Europace Advance Access published August 20, 2014
CLINICAL RESEARCH
Europace doi:10.1093/europace/eut401
Sinus rhythm restoration affects collagen turnover in patients with persistent atrial fibrillation Eleftherios M. Kallergis 1*, Christos A. Goudis 1, Emmanuel M. Kanoupakis 1, Hercules E. Mavrakis 1, Niki E. Maliaraki 2, Nikos Tzanakis 3, and Panos E. Vardas 1
Received 11 September 2013; accepted after revision 22 November 2013
Aims
Collagen turnover and atrial fibrosis have been implicated in the generation and perpetuation of atrial fibrillation (AF). We evaluated the importance of serum markers of collagen turnover in predicting the outcome of electrical cardioversion (CV) of persistent AF and the relationship between AF and fibrosis. ..................................................................................................................................................................................... Methods Serum C-terminal pro-peptide of collagen type-I (CICP) and C-terminal telopeptide of collagen type-I (CITP) were meaand results sured in 164 patients with AF before and 2 months after CV. All the patients were successfully cardioverted to sinus rhythm (SR) although in 38 of them AF recurred. Baseline CICP levels were comparable in patients in SR 60 days after CV and in those who experienced a relapse of AF (85.08 + 16.99 vs. 87.55 + 10.43 ng/mL, respectively, P ¼ ns). Baseline CITP levels were significantly higher in patients with AF recurrence compared with those who remained in SR (0.48 + 0.16 vs. 0.32 + 0.17 ng/mL, respectively, P , 0.0001). In the 126 patients who maintained the SR, CICP levels were significantly lower at the end of the study as compared with the baseline (63.74 + 15.92 vs. 85.08 + 16.99 ng/mL P ¼ 0.003), while there was a mild increase in plasma CITP levels (0.36 + 0.21 vs. 0.32 + 0.17 ng/mL, respectively, P ¼ 0.03). ..................................................................................................................................................................................... Conclusion Atrial fibrillation can result in alterations in atrial structure and architecture that make the atrial myocardium more susceptible to the maintenance of the arrhythmia. Sinus rhythm restoration could affect the fibrotic process occurring or exacerbating during AF course.
----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords
Atrial fibrillation † Collagen † Fibrosis † Cardioversion † Recurrence
Introduction Atrial fibrillation (AF) has a progressive nature.1 – 4 It has been increasingly recognized that there is a tendency for AF to become sustained over time and more resistant to cardioversion (CV). 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 remodelling.1 – 4 In fact, both electrophysiological alterations and anatomical substrates are critical for AF generation, maintenance, and perpetuation.1 – 4 However, pure electrical remodelling could not explain the development of sustained AF.4 – 7 Since AF-induced effective refractory period alterations recover completely before AF become sustained, other factors must be involved in the perpetuation of the arrhythmia,
suggesting that structural, rather than ionic, remodelling may be the primary contributor to AF stabilization.3 – 7 Atrial fibrosis, a factor with a slower timecourse, could be involved in stabilizing AF.8 – 10 Collagens are the major extracellular matrix (ECM) proteins in the heart.11 Of the five different collagen isoforms found in the myocardium, fibrillar collagen type I is the major collagenous product of cardiac fibroblasts; representing 80% of the total cardiac collagen content.11 We have recently found that the serum levels of C-terminal propeptide of collagen type-I (CICP) and C-terminal telopeptide of collagen type-I (CITP), markers of collagen type I synthesis and degradation, respectively, were significantly higher in patients with AF than in the control subjects and there was a general tendency for higher levels of these indices in patients with persistent rather than paroxysmal AF.12
* Corresponding author. Tel: +30 2810392706; fax: +30 2810542055, E-mail: [email protected] & Crown copyright 2014.
Downloaded from http://europace.oxfordjournals.org/ at Columbia University Libraries on August 21, 2014
1 Department of Cardiology, University Hospital of Heraklion, 71100 Heraklion, Crete, Greece; 2Laboratory of Biochemistry, University Hospital of Heraklion, 71100 Heraklion, Crete, Greece; and 3Biostatistics Laboratory, University Hospital of Heraklion, 71100 Heraklion, Crete, Greece
Page 2 of 5
What’s new? † Atrial fibrillation (AF) per se may cause extracellular matrix (ECM) remodelling or may exaggerate ECM remodelling due to the underlying heart diseases. † The development of non-invasive methods to indicate the presence and monitor the process of myocardial fibrosis in AF and in other clinical conditions as well is particularly attractive and could have a broader application.
Methods Patient selection The Ethics Committee of our Institution approved the study. This investigation conforms to the principles outlined in the Declaration of Helsinki. Signed informed written consent was obtained from all the subjects before their participation in the study. The study population consisted of 166 ambulatory patients who suffered from their first episode of persistent, lone AF, defined as AF without clinical or echocardiographic evidence of cardiopulmonary disease, including hypertension, and had been referred to our department for CV. The arrhythmia was considered persistent when it was documented on sequential 12-lead electrocardiograms, without any intervening periods of sinus rhythm (SR). All the patients underwent another electrocardiogram immediately before electrical CV to confirm the persistence of AF. The subjects were excluded if they had any history of coronary artery disease, significant valvular disease, cardiomyopathy, or hyperthyroidism that preceded the history of AF. Conditions associated with elevated serum concentrations of myocardial or tissue fibrosis markers were also excluded. Thus, subjects with chronic liver disease or hepatic dysfunction, renal impairment, pulmonary fibrosis, extensive wounds, metabolic bone disease, malignancy, connective tissue disorders, chronic obstructive pulmonary disease, chronic inflammatory disease, and recent infection or surgery, were not allowed to participate in the study. Patients who were over 75 years old or had a pacemaker/implantable cardioverter-defibrillator were also excluded.
Study design and data collection All the subjects underwent a comprehensive clinical evaluation, which included medical history, physical examination, routine laboratory checks, thyroid function tests, and chest X-ray, on their entry into the study. Maximal left atrial (LA) diameter and left ventricular ejection fraction (LVEF) were also assessed in all the participants by two-dimensional transthoracic echocardiography. Since our purpose was to investigate whether the baseline CICP and CITP levels could predict AF recurrence and to evaluate the timecourse of these markers after CV, blood samples for the CICP and the CITP measurement were drawn immediately prior to sedation for CV and at the end of the 2-month follow-up period.
Blood samples and laboratory assays Routine biochemical measurements were taken and analysed in the standard fashion. Blood samples to determine the serum concentration of the markers of collagen type I turnover were obtained from all the patients at the time of the clinical studies by direct puncture of an antecubital vein after 30 min of supine rest. The blood samples were immediately placed on ice and centrifuged within 1 h. The specimens were stored at 2808C until analysis. The samples were not thawed and refrozen. Measurements were performed by personnel blinded to the patient’s clinical details. Serum CICP levels were determined by a sandwich enzyme immunoassay with a commercially available kit (Metra,TM CICP, EIA), while the CITP was measured by using the Elecsys b-CrossLaps/serum assay which is specific for cross-linked isomerized type I collagen fragments. The intra- and the inter-assay coefficients of the variation of all the assays were ,8 and ,10%, respectively, in our laboratory.
Cardioversion protocol Electrical conversion to the SR was attempted after patients with AF were properly anticoagulated by oral treatment with acenocoumarol that resulted in an international normalized ratio between 2.0 and 3.0 for at least 4 weeks. Anticoagulation had to be continued for at least 1 month after the procedure, regardless of its outcome. Cardioversion was performed in the electrophysiology laboratory under short-lasting general anaesthesia with intravenous propofol, by using a biphasic defibrillator with single synchronized shock energy of 200 J in the anteroposterior paddle position. The electrocardiogram was monitored continuously throughout the procedure and the recordings were stored on an optical disc (EP Lab, Quinton, Inc.).
Statistical analysis Summary descriptive data are presented as mean (+SD) or counts (%), as appropriate. Categorical variables were compared between the groups with Fisher’s exact or x2 tests, as appropriate. Continuous variables were compared between the relapsed and the non-relapsed groups with the t-test or Mann– Whitney U test, while changes within the groups were assessed by paired t-test. Stepwise logistic regression was used to determine independent predictors for relapse. The thresholds for entry into and removal from the model were 5 and 10%, respectively. All the tests were performed at the 5% level of significance. SPPS 18 was used for statistical analysis.
Results Patients The patients included 107 men and 57 women, aged between 24 and 75 years, who suffered from persistent, lone AF, lasting more than 3 months, with no known underlying disease. All the subjects had normal LVEF, as assessed by two-dimensional echocardiography. Diltiazem and beta-blockers were allowed for ventricular rate control and all the AF patients received the indicated antithrombotic treatment according to the international guidelines.
Clinical outcome and serum markers of extracellular collagen type I turnover Of the 166 patients with persistent AF who participated in the study all were successfully cardioverted to SR, but the arrhythmia recurred immediately in two of them who were excluded from any further analysis. Atrial fibrillation recurred within the study period in 38
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Since a relationship between the serum markers of collagen turnover and AF has been suggested, we planned to evaluate the importance of the baseline CICP and CITP levels in predicting the outcome of electrical CV in patients with persistent AF. Moreover, to clarify the relationship between AF and fibrosis, we evaluated the timecourse of these markers after CV.
E.M. Kallergis et al.
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SR restoration affects collagen turnover in patients with AF
Table 1 The baseline characteristics and the serum markers of collagen type I turnover in patients with and without AF recurrence after CV
120
150
P < 0.0001 NS
P = 0.003
P = 0.001
P value
Age (year) Sex (male/female) LVEF (%)
61.4 + 9.7
59.4 + 10.6
NS
84/42 55.1 + 4.1
NS ,0.001
41.5 + 3.0
45.4 + 2.5
,0.001
BMI (kg/m ) SBP (mmHg)
26.8 + 1.7 134.1 + 9.7
27.0 + 1.8 139.0 + 9.3
NS 0.007
DBP (mmHg)
84.5 + 4.3
85.0 + 4.4
NS
CICP (ng/mL) CITP (ng/mL)
85.1 + 17.0 0.32 + 0.18
87.6 + 10.4 0.49 + 0.16
NS ,0.0001
LA (mm) 2
AF, atrial fibrillation; BMI, body mass index; CICP, C-terminal pro-peptide of collagen type-I; CITP, C-terminal telopeptide of collagen type-I; DBP, diastolic blood pressure; LA, left atrial diameter; LVEF, left ventricular ejection fraction; and SBP, systolic blood pressure.
additional patients (total recurrence rate 24.09%). All the patients tolerated the procedures well without experiencing any complication during follow-up. The clinical characteristics of the patients who experienced AF relapse compared with those who remained in the SR are listed in Table 1. Age, gender, body mass index, and diastolic blood pressures were similar in these two groups. Besides, patients with AF recurrence during the follow-up period had greater LA diameters and lower ejection fraction (EF) than those who maintained the SR (45.4 + 2.5 vs. 41.5 + 3 mm, P , 0.001, and 55.1 + 4.1 vs. 59.3 + 5.7, P , 0.001, respectively). In the AF patients, baseline CICP levels, determined prior to the CV, were comparable in patients still in the SR 60 days after the CV and in those who experienced a relapse of AF (85.08 + 16.99 vs. 87.55 + 10.43 ng/mL, respectively, P ¼ ns), (Figure 1). However, patients with AF recurrence had significantly higher baseline CITP levels compared with those who remained in the SR after the CV (0.49 + 0.16 vs. 0.32 + 0.18 ng/mL, respectively, P , 0.0001; Figure 2). The restoration and the maintenance of the SR resulted in a decrease of the CICP but not of the CITP levels. In the 126 patients who were successfully cardioverted and maintained the SR, the plasma CICP levels were significantly lower at the end of the study as compared with the baseline (63.74 + 15.92 vs. 85.08 + 16.99 ng/mL P ¼ 0.003; Figure 1), while they had mild higher values of the CITP on final compared with the initial evaluation, (0.36 + 0.21 vs. 0.32 + 0.17 ng/mL, respectively, P ¼ 0.03; Figure 2). In addition, in patients with AF recurrence both the CICP and the CITP levels decreased. Moreover, in all the patients taken together, we found that there was a positive correlation between the pre-CV CITP levels and the LA dimension (r ¼ 0.268, P ¼ 0.008), while there was a reverse relationship between the CITP and the LVEF (r ¼ 20.222, P ¼ 0.028;
80
60
30 60 0
Pre Post Relapsed
Pre
Post Non-relapsed
0
Figure 1 C-terminal pro-peptide of collagen type I (CICP) for relapsed and non-relapsed patients. In AF patients, baseline CICP levels, determined prior to the CV, were comparable in patients still in the SR 60 days after the CV and in those who experienced a relapse of the AF. In the 126 patients who were successfully cardioverted and maintained the SR, the plasma CICP levels were significantly lower at the end of the study as compared with the baseline.
1.2
NS P = 0.001
P = 0.03
P = 0.003
0.9
0.6
0.3
0.0
Pre
Post Relapsed
Pre
Post Non-relapsed
Figure 2 C-terminal telopeptide of collagen type I (CITP) for relapsed and non-relapsed patients. Patients with the AF recurrence had significantly higher baseline CITP levels compared with those who remained in the SR after the CV, while those who remained in the SR by the end of the follow-up period had mild higher values of CITP on final compared with the initial evaluation. Figure 3). In addition, the LA dimension was reversed and EF was positively correlated with the CITP alterations (dCITP) during the study period. These data could suggest that the AF patients with larger LA sizes, probably as a result of longer duration of the arrhythmia, may have augmented collagen degradation due to the increased fibrotic process.
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23/15 59.3 + 5.7
90
CICP (ng/mL)
................................................................................
120
100 CICP (ng/mL)
Recurrence (38 patients)
CITP (ng/mL)
No recurrence (126 patients)
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E.M. Kallergis et al.
1.0
1.0 r = 0.222 P = 0.028
r = 0.268 P = 0.008 CITP (pre) (ng/mL)
CITP (pre) (ng/mL)
0.8 0.6 0.4 0.2
0.8 0.6 0.4 0.2
0.0
0.0 30
35
50
55
40
50
60 LVEF (%)
70
80
Figure 3 Linear regression line between the baseline CITP levels, LA (mm), and LVEF (%). Closed circles represent relapsed cases. There was a positive correlation between the pre-CV CITP levels and the LA dimension (r ¼ 0.268, P ¼ 0.008), while there was a reverse relationship between the CITP and the LVEF (r ¼ 20.222, P ¼ 0.028).
Stepwise logistic regression analysis revealed that the baseline LA (b ¼ 0.386, P , 0.001) followed by CITP (b ¼ 3.251, P ¼ 0.032) were independent predictors for AF recurrence within 2 months after CV to SR.
Discussion In a population with persistent, lone AF we found that successful CV of AF led to a significant decrease in the plasma CICP but not the CITP levels. Besides, the CITP levels were a predictor of arrhythmia recurrence during the follow-up period. In a recent study, we found that the serum levels of the CICP and the CITP, markers of collagen type I synthesis and degradation, respectively, were significantly higher in patients with AF than in the control subjects and there was a general tendency for higher levels of these indices in patients with persistent rather than paroxysmal AF.12 In the present study, we assessed the amount of fibrosis in the study population by using serum analysis of peptides derived from the tissue synthesis and the degradation of fibrillar collagen type I. According to our results, the SR restoration is associated with the inhibition of collagen synthesis (CICP) while it does not affect the collagen degradation (CITP) which is elevated in the setting of persistent AF. In addition, since the baseline CITP levels were associated with the AF recurrence we could speculate that the collagen type I degradation could play an important role in AF persistence. Furthermore, this finding supports further the hypothesis that the circulating levels of a collagen degradation marker could be associated with AF maintenance. In some cases, increased collagen synthesis over degradation occurs while in others degradation predominates over synthesis. These two different processes could coexist at variable degrees within the same myocardium depending on the disease course. This study is the first to document that the maintenance of the SR was associated with a decrease of the rate of collagen synthesis during the post-CV period, while the AF recurrence had a different effect. In addition, since the restoration of the SR slightly increased the rate of
collagen degradation, we could assume a decrease of fibrosis at least at the first period following the CV. These changes of the CICP and the CITP, as observed in our study, probably reflect the recovery of the atrial haemodynamics from both mechanical and biochemical remodelling. Indeed, during the AF the rapid asynchronous atrial contractions and the decline in the cardiac output contribute to an increased mean atrial pressure and atrial dilatation. The increasing compliance of the fibrillating atrium and the mechanical atrial stretch induce the activation of numerous signalling pathways leading to fibroblast proliferation, collagen synthesis, and tissue fibrosis, along with increased angiotensin II and transforming growth factor-beta 1 expression in cardiac fibroblasts, contributing to the domestication of AF.3,13,14 Both adverse atrial haemodynamics and biochemical changes should return to the previous baseline following reversion to the SR resulting in the elimination of the factors that trigger interstitial fibrotic processes. Several experimental models and human studies demonstrated that atrial fibrosis and inflammation could be the substrate that promotes and maintains AF.2,9,13,15,16 Indeed, augmented interstitial fibrosis could modify the pattern of myocyte apposition, rearranging the atrial myocyte connections and altering the cell-to-cell interaction and communication. The combination of the normal and the diseased atrial fibres in conjunction with the local fibrosis could result in the spatial dispersion of the atrial refractoriness and could cause inhomogeneous localized conduction abnormalities, including intra-atrial conduction block and slow conduction.2,9,13,16 Moreover, fibrosis of the atrial tissue could facilitate the development of ectopic pacemakers and late potentials as a result of inhomogeneous stimulus conduction, and could also lead to fluctuations in the membrane potential.2,9,13,16 Thus, atrial fibrosis can change a homogeneously activated atrium into a discontinuous and fragmented architecture. Consequently, augmented interstitial fibrosis may increase atrial susceptibility to the AF, since it is known that atrial reentrant arrhythmias are facilitated by impairment of the atrial conduction and by the presence of adjacent atrial regions with disparate refractory periods.13,16 Furthermore, according to the dominant concept of the AF, it results
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40 45 LA (mm)
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SR restoration affects collagen turnover in patients with AF
Conclusion Although AF has been considered an electrical disease, it can result in alterations in the atrial structure and the architecture that make the atrial myocardium more susceptible to the maintenance of the arrhythmia. Sinus rhythm restoration could affect the fibrotic process occurring or exacerbating during the AF course. However, further investigation and randomized trials are needed to elucidate the exact role of the fibrosis in the AF and to evaluate the clinical importance and the value of the biochemical monitoring of the collagen turnover in this clinical setting. Conflict of interest: none declared.
References 1. Wijffels MC, Kirchhof CJ, Dorland R, Allessie MA. Atrial fibrillation begets atrial fibrillation. A study in awake chronically instrumented goats. Circulation 1995;92: 1954 –68. 2. Allessie MA, Boyden PA, Camm AJ, Kle´ber AG, Lab MJ, Legato MJ et al. Pathophysiology and prevention of atrial fibrillation. Circulation 2001;103:769–77 14. 3. Burstein B, Nattel S. Atrial fibrosis: mechanisms and clinical relevance in atrial fibrillation. J Am Coll Cardiol 2008;51:802–9. 4. Allessie M, Ausma J, Schotten U. Electrical, contractile and structural remodeling during atrial fibrillation. Cardiovasc Res 2002;54:230 –46. 5. Yu WC, Lee SH, Tai CT, Tsai CF, Hsieh MH, Chen CC et al. Reversal of atrial electrical remodeling following cardioversion of long-standing atrial fibrillation in man. Cardiovasc Res 1999;42:470 – 6. 6. Manning WJ, Silverman DI, Katz SE, Riley MF, Come PC, Doherty RM et al. Impaired left atrial mechanical function after cardioversion: relation to the duration of atrial fibrillation. J Am Coll Cardiol 1994;23:1535 –40. 7. Manning WJ, Silverman DI, Katz SE, Riley MF, Doherty RM, Munson JT et al. Temporal dependence of the return of atrial mechanical function on the mode of cardioversion of atrial fibrillation to sinus rhythm. Am J Cardiol 1995;75:624 – 6. 8. Bishop JE. Regulation of cardiovascular collagen deposition by mechanical forces. Mol Med Today 1998;4:69 –75. 9. Kostin S, Klein G, Szalay Z, Hein S, Bauer E, Schaper J. Structural correlate of atrial fibrillation in human patients. Cardiovasc Res 2002;54:361 –79. 10. Janse MJ. Why does atrial fibrillation occur? Eur Heart J 1997;18:12 –8. 11. Bashey RI, Donnelly M, Insinga F, Jimenez SA. Growth properties and biochemical characterization of collagens synthesized by adult rat heart fibroblasts in culture. J Mol Cell Cardiol 1992;24:691 –700. 12. Kallergis EM, Manios EG, Kanoupakis EM, Mavrakis HE, Arfanakis DA, Maliaraki NE et al. Extracellular matrix alterations in patients with paroxysmal and persistent atrial fibrillation: biochemical assessment of collagen type-I turnover. J Am Coll Cardiol 2008;52:211 –5. 13. Schotten U, Neuberger HR, Allessie MA. The role of atrial dilatation in the domestication of atrial fibrillation. Prog Biophys Mol Biol 2003;82:151–62. 14. Kiryu M, Niwano S, Niwano H, Kishihara J, Aoyama Y, Fukaya H et al. Angiotensin II-mediated up-regulation of connective tissue growth factor promotes atrial tissue fibrosis in the canine atrial fibrillation model. Europace 2012;14:1206 –14. 15. Smit MD, Maass AH, De Jong AM, Muller Kobold AC, Van Veldhuisen DJ, Van Gelder IC. Role of inflammation in early atrial fibrillation recurrence. Europace 2012;14:810 –7. 16. Lammers WJEP, Schalij MJ, Kirchhof CJHJ, Allessie MA. Quantification of spatial inhomogeneity in conduction and initiation of reentrant atrial arrhythmias. Am J Physiol 1990;259:H1254 –63. 17. King JH, Huang CL, Fraser JA. Determinants of myocardial conduction velocity: implications for arrhythmogenesis. Front Physiol 2013;4:154.
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from multiple reentrant circuits of various size and conduction velocities that propagate randomly through the atria and can be initiated by triggers, such as premature atrial contractions arising from focally discharging cells located most commonly in the myocardial sleeves extending from the LA into the pulmonary veins.1,2,4,16,17 The ability of the single extrastimuli to initiate the AF has been associated with the existence of a susceptible underlying substrate, which is characterized by local inhomogeneities in conduction and refractoriness.1,2,4,16,17 Changes in the ECM composition may underlie these electrophysiological alterations, facilitating the induction of the AF by the appropriate triggers. Taken together, these recently published data and the results of the present study could suggest that AF per se may cause ECM remodelling or may exaggerate ECM remodelling due to the underlying heart diseases. Indeed, since our only intervention was the successful CV, we assume that the fibrotic processes are at least exacerbated by the persistence of the AF. It is thus possible that the AF is associated with atrial structural changes that may provoke fibrosis or may have a fibrotic basis. Hence, the AF may induce or be aggravated by fibrosis, which in turn may promote the persistence of the AF. It is therefore likely that in the AF a vicious circle is set in motion, whereby the arrhythmia causes atrial structural remodelling that triggers a low-grade fibrotic response, which in its turn aggravates the AF, and so on. Some limitations of the present study should be taken into consideration. Although the serum markers of collagen turnover are not exclusively heart-specific, we made strenuous efforts to exclude subjects with certain conditions associated with fibrosis, such as hepatic dysfunction, renal impairment, and pulmonary fibrosis. We did not take the duration of the AF into consideration in our analysis because of the lack of reliable data on when the arrhythmia first occurred. Although its duration was more than 3 months in all our patients, the data concerning the AF duration previous to the 3 month point were unavailable in certain of them. In addition, drugs taken by the patients could have affected the serum concentrations of the evaluated markers. In any case, this is the first study to accept these limitations and report that the SR restoration and the maintenance are associated with an attenuation of the fibrotic processes existing during persistent AF, at least at the initial phases of the arrhythmia, demonstrating a potential role for the serum markers of collagen turnover in AF evaluation. The potential for reversing the fibrosis is of particular clinical interest especially when the rhythm control strategies are evaluated as the first choice treatment of AF. In addition, the development of non-invasive methods to indicate the presence and monitor the process of myocardial fibrosis in the AF and in other clinical conditions as well is particularly attractive and could have a broader application. Atrial fibrillation per se may cause ECM remodelling or may exaggerate ECM remodelling due to the underlying heart diseases.
CLINICAL RESEARCH
Europace doi:10.1093/europace/eut401
Sinus rhythm restoration affects collagen turnover in patients with persistent atrial fibrillation Eleftherios M. Kallergis 1*, Christos A. Goudis 1, Emmanuel M. Kanoupakis 1, Hercules E. Mavrakis 1, Niki E. Maliaraki 2, Nikos Tzanakis 3, and Panos E. Vardas 1
Received 11 September 2013; accepted after revision 22 November 2013
Aims
Collagen turnover and atrial fibrosis have been implicated in the generation and perpetuation of atrial fibrillation (AF). We evaluated the importance of serum markers of collagen turnover in predicting the outcome of electrical cardioversion (CV) of persistent AF and the relationship between AF and fibrosis. ..................................................................................................................................................................................... Methods Serum C-terminal pro-peptide of collagen type-I (CICP) and C-terminal telopeptide of collagen type-I (CITP) were meaand results sured in 164 patients with AF before and 2 months after CV. All the patients were successfully cardioverted to sinus rhythm (SR) although in 38 of them AF recurred. Baseline CICP levels were comparable in patients in SR 60 days after CV and in those who experienced a relapse of AF (85.08 + 16.99 vs. 87.55 + 10.43 ng/mL, respectively, P ¼ ns). Baseline CITP levels were significantly higher in patients with AF recurrence compared with those who remained in SR (0.48 + 0.16 vs. 0.32 + 0.17 ng/mL, respectively, P , 0.0001). In the 126 patients who maintained the SR, CICP levels were significantly lower at the end of the study as compared with the baseline (63.74 + 15.92 vs. 85.08 + 16.99 ng/mL P ¼ 0.003), while there was a mild increase in plasma CITP levels (0.36 + 0.21 vs. 0.32 + 0.17 ng/mL, respectively, P ¼ 0.03). ..................................................................................................................................................................................... Conclusion Atrial fibrillation can result in alterations in atrial structure and architecture that make the atrial myocardium more susceptible to the maintenance of the arrhythmia. Sinus rhythm restoration could affect the fibrotic process occurring or exacerbating during AF course.
----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords
Atrial fibrillation † Collagen † Fibrosis † Cardioversion † Recurrence
Introduction Atrial fibrillation (AF) has a progressive nature.1 – 4 It has been increasingly recognized that there is a tendency for AF to become sustained over time and more resistant to cardioversion (CV). 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 remodelling.1 – 4 In fact, both electrophysiological alterations and anatomical substrates are critical for AF generation, maintenance, and perpetuation.1 – 4 However, pure electrical remodelling could not explain the development of sustained AF.4 – 7 Since AF-induced effective refractory period alterations recover completely before AF become sustained, other factors must be involved in the perpetuation of the arrhythmia,
suggesting that structural, rather than ionic, remodelling may be the primary contributor to AF stabilization.3 – 7 Atrial fibrosis, a factor with a slower timecourse, could be involved in stabilizing AF.8 – 10 Collagens are the major extracellular matrix (ECM) proteins in the heart.11 Of the five different collagen isoforms found in the myocardium, fibrillar collagen type I is the major collagenous product of cardiac fibroblasts; representing 80% of the total cardiac collagen content.11 We have recently found that the serum levels of C-terminal propeptide of collagen type-I (CICP) and C-terminal telopeptide of collagen type-I (CITP), markers of collagen type I synthesis and degradation, respectively, were significantly higher in patients with AF than in the control subjects and there was a general tendency for higher levels of these indices in patients with persistent rather than paroxysmal AF.12
* Corresponding author. Tel: +30 2810392706; fax: +30 2810542055, E-mail: [email protected] & Crown copyright 2014.
Downloaded from http://europace.oxfordjournals.org/ at Columbia University Libraries on August 21, 2014
1 Department of Cardiology, University Hospital of Heraklion, 71100 Heraklion, Crete, Greece; 2Laboratory of Biochemistry, University Hospital of Heraklion, 71100 Heraklion, Crete, Greece; and 3Biostatistics Laboratory, University Hospital of Heraklion, 71100 Heraklion, Crete, Greece
Page 2 of 5
What’s new? † Atrial fibrillation (AF) per se may cause extracellular matrix (ECM) remodelling or may exaggerate ECM remodelling due to the underlying heart diseases. † The development of non-invasive methods to indicate the presence and monitor the process of myocardial fibrosis in AF and in other clinical conditions as well is particularly attractive and could have a broader application.
Methods Patient selection The Ethics Committee of our Institution approved the study. This investigation conforms to the principles outlined in the Declaration of Helsinki. Signed informed written consent was obtained from all the subjects before their participation in the study. The study population consisted of 166 ambulatory patients who suffered from their first episode of persistent, lone AF, defined as AF without clinical or echocardiographic evidence of cardiopulmonary disease, including hypertension, and had been referred to our department for CV. The arrhythmia was considered persistent when it was documented on sequential 12-lead electrocardiograms, without any intervening periods of sinus rhythm (SR). All the patients underwent another electrocardiogram immediately before electrical CV to confirm the persistence of AF. The subjects were excluded if they had any history of coronary artery disease, significant valvular disease, cardiomyopathy, or hyperthyroidism that preceded the history of AF. Conditions associated with elevated serum concentrations of myocardial or tissue fibrosis markers were also excluded. Thus, subjects with chronic liver disease or hepatic dysfunction, renal impairment, pulmonary fibrosis, extensive wounds, metabolic bone disease, malignancy, connective tissue disorders, chronic obstructive pulmonary disease, chronic inflammatory disease, and recent infection or surgery, were not allowed to participate in the study. Patients who were over 75 years old or had a pacemaker/implantable cardioverter-defibrillator were also excluded.
Study design and data collection All the subjects underwent a comprehensive clinical evaluation, which included medical history, physical examination, routine laboratory checks, thyroid function tests, and chest X-ray, on their entry into the study. Maximal left atrial (LA) diameter and left ventricular ejection fraction (LVEF) were also assessed in all the participants by two-dimensional transthoracic echocardiography. Since our purpose was to investigate whether the baseline CICP and CITP levels could predict AF recurrence and to evaluate the timecourse of these markers after CV, blood samples for the CICP and the CITP measurement were drawn immediately prior to sedation for CV and at the end of the 2-month follow-up period.
Blood samples and laboratory assays Routine biochemical measurements were taken and analysed in the standard fashion. Blood samples to determine the serum concentration of the markers of collagen type I turnover were obtained from all the patients at the time of the clinical studies by direct puncture of an antecubital vein after 30 min of supine rest. The blood samples were immediately placed on ice and centrifuged within 1 h. The specimens were stored at 2808C until analysis. The samples were not thawed and refrozen. Measurements were performed by personnel blinded to the patient’s clinical details. Serum CICP levels were determined by a sandwich enzyme immunoassay with a commercially available kit (Metra,TM CICP, EIA), while the CITP was measured by using the Elecsys b-CrossLaps/serum assay which is specific for cross-linked isomerized type I collagen fragments. The intra- and the inter-assay coefficients of the variation of all the assays were ,8 and ,10%, respectively, in our laboratory.
Cardioversion protocol Electrical conversion to the SR was attempted after patients with AF were properly anticoagulated by oral treatment with acenocoumarol that resulted in an international normalized ratio between 2.0 and 3.0 for at least 4 weeks. Anticoagulation had to be continued for at least 1 month after the procedure, regardless of its outcome. Cardioversion was performed in the electrophysiology laboratory under short-lasting general anaesthesia with intravenous propofol, by using a biphasic defibrillator with single synchronized shock energy of 200 J in the anteroposterior paddle position. The electrocardiogram was monitored continuously throughout the procedure and the recordings were stored on an optical disc (EP Lab, Quinton, Inc.).
Statistical analysis Summary descriptive data are presented as mean (+SD) or counts (%), as appropriate. Categorical variables were compared between the groups with Fisher’s exact or x2 tests, as appropriate. Continuous variables were compared between the relapsed and the non-relapsed groups with the t-test or Mann– Whitney U test, while changes within the groups were assessed by paired t-test. Stepwise logistic regression was used to determine independent predictors for relapse. The thresholds for entry into and removal from the model were 5 and 10%, respectively. All the tests were performed at the 5% level of significance. SPPS 18 was used for statistical analysis.
Results Patients The patients included 107 men and 57 women, aged between 24 and 75 years, who suffered from persistent, lone AF, lasting more than 3 months, with no known underlying disease. All the subjects had normal LVEF, as assessed by two-dimensional echocardiography. Diltiazem and beta-blockers were allowed for ventricular rate control and all the AF patients received the indicated antithrombotic treatment according to the international guidelines.
Clinical outcome and serum markers of extracellular collagen type I turnover Of the 166 patients with persistent AF who participated in the study all were successfully cardioverted to SR, but the arrhythmia recurred immediately in two of them who were excluded from any further analysis. Atrial fibrillation recurred within the study period in 38
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Since a relationship between the serum markers of collagen turnover and AF has been suggested, we planned to evaluate the importance of the baseline CICP and CITP levels in predicting the outcome of electrical CV in patients with persistent AF. Moreover, to clarify the relationship between AF and fibrosis, we evaluated the timecourse of these markers after CV.
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SR restoration affects collagen turnover in patients with AF
Table 1 The baseline characteristics and the serum markers of collagen type I turnover in patients with and without AF recurrence after CV
120
150
P < 0.0001 NS
P = 0.003
P = 0.001
P value
Age (year) Sex (male/female) LVEF (%)
61.4 + 9.7
59.4 + 10.6
NS
84/42 55.1 + 4.1
NS ,0.001
41.5 + 3.0
45.4 + 2.5
,0.001
BMI (kg/m ) SBP (mmHg)
26.8 + 1.7 134.1 + 9.7
27.0 + 1.8 139.0 + 9.3
NS 0.007
DBP (mmHg)
84.5 + 4.3
85.0 + 4.4
NS
CICP (ng/mL) CITP (ng/mL)
85.1 + 17.0 0.32 + 0.18
87.6 + 10.4 0.49 + 0.16
NS ,0.0001
LA (mm) 2
AF, atrial fibrillation; BMI, body mass index; CICP, C-terminal pro-peptide of collagen type-I; CITP, C-terminal telopeptide of collagen type-I; DBP, diastolic blood pressure; LA, left atrial diameter; LVEF, left ventricular ejection fraction; and SBP, systolic blood pressure.
additional patients (total recurrence rate 24.09%). All the patients tolerated the procedures well without experiencing any complication during follow-up. The clinical characteristics of the patients who experienced AF relapse compared with those who remained in the SR are listed in Table 1. Age, gender, body mass index, and diastolic blood pressures were similar in these two groups. Besides, patients with AF recurrence during the follow-up period had greater LA diameters and lower ejection fraction (EF) than those who maintained the SR (45.4 + 2.5 vs. 41.5 + 3 mm, P , 0.001, and 55.1 + 4.1 vs. 59.3 + 5.7, P , 0.001, respectively). In the AF patients, baseline CICP levels, determined prior to the CV, were comparable in patients still in the SR 60 days after the CV and in those who experienced a relapse of AF (85.08 + 16.99 vs. 87.55 + 10.43 ng/mL, respectively, P ¼ ns), (Figure 1). However, patients with AF recurrence had significantly higher baseline CITP levels compared with those who remained in the SR after the CV (0.49 + 0.16 vs. 0.32 + 0.18 ng/mL, respectively, P , 0.0001; Figure 2). The restoration and the maintenance of the SR resulted in a decrease of the CICP but not of the CITP levels. In the 126 patients who were successfully cardioverted and maintained the SR, the plasma CICP levels were significantly lower at the end of the study as compared with the baseline (63.74 + 15.92 vs. 85.08 + 16.99 ng/mL P ¼ 0.003; Figure 1), while they had mild higher values of the CITP on final compared with the initial evaluation, (0.36 + 0.21 vs. 0.32 + 0.17 ng/mL, respectively, P ¼ 0.03; Figure 2). In addition, in patients with AF recurrence both the CICP and the CITP levels decreased. Moreover, in all the patients taken together, we found that there was a positive correlation between the pre-CV CITP levels and the LA dimension (r ¼ 0.268, P ¼ 0.008), while there was a reverse relationship between the CITP and the LVEF (r ¼ 20.222, P ¼ 0.028;
80
60
30 60 0
Pre Post Relapsed
Pre
Post Non-relapsed
0
Figure 1 C-terminal pro-peptide of collagen type I (CICP) for relapsed and non-relapsed patients. In AF patients, baseline CICP levels, determined prior to the CV, were comparable in patients still in the SR 60 days after the CV and in those who experienced a relapse of the AF. In the 126 patients who were successfully cardioverted and maintained the SR, the plasma CICP levels were significantly lower at the end of the study as compared with the baseline.
1.2
NS P = 0.001
P = 0.03
P = 0.003
0.9
0.6
0.3
0.0
Pre
Post Relapsed
Pre
Post Non-relapsed
Figure 2 C-terminal telopeptide of collagen type I (CITP) for relapsed and non-relapsed patients. Patients with the AF recurrence had significantly higher baseline CITP levels compared with those who remained in the SR after the CV, while those who remained in the SR by the end of the follow-up period had mild higher values of CITP on final compared with the initial evaluation. Figure 3). In addition, the LA dimension was reversed and EF was positively correlated with the CITP alterations (dCITP) during the study period. These data could suggest that the AF patients with larger LA sizes, probably as a result of longer duration of the arrhythmia, may have augmented collagen degradation due to the increased fibrotic process.
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23/15 59.3 + 5.7
90
CICP (ng/mL)
................................................................................
120
100 CICP (ng/mL)
Recurrence (38 patients)
CITP (ng/mL)
No recurrence (126 patients)
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E.M. Kallergis et al.
1.0
1.0 r = 0.222 P = 0.028
r = 0.268 P = 0.008 CITP (pre) (ng/mL)
CITP (pre) (ng/mL)
0.8 0.6 0.4 0.2
0.8 0.6 0.4 0.2
0.0
0.0 30
35
50
55
40
50
60 LVEF (%)
70
80
Figure 3 Linear regression line between the baseline CITP levels, LA (mm), and LVEF (%). Closed circles represent relapsed cases. There was a positive correlation between the pre-CV CITP levels and the LA dimension (r ¼ 0.268, P ¼ 0.008), while there was a reverse relationship between the CITP and the LVEF (r ¼ 20.222, P ¼ 0.028).
Stepwise logistic regression analysis revealed that the baseline LA (b ¼ 0.386, P , 0.001) followed by CITP (b ¼ 3.251, P ¼ 0.032) were independent predictors for AF recurrence within 2 months after CV to SR.
Discussion In a population with persistent, lone AF we found that successful CV of AF led to a significant decrease in the plasma CICP but not the CITP levels. Besides, the CITP levels were a predictor of arrhythmia recurrence during the follow-up period. In a recent study, we found that the serum levels of the CICP and the CITP, markers of collagen type I synthesis and degradation, respectively, were significantly higher in patients with AF than in the control subjects and there was a general tendency for higher levels of these indices in patients with persistent rather than paroxysmal AF.12 In the present study, we assessed the amount of fibrosis in the study population by using serum analysis of peptides derived from the tissue synthesis and the degradation of fibrillar collagen type I. According to our results, the SR restoration is associated with the inhibition of collagen synthesis (CICP) while it does not affect the collagen degradation (CITP) which is elevated in the setting of persistent AF. In addition, since the baseline CITP levels were associated with the AF recurrence we could speculate that the collagen type I degradation could play an important role in AF persistence. Furthermore, this finding supports further the hypothesis that the circulating levels of a collagen degradation marker could be associated with AF maintenance. In some cases, increased collagen synthesis over degradation occurs while in others degradation predominates over synthesis. These two different processes could coexist at variable degrees within the same myocardium depending on the disease course. This study is the first to document that the maintenance of the SR was associated with a decrease of the rate of collagen synthesis during the post-CV period, while the AF recurrence had a different effect. In addition, since the restoration of the SR slightly increased the rate of
collagen degradation, we could assume a decrease of fibrosis at least at the first period following the CV. These changes of the CICP and the CITP, as observed in our study, probably reflect the recovery of the atrial haemodynamics from both mechanical and biochemical remodelling. Indeed, during the AF the rapid asynchronous atrial contractions and the decline in the cardiac output contribute to an increased mean atrial pressure and atrial dilatation. The increasing compliance of the fibrillating atrium and the mechanical atrial stretch induce the activation of numerous signalling pathways leading to fibroblast proliferation, collagen synthesis, and tissue fibrosis, along with increased angiotensin II and transforming growth factor-beta 1 expression in cardiac fibroblasts, contributing to the domestication of AF.3,13,14 Both adverse atrial haemodynamics and biochemical changes should return to the previous baseline following reversion to the SR resulting in the elimination of the factors that trigger interstitial fibrotic processes. Several experimental models and human studies demonstrated that atrial fibrosis and inflammation could be the substrate that promotes and maintains AF.2,9,13,15,16 Indeed, augmented interstitial fibrosis could modify the pattern of myocyte apposition, rearranging the atrial myocyte connections and altering the cell-to-cell interaction and communication. The combination of the normal and the diseased atrial fibres in conjunction with the local fibrosis could result in the spatial dispersion of the atrial refractoriness and could cause inhomogeneous localized conduction abnormalities, including intra-atrial conduction block and slow conduction.2,9,13,16 Moreover, fibrosis of the atrial tissue could facilitate the development of ectopic pacemakers and late potentials as a result of inhomogeneous stimulus conduction, and could also lead to fluctuations in the membrane potential.2,9,13,16 Thus, atrial fibrosis can change a homogeneously activated atrium into a discontinuous and fragmented architecture. Consequently, augmented interstitial fibrosis may increase atrial susceptibility to the AF, since it is known that atrial reentrant arrhythmias are facilitated by impairment of the atrial conduction and by the presence of adjacent atrial regions with disparate refractory periods.13,16 Furthermore, according to the dominant concept of the AF, it results
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40 45 LA (mm)
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SR restoration affects collagen turnover in patients with AF
Conclusion Although AF has been considered an electrical disease, it can result in alterations in the atrial structure and the architecture that make the atrial myocardium more susceptible to the maintenance of the arrhythmia. Sinus rhythm restoration could affect the fibrotic process occurring or exacerbating during the AF course. However, further investigation and randomized trials are needed to elucidate the exact role of the fibrosis in the AF and to evaluate the clinical importance and the value of the biochemical monitoring of the collagen turnover in this clinical setting. Conflict of interest: none declared.
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from multiple reentrant circuits of various size and conduction velocities that propagate randomly through the atria and can be initiated by triggers, such as premature atrial contractions arising from focally discharging cells located most commonly in the myocardial sleeves extending from the LA into the pulmonary veins.1,2,4,16,17 The ability of the single extrastimuli to initiate the AF has been associated with the existence of a susceptible underlying substrate, which is characterized by local inhomogeneities in conduction and refractoriness.1,2,4,16,17 Changes in the ECM composition may underlie these electrophysiological alterations, facilitating the induction of the AF by the appropriate triggers. Taken together, these recently published data and the results of the present study could suggest that AF per se may cause ECM remodelling or may exaggerate ECM remodelling due to the underlying heart diseases. Indeed, since our only intervention was the successful CV, we assume that the fibrotic processes are at least exacerbated by the persistence of the AF. It is thus possible that the AF is associated with atrial structural changes that may provoke fibrosis or may have a fibrotic basis. Hence, the AF may induce or be aggravated by fibrosis, which in turn may promote the persistence of the AF. It is therefore likely that in the AF a vicious circle is set in motion, whereby the arrhythmia causes atrial structural remodelling that triggers a low-grade fibrotic response, which in its turn aggravates the AF, and so on. Some limitations of the present study should be taken into consideration. Although the serum markers of collagen turnover are not exclusively heart-specific, we made strenuous efforts to exclude subjects with certain conditions associated with fibrosis, such as hepatic dysfunction, renal impairment, and pulmonary fibrosis. We did not take the duration of the AF into consideration in our analysis because of the lack of reliable data on when the arrhythmia first occurred. Although its duration was more than 3 months in all our patients, the data concerning the AF duration previous to the 3 month point were unavailable in certain of them. In addition, drugs taken by the patients could have affected the serum concentrations of the evaluated markers. In any case, this is the first study to accept these limitations and report that the SR restoration and the maintenance are associated with an attenuation of the fibrotic processes existing during persistent AF, at least at the initial phases of the arrhythmia, demonstrating a potential role for the serum markers of collagen turnover in AF evaluation. The potential for reversing the fibrosis is of particular clinical interest especially when the rhythm control strategies are evaluated as the first choice treatment of AF. In addition, the development of non-invasive methods to indicate the presence and monitor the process of myocardial fibrosis in the AF and in other clinical conditions as well is particularly attractive and could have a broader application. Atrial fibrillation per se may cause ECM remodelling or may exaggerate ECM remodelling due to the underlying heart diseases.
