Extracellular Matrix Alterations in Patients With Paroxysmal and Persistent Atrial Fibrillation

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1 Journal of the American College of Cardiology Vol. 52, No. 3, by the American College of Cardiology Foundation ISSN /08/$34.00 Published by Elsevier Inc. doi: /j.jacc Heart Rhythm Disorders Extracellular Matrix Alterations in Patients With Paroxysmal and Persistent Atrial Fibrillation Biochemical Assessment of Collagen Type-I Turnover Eleftherios M. Kallergis, MD,* Emmanuel G. Manios, MD,* Emmanuel M. Kanoupakis, MD,* Hercules E. Mavrakis, MD,* Dimitris A. Arfanakis, MD,* Niki E. Maliaraki, MD, Chrisovalantis E. Lathourakis, MD,* Gregory I. Chlouverakis, PHD,* Panos E. Vardas, MD, PHD, FESC, FACC* Crete, Greece Objectives Background Methods Results Conclusions We investigated whether the serum markers of collagen turnover differed in various forms of atrial fibrillation (AF) and in sinus rhythm (SR) in humans. Structural alterations and fibrosis have been implicated in the generation and perpetuation of AF. Serum C-terminal propeptide of collagen type-i (CICP), C-terminal telopeptide of collagen type-i (CITP), matrix metalloproteinase-1, and tissue inhibitor of matrix metalloproteinases-1 were measured as markers of collagen synthesis and degradation in 70 patients with AF and 20 healthy control subjects in SR. C-terminal propeptide of collagen type-i and CITP were significantly higher in AF patients than in control subjects (91 27 ng/ml vs ng/ml, p and ng/ml vs ng/ml, p 0.001, respectively). Persistent AF patients had higher levels of CICP ( ng/ml vs ng/ml, p 0.001), but not CITP, compared with those with paroxysmal AF. Patients with persistent AF had lower levels of matrix metalloproteinase-1 but increased levels of tissue inhibitor of matrix metalloproteinases-1 compared with patients with paroxysmal AF ( ng/ml vs ng/ml, p 0.03 and ng/ml vs ng/ml, p 0.001, respectively). Tissue inhibitor of matrix metalloproteinases-1 levels were significantly lower in control subjects compared with those in both paroxysmal and persistent AF patients ( ng/ml vs ng/ml vs ng/ml, respectively, p 0.001). Serum markers of collagen type-i turnover differed significantly between patients with AF and SR. Furthermore, these markers also differed significantly between paroxysmal and persistent AF patients, suggesting that the intensity of the extracellular synthesis and degradation of collagen type-i may be related to the burden or type of AF. (J Am Coll Cardiol 2008;52:211 5) 2008 by the American College of Cardiology Foundation Recent data from cardiac biopsies of patients with atrial fibrillation (AF) have demonstrated a substantial increase in collagen deposition in the atria of humans with AF as opposed to those in sinus rhythm (SR) (1 3). Preliminary experimental and clinical data suggest that biochemical markers of collagen turnover correlate significantly with fibrosis in endomyocardial biopsies in hypertensive patients (4). However, although such markers have been shown to differ between hypertensive and normotensive individuals, studies of their association with hypertrophy or other echocardiographic parameters have produced inconsistent results (5,6). From the *Department of Cardiology and Laboratory of Biochemistry, University Hospital of Heraklion, Crete, Greece. Manuscript received October 29, 2007; revised manuscript received February 13, 2008, accepted March 18, Collagen type-i is the major collagenous product of cardiac fibroblasts (7). We assessed the amount of fibrosis in patients with paroxysmal or persistent lone AF using serum analysis of peptides derived from the tissue synthesis (Cterminal propeptide of collagen type-i [CICP]) and degradation (C-terminal telopeptide of collagen type-i [CITP]) of collagen type-i (4). Enzymes that control collagen type-i turnover, specifically matrix metalloproteinase (MMP)-1 and tissue inhibitor of matrix metalloproteinases (TIMP)-1, were also measured (8). Methods The ethics committee of our institution approved the study, which conformed to the principles outlined in the Declaration of Helsinki. Informed written consent was obtained from all subjects.

2 212 Kallergis et al. JACC Vol. 52, No. 3, 2008 Fibrosis and AF July 15, 2008:211 5 Abbreviations and Acronyms AF atrial fibrillation CICP C-terminal propeptide of collagen type-i CITP C-terminal telopeptide of collagen type-i LA left atrial LVEF left ventricular ejection fraction MMP matrix metalloproteinase SR sinus rhythm TIMP tissue inhibitor of matrix metalloproteinase The study population consisted of 70 ambulatory patients (age 24 to 78 years) who suffered from lone AF, defined as AF without clinical or echocardiographic evidence of cardiopulmonary disease, including hypertension. The arrhythmia was considered paroxysmal when it was self-terminating in 24 h and persistent when it was documented on sequential 12-lead electrocardiograms, without any intervening periods of SR, for at least 3 months preceding enrollment. Patients with persistent AF had been referred to our department for electrical cardioversion; none had undergone such a procedure before. No patient had permanent AF, defined as sustained arrhythmia despite cardioversion. Twenty healthy subjects in SR with no history of atrial arrhythmias served as a control group. Exclusion criteria were conditions associated with elevated serum concentrations of myocardial or tissue fibrosis markers, such as liver disease, renal impairment, pulmonary fibrosis, extensive wounds, metabolic bone disease, malignancy, connective tissue disorders, chronic inflammatory disease, and recent infection or surgery. Patients who were over 80 years old or had a pacemaker/implantable cardioverter-defibrillator were also excluded. Using a case-control study design, serological markers of collagen type-i turnover, echocardiographic maximal left atrial (LA) diameter, and left ventricular ejection fraction (LVEF) were compared in patients and control subjects. Diltiazem and beta-blockers were allowed for ventricular rate control, and all AF patients received the indicated antithrombotic treatment. All patients were in AF at the time of blood sampling. Blood samples were obtained at the time of the clinical studies and were immediately placed on ice and centrifuged within 1 h. Specimens were stored at 80 C until analysis. Serum TIMP-1 and MMP-1 levels were assayed by enzyme-linked immunoadsorbent assay with commercially available kits (Human Biotrack enzyme-linked immunoadsorbent assay system, Amersham Biosciences, Piscataway, New Jersey). C-terminal propeptide of collagen type-i levels were determined by a sandwich enzyme immunoassay with a commercially available kit (enzyme immunoassay, Metra CICP, Quidel, San Diego, California), while CITP was measured using the Elecsys -CrossLaps/serum assay (Roche Diagnostics, Mannheim, Germany). Measurements were performed by personnel blinded to the patients clinical details. The intra- and interassay coefficients of variation of all assays were 8% and 10%, respectively, in our laboratory. Statistical analysis. Summary descriptive statistics for continuous parameters are presented as mean SD. Categorical variables were compared among the persistent, paroxysmal AF, and control groups using the chi-square test. One-way analysis of variance was used to test whether these 3 groups differed with respect to various continuous parameters of interest. Where findings were significant, post-hoc Tukey tests were performed to pinpoint differences. If the homogeneity of variance assumption was violated, the nonparametric Kruskal-Wallis test was used instead. Stepwise logistic regression analysis was used to assess whether age, gender, LA, and fibrosis markers were independently associated with AF. Age, LA, and fibrosis markers were treated as continuous variables in the model. The thresholds for entry into and removal from the model were 5% and 10%, respectively. Associations between continuous variables of interest were assessed with correlation and linear regression techniques. Values of p 0.05 were considered statistically significant. Results Patients. The baseline clinical and demographic features of the study population are shown in Table 1. Thirty-two patients had persistent and 38 paroxysmal AF. There were no significant differences in gender (p 0.40) or age (p 0.058) between the AF groups. Control subjects were younger than AF patients (p 0.001), but were comparable in terms of gender distribution. Patients with persistent AF had lower LVEF (p 0.038) and a larger LA (p 0.001) compared with paroxysmal AF patients. Serum markers of collagen turnover. The findings are summarized in Table 1. Both CICP and CITP levels were significantly higher in persistent AF patients than in control subjects (p 0.016). Persistent AF patients also had higher CICP levels compared with those with paroxysmal AF (p 0.001), whereas there was no significant difference in CITP levels (p 0.57) (Table 1, Figs. 1 and 2). Patients with persistent AF had lower levels of MMP-1 (p 0.026) but higher levels of TIMP-1 (p 0.013) compared with patients with paroxysmal AF (Table 1, Figs. 3 and 4). Tissue inhibitor of matrix metalloproteinases-1 levels were significantly lower (p 0.001) in control subjects compared with both paroxysmal and persistent AF patients (Fig. 4). Plasma MMP-1 levels did not differ significantly (p 0.20) between persistent AF patients and control subjects (Fig. 3). Finally, in all AF patients taken together, there was a positive correlation between both CICP and TIMP levels and LA dimension (r 0.635, p and r 0.563, p 0.001, respectively) (Fig. 5), while there was a weak, inverse relation between CICP and TIMP levels and LVEF (r 0.234, p 0.05 and r 0.278, p 0.020, respectively). A positive correlation was also observed between MMP-1 levels and LVEF (r 0., p 0.012) while there was an inverse relation between MMP-1 levels

3 JACC Vol. 52, No. 3, 2008 July 15, 2008:211 5 Baseline Patients Characteristics With Persistent andorserum Paroxysmal Markers AFof and Collagen HealthyType-I Control Turnover Subjects in SR Table 1 Baseline Characteristics and Serum Markers of Collagen Type-I Turnover in Patients With Persistent or Paroxysmal AF and Healthy Control Subjects in SR Kallergis et al. Fibrosis and AF 213 Persistent AF (n 32) Paroxysmal AF (n 38) Control Subjects (n 20) p Value Age (yrs) Gender (M/F) 23/9 27/11 12/ LVEF (%) LA (mm) BMI (kg/m 2 ) SBP (mm Hg) DBP (mm Hg) CICP CITP MMP TIMP Values are expressed as mean SD. Post-hoc analysis showed that: 1) age and left atrial diameter (LA) differed significantly between control subjects and persistent atrial fibrillation (AF) patients; 2) C-terminal propeptide of collagen type-i (CICP) levels in persistent AF patients differed significantly compared with both paroxysmal and control subjects; 3) C-terminal telopeptide of collagen type-i (CITP) levels in control subjects differed significantly compared with both paroxysmal and persistent AF patients; 4) matrix metalloproteinase (MMP)-1 levels differed significantly between persistent and paroxysmal AF patients; and 5) tissue inhibitor of matrix metalloproteinase (TIMP)-1 levels differed significantly in all intergroup comparisons. BMI body mass index; DBP diastolic blood pressure; LVEF left ventricular ejection fraction; SBP systolic blood pressure; SR sinus rhythm. and LA dimension (r 0.615, p 0.001). Logistic regression analysis showed that, among age, gender, LA and collagen markers, TIMP-1, MMP-1, age, and CICP were independently associated with AF (Table 2). Discussion Patients initially presenting with paroxysmal AF often progress to persistent and eventually develop permanent AF. Although the exact pathophysiological mechanisms are poorly understood, persistence of AF is thought to result from atrial remodeling (9,10). However, pure electrical remodeling cannot explain the development of sustained AF (10,11). Atrial fibrosis, a factor with a slower time course, could be involved (3,12,13). In this study we demonstrated an elevation in CICP and CITP in the AF cohort taken as a whole compared with those in SR. Interestingly, persistent AF patients had the highest serum concentrations of CICP, whereas there was no difference in CITP levels between patients with persistent and paroxysmal AF. Thus, CICP demonstrated a gradual increase from control subjects to paroxysmal and then to persistent AF, but CITP did not, suggesting that the intensity of the extracellular degradation of collagen type-i was insufficient to compensate for its increased synthesis, resulting in augmented fibrosis in patients with persistent AF. Furthermore, in patients with persistent AF, MMP-1 levels were decreased, whereas TIMP-1 levels were increased compared with paroxysmal AF patients. Levels of TIMP-1 were also higher in paroxysmal AF patients than in control subjects. In addition, control subjects had lower levels of MMP-1 than paroxysmal AF patients but higher CICP CITP Figure 1 Comparative Levels of Serum Concentration of CICP Figure 2 Box Plot of Serum Concentrations of CITP C-terminal propeptide of collagen type-i (CICP) showed a gradual increase from control subjects to paroxysmal and then to persistent atrial fibrillation (AF) patients as shown in this box plot. C-terminal telopeptide of collagen type-i (CITP) in control subjects differed significantly compared with that in both paroxysmal and persistent atrial fibrillation (AF) patients.

4 214 Kallergis et al. JACC Vol. 52, No. 3, 2008 Fibrosis and AF July 15, 2008:211 5 MMP CICP Persistent AF Paroxysmal AF 5 0 Controls Persistent AF Paroxysmal AF LA (mm) Figure 3 Intergroup Serum Concentrations of MMP-1 This box plot depicts matrix metalloproteinase (MMP)-1 levels that differed significantly between persistent and paroxysmal atrial fibrillation (AF) patients. levels than persistent AF patients (although the differences did not reach statistical significance). That seems paradoxical, but it could be the result of the activation manner of MMP-1, which depends on the nature of the stimulus and differs in the setting of acute or chronic stimulation (14). Thus, short-lasting paroxysmal AF could result acutely in pressure or volume overload, activating the MMP-1 system, which then becomes compromised with the prolongation and stabilization of the stimulus. Another interesting finding was that both CICP and TIMP levels positively correlated with LA diameter and inversely related to LVEF, whereas AF patients with a larger LA and a smaller LVEF, probably as a result of a longer arrhythmia duration, had lower MMP-1 levels. Since our study included patients with lone AF only, we can speculate that the aforementioned alterations were TIMP1 Figure Box Plot Depiction of the Differences in Levels of Serum TIMP-1 Tissue inhibitor of matrix metalloproteinase (TIMP)-1 levels differed significantly in all intergroup comparisons. AF atrial fibrillation. Figure 5 Direct Correlation Between Serum Concentrations of CICP and LA Diameter Dimensions In all AF patients taken together, there was a positive correlation between CICP and left atrial diameter (LA) dimension (r 0.635, p 0.001). Abbreviations as in Figure 1. attributable to the arrhythmia itself, and not to the presence or absence of any confounding factor, with a progressive increase in fibrosis from paroxysmal to persistent AF. In addition, the augmented fibrosis, especially in patients with the persistent form of AF, could further suggest that if collagen modification plays a causal role, this relates to both the initiation and the maintenance of AF. Study limitations and clinical implications. Serum markers of collagen turnover are not heart specific. In addition, we did not support our findings with atrial tissue biopsy data or coronary sinus sampling. However, we made strenuous efforts to exclude subjects with conditions associated with fibrosis. Despite our efforts to match control subjects with AF patients, the latter were older. However, collagen markers differed significantly between patients with persistent and paroxysmal AF, despite the lack of any significant difference in age, suggesting that age differences did not influence the interpretation of our results. Serial measurements of collagen indexes after SR restoration to evaluate the potential temporal alterations of collagen turnover are not available, although these data would undoubtedly have been a valuable addition to our study and could have reinforced our findings. Independent to Stepwise Logistic Predictors Regression of AF According Analysis Table 2 Independent Predictors of AF According to Stepwise Logistic Regression Analysis Variable Odds Ratio 95% CI p Value TIMP MMP Age CICP CI confidence interval; other abbreviations as in Table 1.

5 JACC Vol. 52, No. 3, 2008 July 15, 2008:211 5 Kallergis et al. Fibrosis and AF 215 Finally, the small patient sample size does not allow major conclusions regarding the relationship between systemic fibrosis and AF. Given these limitations, this is the first study to estimate the relationship between AF burden and serum markers of collagen type-i synthesis and degradation, demonstrating a potential role for these markers in AF evaluation. Conclusions Serum markers of collagen type-i turnover may provide a noninvasive method of documenting and monitoring both the extent and the mechanisms of myocardial fibrosis in AF patients and of evaluating pharmacological measures designed to treat this arrhythmia. However, further investigation and randomized trials are needed to elucidate the exact role of fibrosis in AF and to evaluate the clinical importance and value of biochemical monitoring of collagen turnover in this clinical setting. Although cardiac biopsy is the gold standard for documenting and monitoring myocardial fibrosis, noninvasive methods offer a particularly attractive alternative that could have a broader application. Reprint requests and correspondence: Dr. Panos E. Vardas, Department of Cardiology, Heraklion University Hospital, 71100, Heraklion, Crete, Greece. cardio@med.uoc.gr. REFERENCES 1. Frustaci A, Chimenti C, Bellocci F, Morgante E, Russo MA, Maseri A. Histological substrate of atrial biopsies in patients with lone atrial fibrillation. Circulation 1997;96: Boldt A, Wetzel U, Lauschke J, et al. Fibrosis in left atrial tissue of patients with atrial fibrillation with and without underlying mitral valve disease. Heart 2004;90: Bishop JE. Regulation of cardiovascular collagen deposition by mechanical forces. Mol Med Today 1998;4: López B, González A, Varo N, Laviades C, Querejeta R, Díez J. Biochemical assessment of myocardial fibrosis in hypertensive heart disease. Hypertension 2001;38: Laviades C, Varo N, Fernandez J, et al. Abnormalities of the extracellular degradation of collagen type-i in essential hypertension. Circulation 1998;8: Lindsay MM, Maxwell P, Dunn FG. TIMP-1: a marker of left ventricular diastolic dysfunction and fibrosis in hypertension. Hypertension 2002;40: Lijnen P, Petrov V, Fagard R. Induction of cardiac fibrosis by transforming growth factor-b1. Mol Genet Metab 2000;71: Li YY, McTiernan CF, Feldman AM. Interplay of matrix metalloproteinases, tissue inhibitors of metalloproteinases and their regulators in cardiac matrix remodeling. Cardiovasc Res 2000;46: Wijffels MC, Kirchhof CJ, Dorland R, Allessie MA. Atrial fibrillation begets atrial fibrillation. A study in awake chronically instrumented goats. Circulation 1995;92: Allessie M, Ausma J, Schotten U. Electrical, contractile and structural remodeling during atrial fibrillation. Cardiovasc Res 2002;54: Yu WC, Lee SH, Tai CT, et al. Reversal of atrial electrical remodeling following cardioversion of long-standing atrial fibrillation in man. Cardiovasc Res 1999;42: Kostin S, Klein G, Szalay Z, Hein S, Bauer E, Schaper J. Structural correlate of atrial fibrillation in human patients. Cardiovasc Res 2002;54: Janse MJ. Why does atrial fibrillation occur? Eur Heart J 1997; 18: Nagatomo Y, Carabello BA, Coker ML, et al. Differential effects of pressure or volume overload on myocardial MMP levels and inhibitory control. Am J Physiol 2000;278:H Key Words: atrial fibrillation y collagen y fibrosis.