2

The reported prevalence of LAF has ranged widelybetween 2% and 30%, presumably reflecting the ambiguous

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Heart Rhythm, Vol 0, No 0, Month 20142

definition and uncertain diagnostic criteria for this supra-ventricular arrhythmia.3 However, new and more accurateclinical tests, as well as the discovery of new etiologies, haveprogressively reduced the number of LAF cases with noknown cause.4

Patients with LAF do not represent a homogeneous groupin terms of cardiovascular and thromboembolic risks.Instead, these patients should be divided into low-risk andhigh-risk categories, based on the chronicity of theirarrhythmia and/or left atrial size, and treated accordingly.5,6

In this regard, an emerging parameter for a more accurate andindividualized prognostic definition of LAF involves non-invasive assessment of atrial myocardial structural remodel-ing.7 Of the various histopathologic features of structuralremodeling in AF substrates, myocardial fibrosis seems to beone of the most important, especially in view of its apparentstructural stability and negligible regression after restorationof sinus rhythm.8,9

An interesting subset of LAF patients consists of casesthat have progressed from paroxysmal to persistent AF in theabsence of an underlying associated cardiopulmonary dis-ease.10 To the best of our knowledge, no clinicopathologicstudies on such patients have been reported in the Englishlanguage literature. A thorough understanding of the struc-tural and molecular state characterizing the LAF atrialmyocardium certainly could represent the basis for futureinvestigations focusing on structural remodeling preventionin these individuals.

Based on recent advances regarding morphologic changesin AF and our own experience in analyzing the histopathol-ogy of AF atrial samples, the purpose of this retrospectiveinvestigation was to characterize the histopathology of tissueremodeling, quantify expression of molecular markers ofoxidative stress and inflammation in left and right atrialendomyocardial biopsies, and correlate these changes withclinical characteristics in patients with persistent LAF under-going surgical radiofrequency ablation.8,11–14

MethodsPatient population and controlsWe studied 19 consecutive cases of LAF whose currentarrhythmic episode lasted 47 days (“persistent AF” accord-ing to the current AF classification). Among these cases,patients whose present AF occurrence lasted Z1 year werefurther classified as having “long-lasting persistent AF.”1,3

As controls, we used 15 consecutive autopsied hearts fromage-matched patients. The study was approved by the SanRaffaele Hospital Institutional Review Board. All studypatients gave written informed consent.

Atrial myocardial samplesIn each patient, myocardial samples (about 5 � 5 � 2 mm)were excised from the left atrial posterior wall (adjacent tothe atriotomy line) and the right atrial free wall (between the2 caval vein outlets).12 All of the biopsy sites were far fromany previous transcatheter ablation lines. Furthermore,

biopsies were obtained before any new radiofrequencyablation lines were produced through the atrial myocar-dium.12 Control autopsy tissues were sampled in the sameway as those obtained surgically.

Each left and right atrial sample was immediately dividedinto 2 fragments: 1 was fixed in 10% buffered formalin for 24hours for histopathologic evaluations, and the other wasfrozen in liquid nitrogen and stored at –801C for subsequentmolecular analyses.

Other methodsMethods for histologic, morphometric, and immunofluores-cence studies, enzyme-linked immunosorbent assay, andmeasurements of thiobarbituric acid reactive substance(TBARS), 3-nitrotyrosine levels, and heme oxygenase-1(HO-1) mRNA are detailed in the Online SupplementaryData.

Statistical analysisNormality of data was assessed by the Shapiro-Wilks test. Ifthere was no evidence against normality, data are expressedas mean � SD. Comparisons were made using independent(AF patients vs controls) or repeated measures (left atrium vsright atrium comparisons within each single patient orcontrol case) Student t tests. When there were significantdeviations from normality, data are expressed as median(with interquartile range), and nonparametric tests wereperformed (Mann-Whitney U test for independent samplesand Wilcoxon test for repeated measures). The Pearsoncorrelation test was applied when no outliers were present.Otherwise, the Spearman correlation test was used. Differ-ences in connexin43 (Cx43) immunoreactive signal distri-bution were tested using the χ2 test. P o.05 was consideredsignificant. Statistical analysis was performed using SPSS(version 21, IBM, Armonk, NY).

ResultsPatient population and controlsNineteen patients (7.6% of the total AF patients undergoingsurgical ablation at San Raffaele Hospital in the same period)fulfilled criteria for the diagnosis of persistent LAF and wereenrolled in this study. According to their current AF episodeduration, 11 patients (58%) had long-lasting persistent AF.3

General clinical data and preoperative echocardiographicmeasurements of the LAF patients are listed in Table 1. Theaverage age of the LAF patients was 49 years. All but twowere younger than 60 years, consistent with previous studiesshowing younger age of subjects with LAF than those withAF related to other conditions. There was a striking malepredominance (17/19 cases [90%]), as also previouslyreported for LAF.4 Echocardiographic measures (Table 1)all were within normal limits except for left and right atrialvolumes, which were above the reference values.15 Preop-eratively, 5 patients had asymptomatic AF (New York HeartAssociation [NYHA] class I), whereas 14 had AF withpalpitations (and on this basis only were considered as being

Table 1 General patient characteristics and echocardiographic data

LAF Controls P value

No. of cases 19 15Age (y) 49 � 9 (31–66) 48 � 9 (33–60) NSGender (M/F) 17/2 12/3 NSBody mass index (kg/m2) 27 � 3 (18–29) 26 � 3 (16–29) NSBSA (m2) 2.00 � 0.22 (1.44–2.40) 1.95 � 0.12 (1.38–2.05) NSTotal heart weight (g) 341 � 40 (270–390)Left atrial volume/BSA (mL/m2)* 42 � 13 (24–77)Right atrial volume/BSA (mL/m2) 34 � 15 (22–67)Left ventricular end-diastolic diameter (mm) 50 � 4 (39–5)left ventricular end-systolic diameter (mm) 29 � 3 (24–35)Left ventricular end-diastolic volume (mL) 95 � 10 (62–110)Left ventricular end-systolic volume (mL) 40 � 6 (25–52)Left ventricular ejection fraction (%) 58 � 4 (53–68)Interventricular septum thickness (mm) 10 � 1 (9–11)Left ventricular posterior wall thickness (mm) 9 � 1 (8–10)Pulmonary artery systolic pressure (mm Hg) 25 � 4 (20–30)Total LAF disease duration (mo) 91 � 69 (24–240)Current LAF episode duration (mo) 17 � 19 (2–84)No. of previous transcatheter ablation procedures (0/1/2/3/4/5) 3/4/4/4/3/1New York Heart Association functional class (I/II/III/IV) 5/14/0/0

Data are expressed as frequency or mean � SD (range).BSA ¼ body surface area; LAF ¼ lone atrial fibrillation.

*Left atrial volume calculated using biplane Simpson method.

3Corradi et al Lone Atrial Fibrillation

in NYHA class II). All patients underwent the Maze IIIsurgical ablation procedure (13 through minithoracotomyand 6 through sternotomy). Sixteen patients (84%) hadpreviously undergone a variable number (from 1–5,Table 1) of unsuccessful (nonsurgical) catheter ablations.After undergoing surgical AF ablation, all but 1 patientreverted to sinus rhythm. The preoperative pharmacologictreatments are listed in Table 2. All of the patients ate astandard Mediterranean-type diet. In accordance with theirNYHA functional classification, all subjects performed mild-to-moderate physical activity.

General data regarding the control (autopsy) populationare also listed in Table 1. Causes of death were traumaticinjuries in 12 and nontraumatic cerebral hemorrhage in 3.Prior clinical measurements of atrial volume were notavailable for the autopsied controls. However, carefulevaluation by experienced cardiopathologists indicated thatatrial sizes in all control subjects fell within the normal rangeas previously described.16

Table 2 Preoperative treatment

n (%)

Acetylsalicylic acid 1 (5)Allopurinol 1 (5)Amiodarone 1 (5)Angiotensin-converting enzyme inhibitor 1 (5)Angiotensin receptor antagonist 7 (37)Beta-blocker 8 (42)Calcium channel blocker 2 (10)Gastric acid pump inhibitor 2 (10)Oral anticoagulant 14 (74)Statin 1 (5)

Data are expressed as no. of patients (percentage).

General histopathology and morphometricevaluationAtrial myocardium from patients with LAF showed signifi-cant pathologic changes consisting of hypertrophy, myocy-tolytic degeneration and glycogen accumulation incardiomyocytes, remodeling of cardiomyocyte bundle ori-entation, and accumulation of interstitial and perivascularfibrosis. Representative images are shown in Figure 1, andquantitative morphometric data are given in Table 3. Atrialcardiomyocytes were sometimes arranged in irregular bun-dles (Figure 1A) compared to controls (Figure 1B). Myocy-tolytic degeneration was characterized by perinuclear loss ofsarcomeres appearing as a clear vacuole extending eccentri-cally into the adjacent sarcoplasm (Figure 1C). Thesevacuoles were inconstantly filled with glycogen (seen asreddish material in the periodic acid–Schiff [PAS]-stainedhistologic sections in Figure 1D). Other cardiomyocyteswithout apparent loss of sarcomeres also showed moderatelyincreased punctate sarcoplasmic PAS positivity, presumablyrepresenting intermyofibrillar glycogen accumulation secon-dary to cell metabolic changes. Atrial myocardial architec-ture in AF samples was typically modified by pathologicfibrosis between muscle cells (interstitial fibrosis; Figure 1E)and surrounding the adventitia of small intramural bloodvessels (perivascular fibrosis; Figure 1F). In some cases, thesubendocardial layer was thickened as a result of mildfibroelastosis. The pattern of fibrosis observed in AF patientswas typical of what has been reported in previous studies oftissue remodeling in AF and was distinctly different than thedense local fibrosis resulting from previous catheter ablationprocedures.

Quantitative analysis of structural remodeling demon-strated significant differences between tissue features from

Figure 1 Representative examples of left atrial structural remodeling in lone atrial fibrillation (LAF). Atria in LAF (A vs controls in B) show changes incardiomyocyte size and orientation. Some cardiomyocytes show myolysis with characteristic perinuclear loss of sarcomeres (arrowheads in C) and glycogenaccumulation (arrowhead in D). Interstitial fibrosis (arrowheads in A and E) and perivascular fibrosis (arrowheads in F) reflect collagen deposition aroundcardiomyocytes and intramural coronary branches. Bar ¼ 200 μm in A, B, E, and F; bar ¼ 100 μm in C and D.

Table 3 Morphometry

LAF Controls

LA RA LA RA

Interstitial fibrosis (%) 4.6 � 3.9 (1.1–16.9)*† 2.3 � 2.2 (0.9–10.1)† 0 0Cardiomyocyte myocytolysis (%) 5.1 � 5.6 (0–20.0)† 2.8 � 3.3 (0–10.5)† 0.5 � 0.1 (0–2.2) 0.4 � 0.2 (0–1.4)Cardiomyocyte transversediameter (mm)

16.6 � 2.7 (12.9–20.6)† 15.5 � 2.6 (10.9–20.1)† 10.9 � 0.5 (10.4–13.4) 10.3 � 0.8 (10.2–13.5)

Data are expressed as average � SD (range).LA ¼ left atrium; LAF ¼ lone atrial fibrillation; RA ¼ right atrium.

*P o.05 (LA vs RA, in patients).†P o.0001 (patients vs controls).

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5Corradi et al Lone Atrial Fibrillation

AF patients vs controls for all 3 parameters of tissueremodeling (myocytolysis, hypertrophy and fibrosis;Table 3). Controls showed no fibrosis and virtually nomyocytolytic degeneration. PAS-positive material in con-trols consisted of minute particles distributed homogene-ously throughout the sarcoplasm. Changes in cell andtissue structure generally appeared to be more severe in theleft vs the right atrium in patients with AF, although thisdifference was significantly different only for the amountof interstitial fibrosis (Table 3). Cardiomyocyte transversediameters showed statistically significant correlationbetween left atrium and right atrium (Pearson test, r ¼0.61, P o.05). There were no correlations between any ofthe histopathologic parameters and the number of previoustranscatheter ablations. There was also no correlationbetween the amount of atrial fibrosis vs patient’s age orAF duration.

Connexin43 patternStrong immunoreactive signal for Cx43 was seen in 16 of 19control atrial cardiomyocytes both at intercalated diskregions and along the lateral sarcolemma (Figure 2). In the

Figure 2 Connexin43 (Cx43) distribution in representative cases of lone atrial fibat cell–cell junctions and in some cases at lateral membranes. In both LAF sampl

remaining 3 cases, the amount of signal appeared to bereduced. By contrast, Cx43 signal was diminished in 11 of19 of the LAF cases (P o.05 compared with controls;Figure 2). Signal was abundant at intercalated disks insamples from 7 AF cases and lateralized in 1 case. ReducedCx43 signal occurred equally in both atria in 8 of the 11 casesof LAF with reduced signal. Signal was reduced in the leftatrium but normal in the right atrium in 1 case and wasreduced in the right atrium but normal in the left atrium in2 cases.

Molecular markers of oxidative stress andinflammationQuantitative measurements of tissue markers of oxidativestress and inflammation are given in Table 4. Expression ofthe oxidative stress-related protein HO-1 was significantlyincreased in the left and right atrium (10- and 37-fold,respectively) in LAF patients compared to controls.Increased immunoreactive signal for HO-1 was also readilyapparent in atrial cardiomyocytes from LAF patients com-pared to controls (Figure 3), whereas other markers ofoxidative stress or inflammation showed no apparent

rillation (LAF) and controls. Controls showed strong immunoreactive signales, Cx43 signal is significantly depressed. Bar ¼ 40 μm.

Table 4 Proteins, thiobarbituric acid reactive substance, and 3-nytrotyrosine molecular measurements

LAF Controls

LA RA LA RA

HO-1 (μg/g proteins) 8.3 (3.0-19.8)* 14.7 (2.6-24.7)* 0.8 (0.6-4.0) 0.4 (0.2-1.3)SOD-1 (μg/g proteins) 4.6 � 2.3 4.7 � 2.9 5.2 � 3.2 3.5 � 1.3SOD-2 (μg/g proteins) 0.6 (0.3-1.1)† 1.2 (0.5-4.6) 1.7 (0.4-3.1) 0.7 (0.4-1.1)IL-6 (ng/g proteins) 26.4 (14.3-41.2) 41.6 (20.4-75.5) 50.3 (29.0-75.4) 40.6 (28.8-66.3)IL-8 (ng/g proteins) 25.8 � 16.3† 36.1 � 27.4 47.7 � 26.8 40.5 � 27.5IL-10 (ng/g proteins) 1.7 (1.1-5.4)† 4.3 (2.9-9.2) 5.0 (3.2-8.6) 3.6 (2.4-9.5)TNF-α (ng/g proteins) 25.3 � 16.8 37.3 � 26.3† 31.7 � 17.8 19.1 � 9.6TBARS (nmol/g proteins) 16.5 (6.9-26.1)† 19.3 (11.0-79.9) 36.1 (23.0-46.3) 26.2 (18.4-38.4)3-Nitrotyrosine (mmol/mol tyrosine) 62.9 (46.2-89.5)* 73.8 (50.4-188.4)* 28.8 (18.2-39.6) 22.4 (17.0-27.5)

* ¼ p o 0.001, † ¼ p o 0.05. Data are expressed as average � SD or median value (interquartile range).HO ¼ heme oxygenase-1; IL ¼ interleukin; LA ¼ left atrium; LAF ¼ lone atrial fibrillation; RA ¼ right atrium; SOD ¼ superoxide dismutase; TBARS ¼

thiobarbituric acid reactive substance; TNF-α ¼ tumor necrosis factor-α.

Heart Rhythm, Vol 0, No 0, Month 20146

differences in immunostaining compared with controls(data not shown). HO-1 mRNA was detected in both atriaof patients with LAF in roughly equal amounts (0.47 �0.33 vs 0.48 � 0.27 arbitrary units for left and right atria,respectively; P ¼ NS). Increased levels of HO-1 proteinwere associated with significantly greater levels (2- to 3-fold) of the oxidative stress marker 3-nitrotyrosine in LAFtissue compared with controls (Table 4). Otherwise, leftatrial tissue from LAF patients contained lower levelsof superoxide dismutase-2 (SOD-2), interleukin (IL)-8,IL-10, and TBARS than the corresponding left atrialcontrol tissue. Right atrial tissue from LAF patientscontained greater amounts of tumor necrosis factor-α(TNF-α) than the corresponding right atrial control tissue(Table 4). The magnitude of these differences was notnearly as great, however, as that seen for HO-1 and 3-nitrotyrosine.

Figure 3 Heme oxygenase-1 (HO-1) immunoreactive signal (green) is located iactin (SA). HO-1 signal intensity is much greater in the pathologic atrium than in tBar ¼ 40 μm.

DiscussionIn the present study, we found the same type of structural andmolecular remodeling in the atria of patients with LAF aspreviously described in patients with more conventionaldisease-associated AF, thus suggesting that the differenttypes of AF may be associated with the same continuum oflesions.

The definition of LAF is still based largely on the apparentabsence of other disorders that might account for “secon-dary” AF.2 Therefore, the diagnosis depends on the accuracyof clinical evaluation. Advances in such evaluations andidentification of additional risk factors associated with AFhave progressively diminished the fraction of cases thatinitially would have been labeled as “lone,”2 such that somenow regard the designation of lone/idiopathic AF as obso-lete.17 Nevertheless, the effects of the arrhythmia itself onatrial remodeling may be greater in LAF than in AF

n the cardiomyocyte sarcoplasm, identified by red staining for α-sarcomeriche control (C). Yellow color indicates green and red signal superimposition.

7Corradi et al Lone Atrial Fibrillation

associated with conventional risk factors (e.g., mitral valvedisease) in which structural and molecular remodeling likelyprecede onset of AF. In this regard, pacing-induced AF inanimals may be a better model of LAF than AF associatedwith usual risk factors.8

Age 460 years and increased atrial volume havegenerally been regarded as exclusion criteria for LAF, butrecent studies have identified cases of AF without anyevident underlying process in patients older than 60 years,suggesting that this age cutoff may be inappropriate.4,18 Inthe present study, we did not exclude patients with atrialenlargement in the absence of other indications of cardio-vascular disease. In such cases, atrial enlargement may be theresult rather than the cause of AF,10 although at some pointin the natural history atrial enlargement likely is both a causeand a consequence of the arrhythmia.

Structurally, the atrial myocardial architecture of ourpatient population had been altered by changes in cardio-myocytes and the interstitium, including increased cardio-myocyte size, myolysis, and interstitial fibrosis. All of thesemodifications were significantly greater in the LAF patientsthan in the control population but were less severe than thoseseen in our previous studies of AF associated with mitralvalve disease. For example, the proportion of left and rightatrial cardiomyocytes showing myolytic degeneration was5.1% and 4.6%, respectively, in LAF compared with 15.1%to 20.8% and 7.16% to 10.4%, respectively, in non-loneforms of AF. Hypertrophy of atrial cardiomyocytes in LAFwas only slightly less than that seen in valvular AF (16.6 vs18.6–19 μm, respectively).11–14 The left atrium showed moreinterstitial fibrosis than the right atrium in LAF (4.6� 3.9 vs2.3 � 2.2, respectively, P o.05), perhaps related to thedifferent hemodynamic loads of these 2 atrial chambers inAF.19

On the whole, these data confirm the fact that changes inAF structural remodeling are essentially nonspecific andsuggest that, in terms of myocardial architectural distortion,the various forms of AF should be placed in a spectrum ofdisease where the specific effects of the arrhythmia (includ-ing its duration), associated diseases, and aging should beconsidered in addition to other factors.

We found a consistent lack of correlation between AFduration and structural remodeling parameters in our pre-vious studies on valvular AF (even after combining thedifferent cases in a single patient population, unpublisheddata).11–14 The large variability in structural remodeling as amanifestation of myocardial injury likely is related to manyfactors, including genetics, therapy, and aging in addition tothe usual risk factors implicated in AF initiation and/orperpetuation). It is unlikely that structural remodeling ofatrial myocardium develops at the same rate over the naturalhistory of AF. In fact, it has been demonstrated experimen-tally that histopathologic changes in develop progressivelyover an interval of 4 months of pacing-induced AF, by whichtime structural remodeling is fully established.20 Thus, itappears that the atria undergo structural remodeling earlyafter the onset of AF, which might explain the consistent lack

of correlation between the severity of structural remodelingand the duration of AF in patients whose arrhythmia hasoccurred for at least a few months. Lastly, AF durationusually is calculated from the first arrhythmic episode, butthe number of episodes over the clinical history of AF maybe highly variable and often is unknown. The actual diseaseinterval may vary considerably in patients with the sameapparent AF duration.

In contrast to the study by Frustaci et al,21 we did not findinflammatory infiltrates in myocardial samples from LAFpatients. However, the patient populations were not identical,with paroxysmal AF in the previous investigation andpersistent AF in the present study. We cannot definitivelyrule out the possibility that the fibrosis we observed couldhave been caused by previous myocarditis, and resultingsubstrate could have played a role in inducing and/orperpetuating the arrhythmia.

Changes in the amount of immunoreactive signal forconnexins and redistribution of signal from intercalated diskregions to the lateral margins of atrial cardiomyocytes havebeen described in AF.22,23 In our study, the amount of Cx43signal was reduced in significantly more AF samples thancontrols, providing at least some basis for speculating thatreduced coupling may have contributed to AF initiation and/or maintenance. Because the number of LAF cases waslimited, it was not possible to correlate changes in Cx43signal with AF duration, left atrial dimensions, or the otherfeatures of structural and molecular remodeling analyzed inthis study.

Oxidative stress, in which reactive oxygen speciesdamage both structural and functional proteins in cardio-myocytes, has been implicated in the pathogenesis of AF.8 Inthis study, we observed significantly increased expression ofthe protective enzyme HO-1 in LAF subjects compared tocontrols as well as higher levels of the oxidative stressmarker 3-nitrotyrosine, which is formed when reactivenitrogen species (e.g., peroxynitrite, nitryl chloride) interactwith the aromatic ring of tyrosine in soluble amino acids andin proteins.24 Previously, we reported increased HO-1expression (greater in the left atrial posterior wall that inthe left atrial appendage) in 2 different patient populationswith valvular AF.13,14 HO-1 expression has previously beenshown to confer protection in various models of cardiovas-cular injury or disease, including hypertension, atheroscle-rosis, myocardial infarction, and allograft rejection.25 Furtherand more focused investigations are necessary to testwhether HO-1 may serve as a predictive marker in AF. Inthe present study, HO-1 expression was not associated with aparallel increase in SOD-2 or lipid peroxidation (as deter-mined by measurement of TBARS) in the left atrialmyocardium. In fact, TBARS levels were significantlydecreased compared to controls. This might reflect theactivation of antioxidative stress-protective mechanisms, asobserved in in vitro models.26 The significantly increasedlevels of 3-nitrotyrosine also suggest greater atrial nitricoxide levels and ongoing nitric oxide–dependent reactivespecies-induced oxidative stress.27 Increased myocardial

Heart Rhythm, Vol 0, No 0, Month 20148

3-nitrotyrosine has previously been reported in AF associ-ated with mitral valve disease.28

Inflammatory markers such as C-reactive protein, TNF-α,IL-2, IL-6, and IL-8 are thought to play a role in initiatingand perpetuating AF and AF-related thrombosis throughmechanisms involving endothelial damage/dysfunction andplatelet activation.29 Whether AF is the cause or a conse-quence of inflammation is not known, but it is likely thatboth relationships occur. Inflammation may be a potenttrigger of AF, but at the same time AF seems to promote aninflammatory and prothrombotic state.8,29,30 Only limiteddata are available on the systemic or local nature ofinflammation in AF. Liuba et al31 found higher plasmalevels of IL-8 in femoral vein, right atrial, and coronary sinusblood, but not in pulmonary vein samples, consistent with asystemic source of inflammation. Marcus et al30 measuredhigher C-reactive protein levels in the left atrium than in thecorresponding coronary sinus and suggested that transcar-diac cytokine gradients in AF arise by sequestration ofinflammatory cytokines in the heart. The results of thepresent study do not indicate increased local production/accumulation of atrial myocardial IL-6, IL-8, IL-10, andTNF-α in LAF patients.

Study limitations and clinical implicationsLimitations of the present study include small sample sizes,prior catheter ablations, use of autopsy controls, and use bysome patients of drugs such as angiotensin-convertingenzyme inhibitors, angiotensin receptor antagonists, andstatins, which could decrease interstitial collagen depositionby blocking fibroproliferative signaling pathways.8 In addi-tion, the numerically limited patient population could havenegatively influenced some of the statistical results, partic-ularly possible linear correlations between the variousdemographic, clinical, and morphologic parameters (e.g.,patient age vs interstitial fibrosis).

Furthermore, we cannot establish causal relationshipsbetween structural remodeling and arrhythmia, oxidativestress, or inflammation. As in any retrospective clinicopa-thologic study, we can only offer a snapshot of a givenmoment in the disease.

Even with the limitations inherent in a retrospectiveanalysis of patient tissue samples, our study clearly indicatesthat, despite the absence of underlying cardiovascular andpulmonary disease, patients with persistent LAF exhibitsignificant atrial structural remodeling and express molecularmarkers suggestive of atrial oxidative stress. The morpho-logic modifications are similar to but less severe than thoseaffecting patients with mitral valve disease–related AF. Thefact that all but 3 of the LAF patients had previouslyundergone 1 or more unsuccessful catheter ablations sug-gests that atrial structural remodeling had been widespreadand of long duration.32,33 This hypothesis is supported byobservations in a model of pacing-induced AF in goats inwhich atrial structural remodeling developed progressivelyover 16 weeks after atrial burst stimulation.20 In view of the

high stability of collagen fibers, preventing or limitinginterstitial fibrosis appears to be an important way to mitigateAF. In this regard, interstitial fibrosis was the only compo-nent of structural remodeling that had not regressed 4 monthsafter restoration of sinus rhythm in the goal model of pacing-induced AF.9

Clinical and experimental evidence suggests that inter-stitial fibrosis and other components of atrial structuralremodeling interact with AF in a vicious cycle in whichthey act as both a cause and a consequence of each other.8 Inthis view, progression of the abnormal substrate would play apart in the transition from paroxysmal to persistent AF.

In view of limitations associated with antiarrhythmicdrugs and extensive ablative procedure and with increasingevidence of ongoing oxidative stress, attention has turned tothe role of so-called “upstream therapy.” To be of optimalbenefit, such efforts should be undertaken before the onset ofstructural remodeling.34 Noninvasive detection of early signsof structural remodeling (e.g., delayed enhancement mag-netic resonance imaging) may be of particular importance inidentifying those patients at risk for developing further atrialfibrosis.

ConclusionIn patients with LAF, the atria are modified by structuralremodeling and show molecular changes indicative of oxida-tive stress. Based on the literature9,20 and our present data,morphologic changes in LAF seem to occur early after theclinical onset of AF and are qualitatively no different thanthose observed in patients with AF associated to underlyingcardiovascular diseases (e.g., mitral valve dysfunctions). Thesefindings suggest that different types of AF are associated withthe same spectrum of tissue lesions. Therefore, even patientslabeled as having early LAF should undergo careful follow-upto discover early signs of atrial structural remodeling and, ifpossible, prevent the development of diffuse foci of interstitialfibrosis throughout the myocardial tissues.

AcknowledgmentsWe thank Professor Rita Gatti, Department of Biomedical,Biotechnological, and Translational Sciences (S.Bi.Bi.T.),Unit of Histology, University of Parma, for expert confocalmicroscopy, and Ms. Maria Nicastro, Department of Clinicaland Experimental Medicine, University of Parma, fortechnical assistance.

AppendixSupplementary dataSupplementary data associated with this article can be foundin the online version at http://dx.doi.org/10.1016/j.hrthm.2014.02.008.

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