Decreased Apoptosis following Successful Ablation of Atrial Fibrillation

Abstract Objectives: Increased apoptotic processes in tissue samples from hearts in atrial brillation (AF) have been previously documented in animals. Whether the restoration of sinus rhythm is associated with decreased apoptosis is not known. The aim of the present study was to establish whether successful epicardial ablation of AF leads to changes in the con- centration of serum markers of apoptosis. Methods: Twenty- ve patients with AF were prospectively studied. All under- went epicardial isolation of pulmonary veins. The success of the ablation was assessed clinically and with 3 Holter record- ings. Blood samples were drawn before surgery, and at 3 and 6 months after. Serum concentrations of Fas (apoptosis-stim- ulating fragment) and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) were measured using ELISA. Re- sults: AF was successfully ablated in 15 patients (SR group). In the other 10 patients (AF group), AF recurred during fol- low-up. Neither group differed with respect to age, sex, left ventricular ejection fraction, or preoperative concentrations of measured molecules. While Fas decreased in successfully ablated patients, there was no change in the Fas concentration in the AF group. Similarly, the concentrations of TRAIL decreased in the SR group, but remained unchanged in the AF group. Conclusion: The ablation of AF is associated with decreased serum markers for apoptosis. Introduction Atrial brillation (AF) is the most common cardiac arrhythmia and its prevalence is increasing. AF is char- acterized by a complex physiology and is an independent predictor of morbidity and mortality [1]. The treatment of AF is aimed at restoration and maintenance of sinus rhythm (rhythm control strategy) or control of heart rate and prevention of thromboembolic complications (rate control strategy). Currently, rhythm control, non-phar- macologic approaches are preferred due to the poor ef- fectiveness of pharmacologic treatments in symptomatic patients [2]. Besides interventional techniques, minimal- ly invasive epicardial thoracoscopic surgery has been available since 2002 [3, 4]. Tissue brosis, which is present in brillating atria, occurs most commonly as a reparative process to replace degenerating or dead myocardial parenchyma. There-

The aim of the present study was to compare the re- versibility of apoptosis in patients with lone AF. We hy- pothesize that patients with AF will have higher concen- trations of apoptotic markers compared to healthy con- trols and restoration and maintenance of sinus rhythm will be associated with decreased apoptosis.

Methods Patients and Follow-Up Twenty-ve patients with symptomatic paroxysmal or persis- tent AF were prospectively studied. All were symptomatic and resistant to pharmacologic treatment; AF was present despite treatment with at least 1 anti-arrhythmic drug (amiodarone was used in 80% of patients). A written informed consent was ob- tained from each participant and the study was approved by the Ethics Committee of University Hospital Kralovske Vinohrady (Approval No. EK/23/2007, April 11, 2007). Exclusion criteria in- cluded: (1) the presence of signicant valve disease; (2) coronary artery disease without previous complete revascularization; (3) thyrotoxicosis; (4) systolic dysfunction of the left ventricle (i.e. ejection fraction !40%); (5) signicant pericardial effusion; (6) chronic obstructive pulmonary disease, and (7) a history of pneumothorax or history of signicant thoracic surgery. Anticoagulation (warfarin with target INR 2, 03, 0) and anti-arrhyth- mic medication (amiodarone 200 mg/day or sotalol if amiodarone was not tolerated 160 mg/day) was maintained for at least 3 months after the AF ablation. Later warfarin treatment was initi- ated according to the CHADS2 criteria [2]. The success of the ab- lation was assessed clinically and with 3 Holter recordings during the rst 6 months following ablation. One 24-hour Holter record- ing was done 1 month after the procedure and two 48-hour Holt- er recordings were done after 3 and 6 months (Cardiette GiOtto, UK). Because paroxysmal AF immediately after ablation is quite common, its presence during the rst 4 weeks after the procedure was not considered to be a sign of ablation failure. The ablation was considered successful if the patient was AF symptom free during months 26 of the study (i.e. no palpitation or AF symp- toms, which were present before the procedure) and if all Holter recordings were negative for AF. Standard echocardiography evaluation was done before the ablation (Vivid 7, GE Medical Sys- tems, Horten, Norway). Ten healthy adults in sinus rhythm served as controls. Operative Procedure All patients underwent epicardial microwave isolation of pul- monary veins. The procedure was done under general anesthesia, with selective intubation of the left bronchus and selective left lung ventilation. Three ports were inserted in the right hemithorax. Then, after deation of the right lung, pericardiotomy was done above the right phrenic nerve. Next, preparation of the oblique and transverse sinus was performed and a Flex 10 (Guidant, Santa Clara, Calif., USA) catheter was inserted and po- sitioned such that it encircled the pulmonary veins. After verify- ing the correct position of the catheter (i.e. positioned under the auricle of the left atrium), the ablation was performed, usually in 2 cycles of 120 s each, creating a box lesion. After sinus rhythm was restored, perioperative testing of the conduction block be- tween the pulmonary veins and atrial wall was carried out. In patients with brillating atria during surgery, electrophysiologic testing could not be completed during the procedure. All proce- dures were performed in the department of cardiac surgery of the Cardiocenter, University Hospital Kralovske Vinohrady. Blood Drawing Blood samples were drawn before surgery, and at 3 and 6 months after. Serum concentrations of 2 apoptotic markers, apo- ptosis-stimulating fragment (Fas) and tumor necrosis factor-re- lated apoptosis-inducing ligand (TRAIL), both members of the tumor necrosis factor superfamily [9] were measured using com- mercially available ELISA kits (RD Systems, Minn., USA), using ELISA Reader (Elx808, Biotek, Vt., USA). Inter- and intra-assay coefcients of variation were satised (!10% and !5%, respec- tively).

Statistical Analysis Statistical analysis was performed by an experienced statisti- cian using SPSS v. 12 (SPSS, Chicago, Ill., USA) and Sigma STAT (Aspire Software, Ashburn, Va., USA). p ! 0.05 was considered statistically signicant. Categorical variables were tested using 2 analysis or Fishers exact test, as appropriate. Data were tested for normality using the Kolmogorov-Smirnov test. Data sets with a normal (Gaussian) distribution were analyzed using Students t test and those with a non-Gaussian distribution were analyzed using the Mann-Whitney U test. Time-course analyses of the ob- served parameters were done using analysis for repeated mea- surements and for 11 between-subjects factor (including age, sex, AF duration). In multivariate analysis a stepwise logistic regres- sion model was used. Results Clinical Results Twenty-ve patients with AF were enrolled in the study. The mean age of the study population was 59.5 8 8.2 years, there were 19 men and 6 women, and BMI was 26.4 8 1.3. AF was paroxysmal in 9 patients and persis- tent in the other 16 patients. The mean ejection fraction of the left ventricle was 55.8 8 10.4%. In 11 patients, a small mitral insufciency (1/4) was present. Two patients underwent percutaneous coronary intervention before ablation; the other 23 patients underwent coronary angi- ography, which revealed no signicant stenosis in the coronary arteries. In all patients, anti-arrhytmic medica-group, i.e. they were without clinical symptoms and without AF during Holter monitoring, while AF recurred in 10 patients (AF group). Clinical characteristics and dif- ferences between the SR and AF groups are summarized in tables 1 and 2. Both groups were comparable with re- spect to basic clinical characteristics, except for sex (more men in the SR group). Ten healthy adults, 7 men and 3 women, with a mean age of 59.4 8 2.6 years, all in sinus rhythm and without history of any cardiovascular dis- ease and arrhythmia, served as the control group. Paroxysmal versus Persistent Atrial Fibrillation, Duration of Atrial Fibrillation Nine patients suffered from paroxysmal and 16 from persistent AF. The clinical characteristics of both groups are shown in table 3. Except AF duration, which was lon- ger in paroxysmal AF patients, no other clinical param- eters were statistically different. Fas and TRAIL baseline (preoperative) concentrations were not different between the groups. A statistical analysis was done to evaluate the correlation between the duration of AF and the concen- tration of apoptotic markers; however, no correlation was found between the preoperative concentrations of apo- ptotic markers and the duration of AF, The time-course of concentrations of the Fas and TRAIL apoptotic markers are shown in gures 1 and 2. The concentration of Fas decreased in the SR group 3 months after surgery and decreased further at 6 months (both p ! 0.05; g. 1). While the baseline values of the SR group were signicantly higher than values obtained from healthy controls (p ! 0.001), the values 6 months after ablation were not statistically different from con- trols (p = n.s.; g. 1). No decrease in Fas was observed in the AF group. The concentration of TRAIL decreased in the SR group at 3 months, although the decrease did not reach statistical signicance. However, the decrease con- tinued and the values of TRAIL at 6 months were signif- icantly different compared with baseline values and were comparable to values of healthy control subjects. The concentration of TRAIL in the AF group remained ele- vated and unchanged over the entire observational peri- od, compared with healthy controls. Multivariate Analysis

In a multivariate analysis, only male sex in the SR group was associated with a decrease in serum concentra- tion of apoptotic markers. No other clinical or laboratory variables were associated with the decrease of apoptotic markers in the multivariate analysis. Fig. 2. The concentrations of TRAIL before and after ablation. Data are presented as median 8 5 95% condence interval, and are shown as closed triangles (SR group, n = 15), open circles (AF group, n = 10) or vertical box (healthy controls, n = 10). Data sets were analyzed by repeated measures ANOVA and Mann-Whit- ney U test for 3 repeated measurements. a p ! 0.05, within SR group; b p ! 0.05, SF group vs. AF group; c p ! 0.05, healthy con- trols vs. baseline sample of study patients (pooled data of SR and AF groups). Discussion The major nding of our study is that the concentra- tion of apoptosis markers decreased to normal values af- ter successful AF ablation. In cases where ablation was unsuccessful and AF persisted, apoptosis marker values remained unchanged. The hallmark of atrial structural remodeling is atrial tissue brosis [7, 10]. Increased collagen deposition has been documented in lone AF patients compared to sinus rhythm control patients [11]. The composition of atrial extracellular matrix has been correlated with AF persis- tence [12]. Tissue brosis occurs most commonly as a re- parative process to replace degenerating or dead myocardial parenchyma with concomitant reactive brosis. In experimental models, ventricular tachypacing in- duces congestive heart failure in dogs by causing tachy- cardiomyopathy, but also induces AF and atrial intersti- tial brosis (comparable to the atrial pathology seen in experimental lone AF) [7]. Heinke et al. measured the ex- pression of apoptotic molecules (Fas and Fas ligand) in cardiac tissue from the atria and ventricles of dogs, which had undergone rapid ventricular pacing for a period of 4 weeks. They found a signicantly elevated expression of apoptotic inducers compared to samples from control, non-paced hearts [13]. Dr. Aime-Sempe examined human right atrial tissue samples from patients who had undergone cardiac surgery (mainly for mitral valve dis- ease) for the presence of apoptosis associated with atrial brillation. Compared to tissues from patients in sinus rhythm, there was signicantly higher myolysis, nuclear alterations, and activation of apoptosis (determined by Western blot) in brillating atria [8]. Kim et al. analyzed the changes in gene expression in human atrial tissues between patients with permanent AF and those with si- nus rhythm. They found a prominent DNA ladder in the atrial cardiomyocytes in chronic AF, which is a biochem- ical hallmark of apoptosis [14]. In experimental models of pacing-induced AF, apoptosis, leukocyte inltration, and increased cell death occur prior to arhythmogenic struc- tural remodeling [6]. We found elevated levels of soluble serum apoptotic markers (Fas and TRAIL) in AF patients. Further, we ob- served that the concentration of these markers decreased after sinus rhythm restoration. This nding corresponds with the nding of Dr. Aime-Sempe et al. [8]. To the best of our knowledge, soluble Fas, TRAIL, and other measur- able soluble serum or plasma apoptotic markers have nev- er been evaluated in patients with AF, nor have marker concentrations been compared between AF and sinus rhythm. We cannot exclude extra-cardiac origin of apo- ptotic molecules. Neither Fas nor TRAIL are specic for cardiomyocytes; based on concentrations we cannot ass- es the origin of the measured molecules. An invasive car- diac biopsy of atria before (or during) the ablation and during follow-up, with measurement of apoptosis using standardized methods such as Western blot or TUNEL assays, could accurately assess the apoptosis of cardiomyocytes in the atria and conrm the exact origin of sol- uble apoptotic molecules. While this would have been of great scientic interest, we felt the biopsy posed unjusti- able risks to the

patient. There are no other techniques which would allow measurement of apoptosis without tissue sample from the specic organ. On the other hand, our patients were without other signicant comorbidities (such as tumors, systemic dis- ease, or chronic inammatory disease), which are associ- ated with increased apoptosis and can signicantly inu- ence the serum concentration of apoptotic molecules. The only variable during follow-up was whether the sinus rhythm was restored or not; in successfully ablated pa- tients the concentrations decreased to values seen in healthy controls. So, even if not cardiac in origin, the apoptotic markers are nonetheless AF related, and other sources of apoptotic molecules, in otherwise healthy sub- jects, are unlikely. Furthermore, the less invasive assess-ment of apoptosis is readily available in cardiology and some recent papers have used it as an acceptable routine technique. Conclusion

The ablation of AF is associated with decreased serum markers for apoptosis.

Comment ; From this Scientic Article, We can prove hy- pothesize that patients with AF will have higher concen- trations of apoptotic markers compared to healthy con- trols and restoration and maintenance of sinus rhythm will be associated with decreased apoptosis. References; 1. 2. ^ a b c USdict:ptss (American Heritage Dictionary) ^ "About Apoptosis". Archived from the original on 13 November 2009. Retrieved November 2009. "Apoptosis Interest Group, preferred pronunciation of National Institute of Health" ^ Green, Douglas (2011). Means to an End: Apoptosis and other Cell Death Mechanisms. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. ISBN978-0-87969-888-1. ^ Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter (2008). "Chapter 18 Apoptosis: Programmed Cell Death Eliminates Unwanted Cells". Molecular Biology of the Cell (textbook) (5th ed.). Garland Science. p.1115. ISBN978-0-8153-4105-5. Cardiology 2010;116:302307 DOI: 10.1159/000319619

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Scientic Article On Apoptosis

Decreased Apoptosis following Successful Ablation of Atrial Fibrillation

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REGMI, ANIL BABU 12-1-41357 ICMB July 18 2013