apoptopsis

7/30/2019 apoptopsis

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Decreased Apoptosis following

Successful Ablation of Atrial

FibrillationAbstract

Objectives: Increased apoptotic processes in tissue samples from hearts in atrial fibrillation (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 suc-

cessful epicardial ablation of AF leads to changes in the concentration of serum markers of

apoptosis.Methods: Twenty- five patients with AF were prospectively studied. All underwent

epicardial isolation of pulmonary veins. The success of the ablation was assessed clinically and with3 Holter recordings. Blood samples were drawn before surgery, and at 3 and 6 months after. Serum

concentrations of Fas (apoptosis-stimulating 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 follow-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 concentra-

tion 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 fibrillation (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-pharmacologic 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 fibrosis, which is present in fibrillating 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 concentrations of apoptotic markers

compared to healthy controls and restoration and maintenance of sinus rhythm will be associatedwith decreased apoptosis.


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Methods

Patients and Follow-Up

Twenty-five patients with symptomatic paroxysmal or persistent 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 informedconsent was obtained 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 significant 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) significant pericardial effusion; (6) chronic obstructive pulmonary

disease, and (7) a history of pneumothorax or history of significant thoracic surgery. Antico-

agulation (warfarin with target INR 2, 03, 0) and anti-arrhythmic 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 ablation was assessed clinically and with 3 Holter recordings

during the first 6 months following ablation. One 24-hour Holter record- ing was done 1 month afterthe 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 first 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 symptoms, 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 pulmonary 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 hemitho-

rax. Then, after deflation 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 positioned 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 between the

pulmonary veins and atrial wall was carried out. In patients with fibrillating 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, apoptosis-stimulating fragment (Fas) and tumor necrosis factor-related

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 coefficients of variation were

satisfied (!10% and !5%, respec- tively).


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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 significant. 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 anon-Gaussian distribution were analyzed using the Mann-Whitney U test. Time-course analyses of

the observed parameters were done using analysis for repeated measurements 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-five 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 in9 patients and persistent 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 insufficiency (1/4) was present. Two patients

underwent percutaneous coronary intervention before ablation; the other 23 patients underwent

coronary angi- ography, which revealed no significant 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 respect 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 disease 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 parameters 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 concentration of apoptotic markers; however, no correlation was found between the

preoperative concentrations of apoptotic markers and the duration of AF,The time-course ofconcentrations of the Fas and TRAIL apoptotic markers are shown in figures 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; fig. 1). While the baseline values of the SR group were significantly higher

than values obtained from healthy controls (p ! 0.001), the values 6 months after ablation were not

statistically different from controls (p = n.s.; fig. 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 significance. However, the decrease con- tinued and the values of TRAIL at

6 months were significantly different compared with baseline values and were comparable to

values of healthy control subjects. The concentration of TRAIL in the AF group remained elevated

and unchanged over the entire observational period, compared with healthy controls.

Multivariate Analysis


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In a multivariate analysis, only male sex in the SR group was associated with a decrease in serum

concentration 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% confidence 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 repeatedmeasures 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 controls vs. baseline sample of study

patients (pooled data of SR and AF groups).

Discussion

The major finding of our study is that the concentration of apoptosis markers decreased to normal

values after 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 fibrosis [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

fibrosis occurs most commonly as a reparative process to replace degenerating or dead myocar-

dial parenchyma with concomitant reactive fibrosis.

In experimental models, ventricular tachypacing induces congestive heart failure in dogs by

causing tachy- cardiomyopathy, but also induces AF and atrial intersti- tial fibrosis (comparable to

the atrial pathology seen in experimental lone AF) [7]. Heinke et al. measured the expression 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 significantlyelevated 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 disease) for the presence of apoptosis associated with

atrial fibrillation. Compared to tissues from patients in sinus rhythm, there was significantly higher

myolysis, nuclear alterations, and activation of apoptosis (determined by Western blot) in

fibrillating atria [8]. Kim et al. analyzed the changes in gene expression in human atrial tissues

between patients with permanent AF and those with sinus 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 infiltration, and increased

cell death occur prior to arhythmogenic structural remodeling [6].

We found elevated levels of soluble serum apoptotic markers (Fas and TRAIL) in AF patients.

Further, we observed that the concentration of these markers decreased after sinus rhythm

restoration. This finding corresponds with the finding 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 apoptotic

molecules. Neither Fas nor TRAIL are specific for cardiomyocytes; based on concentrations we

cannot ass- es the origin of the measured molecules. An invasive cardiac 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 cardio-

myocytes in the atria and confirm the exact origin of soluble apoptotic molecules. While thiswould have been of great scientific interest, we felt the biopsy posed unjustifi- able risks to the


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patient. There are no other techniques which would allow measurement of apoptosis without tissue

sample from the specific organ.

On the other hand, our patients were without other significant comorbidities (such as tumors,

systemic disease, or chronic inflammatory disease), which are associated with increased

apoptosis and can significantly influ- ence the serum concentration of apoptotic molecules. The only

variable during follow-up was whether the sinus rhythm was restored or not; in successfully ablatedpa- 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 subjects, 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 Scientific Article, We can prove hy- pothesize that patients with AF will have higher

concentrations of apoptotic markers compared to healthy controls and restoration and

maintenance of sinus rhythm will be associated with decreased apoptosis.

References;

1. ^abcUSdict:ptss (American Heritage Dictionary)

2. ^"About Apoptosis". Archived from the original on 13 November 2009. RetrievedNovember 2009. "Apoptosis Interest Group, preferred pronunciation of National

Institute of Health"3. ^ Green, Douglas (2011). Means to an End: Apoptosis and other Cell DeathMechanisms. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.ISBN978-0-87969-888-1.

4. ^ Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith;Walter, Peter (2008). "Chapter 18 Apoptosis: Programmed Cell Death EliminatesUnwanted Cells". Molecular Biology of the Cell (textbook) (5th ed.). GarlandScience. p.1115. ISBN978-0-8153-4105-5.

5. Cardiology 2010;116:302307 DOI: 10.1159/000319619
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Scientific Article

On

Apoptosis Decreased Apoptosis following

Successful Ablation of Atrial

Fibrillation

Submitted By;

REGMI, ANIL BABU

12-1-41357

ICMB

July 18 2013

apoptopsis

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