Activation of the calcineurin-nuclear factor of activated T-cell signal transduction pathway in atrial fibrillation

Activation of the Calcineurin-Nuclear

Factor of Activated T-Cell Signal

Transduction Pathway in Atrial

Fibrillation*

Chih-Chung Lin; Jiunn-Lee Lin, MD; Chich-Sheng Lin, PhD; Mei-Chuan Tsai;

Ming-Jai Su, PhD; Ling-Ping Lai, MD, PhD; and Shoei K. Stephen Huang, MD

Study objectives: The calcineurin-nuclear factor of activated T-cell (NFAT) signal transduction

pathway regulates the expression of a plethora of genes in the myocardium. Cytosolic calcium

overloading occurs in atrial fibrillation (AF), and this fulfills the condition needed for activation

of this pathway. We therefore investigated the NFAT pathway in atrial tissue in a porcine model

of AF.

Methods and results: AF was induced in eight adult pigs by rapid atrial pacing. Investigations on

the calcineurin and NFAT pathway were performed on transmural left atrial tissue obtained 6

weeks after implantation of the pacemaker (pacing for 4 weeks, and AF without pacing for 2

weeks). In the AF group, the left atrial dimension increased significantly (26

ⴞ 4 mm vs 31 ⴞ 4

mm, respectively, p < 0.05 [mean

ⴞ SD]). Calcineurin enzyme activity increased significantly in

pigs with AF (n

ⴝ 8) when compared to control pigs (n ⴝ 6) [0.143 ⴞ 0.034 vs 0.038 ⴞ 0.063 mmol

PO

released, p < 0.01]. We found that both NFAT-c3 and NFAT-c4, the downstream effectors

of calcineurin, increased significantly in the nuclei in AF tissue using immunoblotting.

Translo-cation of NFAT-c3 and NFAT-c4 into the nuclei was also demonstrated in AF tissue microsections

using immunohistochemistry. The electrophoresis mobility shift assay further demonstrated that

nuclear extracts from AF tissue had a significantly larger binding capacity for NFAT-specific

oligonucleotide probes.

Conclusions: Our results demonstrate that calcineurin activity was increased in AF with

subse-quent NFAT-c3 and NFAT-c4 translocation into the nucleus. Activation of this signal transduction

pathway may play an important role in the pathogenesis of AF.

(CHEST 2004; 126:1926 –1932)

Key words: atrial fibrillation; calcineurin; calcium; nuclear factor of activated T cell

Abbreviations: AF⫽ atrial fibrillation; EDTA ⫽ ethylenediaminetetraacetic acid; EMSA ⫽ electrophoresis mobility shift assay; NFAT⫽ nuclear factor of activated T cell; PMSF ⫽ phenylmethylsulfonyl fluoride

A

trial fibrillation (AF) is the most common

ar-rhythmia in humans. It causes palpitations,

de-creased cardiac output, heart failure, and systemic

thromboembolism, and is a major issue in public

health.

_{Current treatment modalities for AF are}

far from satisfactory. Despite aggressive treatment,

the recurrence rate of AF is still high, and

perma-nent AF refractory to any treatment including

elec-trical cardioversion develops in many patients. These

unsatisfactory outcomes are attributed, at least in

part, to the lack of understanding about the

patho-physiology of AF.

There is evidence showing that AF begets AF, and

through this vicious cycle AF becomes incessant.

_It

has been reported that AF causes structural and

functional changes in the atrial tissue, which, in turn,

result in further AF. These changes include a

short-*From the Institute of Pharmacology (Mr. Lin, Ms. Tsai, and Drs. Su and Lai), National Taiwan University, Taipei; Department of Internal Medicine (Drs. J-L. Lin and Huang), National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei; and Department of Biological Science and Technology (Dr. C-S. Lin), National Chiao Tung University, Hsinchu, Taiwan.

This work was supported in part by grant 91–2314-B-002–273 from the National Science Council in Taiwan.

Manuscript received February 27, 2004; revision accepted July 13, 2004.

Reproduction of this article is prohibited without written permis-sion from the American College of Chest Physicians (e-mail: [email protected]).

Correspondence to: Ling-Ping Lai, MD, PhD, No. 1, Jen-Ai Rd, Section 1, Institute of Pharmacology, School of Medicine, Na-tional Taiwan University, Taipei, Taiwan, 100; e-mail: [email protected]. ntu.edu.tw

ening of atrial effective refractory period, a decrease

of L-type calcium current, and changes in receptors,

contractile proteins, and interstitial matrix.

How-ever, the link between atrial rapid depolarization and

these changes remains unknown.

Ca

is a major cation involved in many important

physiologic responses in the heart, including

excita-tion-contraction coupling, secretion, cell-signaling

pathways, and transcription regulation.

Calcineurin-nuclear factor of activated T-cell (NFAT) pathway is

a well-established calcium-dependent pathway in T

cells. There is growing evidence showing the

impor-tance of this pathway in cardiac diseases such as

ventricular hypertrophy.

_{It has also been}

re-ported that the NFAT plays an essential role in the

regulation of many cardiac genes. Sustained

eleva-tion of cytosolic calcium occurs at early stage of AF,

and it is the trigger for calcineurin-NFAT pathway

activation.

_{Therefore, we tested the hypothesis}

that the calcineurin-NFAT pathway was activated in

atrial tissue in AF.

Materials and Methods

Porcine Model of AF

The porcine model of AF has been reported in detail previ-ously.15_{The investigation conforms to the National Institutes of}

Health guidelines for the care and use of laboratory animals. In brief, adult pigs of Yorkshire-Landrace strain weighing 50 to 80 kg were used. After IV anesthesia with ketamine, we implanted a high-speed pacemaker (Itrel III; Medtronic; Minneapolis, MN) to pace the atrium at a rate of 10 Hz (600 per minute). AFter pacing for 4 weeks, the pacemakers were turned off and the pigs were in persistent AF. The pigs were killed 2 weeks after turning off the pacemaker, and the total duration of rapid atrial depolar-ization was 6 weeks (rapid pacing for 4 weeks and AF without pacing for 2 weeks). In the sham (control) group, a pacemaker was implanted but remained off. The control pigs were killed 6 weeks after the implantation. The whole heart was removed from the chest cavity. Transmural left atrial free-wall tissue blocks were obtained and stored in liquid nitrogen for further use. There were eight pigs in the AF group and six pigs in the control group. Transthoracic echocardiography was performed at base-line and 6 weeks after implantation of the pacemaker. Left atrial dimension, left ventricular dimension (systolic and end-diastolic), and left ventricular ejection fraction were measured in two-dimension assisted M-mode in long-axis view.

Calcineurin Activity Assay

Calcineurin phosphatase activity was measured using a syn-thetic phosphopeptide substrate (R-II peptide) as described previously (AK-804 kit; BIOMOL; Plymouth Meeting, PA).16

Tissue samples were homogenized in phosphatase lysis buffer containing 50 mmol/L Tris (pH 7.5), 0.1 mmol/L NaCl, 1 mmol/L dithiothreitol, 1 mmol/L ethylenediamine tetraacetic acid (EDTA), 0.1 mmol/L ethyleneglycol tetra-acetic acid, 1␮mol/L pepstatin A, and protease inhibitor cocktail tablets (Complete; Roche; Mannheim, Germany). Calcineurin enzymatic activity was measured in phosphatase buffer containing 50 mmol/L Tris

(pH 7.5), 100 mmol/L NaCl, 6 mmol/L MgCl2, 1 mmol/L CaCl2,

1 mmol/L dithiothreitol, 0.05% ethylphenyl-polyethylene glycol (NP-40). Phosphatase activity was determined as the dephos-phorylation rate of the R-II peptide. The detection of free phosphate released from R-II peptide was based on the classic Malachite green assay.17

Preparation of Cytosolic Protein Extracts

The samples were homogenized in homogenization buffer containing 25 mmol/L Tris (pH 7.5), 0.5 mmol/L EDTA, 0.5 mmol/L ethyleneglycol tetra-acetic acid, 1 mmol/L phenylmeth-ylsulfonyl fluoride (PMSF), 1 mmol/L dithiothreitol, 25␮g/mL leupeptin, 25 mmol/L NaF, and 1 mmol/L Na3VO4. The

homog-enates were centrifuged at 14,000g for 15 min, and the resulting supernatants were collected as cytosolic proteins for immuno-blotting analysis. Protein concentrations were determined (BCA Protein Assay Reagent Kit; Pierce; Rockford, IL).

Preparation of Nuclear Protein Extracts

The samples were homogenized in buffer A (10 mmol/L hydroxyethyl piperazine-ethanesulfonic acid [pH 7.9], 1.5 mmol/L MgCl₂, 10 mmol/L KCl, 1 mmol/L dithiothreitol, 25 ␮g/mL leupeptin, and 1 mmol/L PMSF). AFter a 10-min incu-bation on ice, the samples were centrifuged at 1,850g for 10 min at 4°C. The pellets were dissolved in buffer B (buffer A⫹ 0.1% Triton X-100), incubated on ice for 10 min, and centrifuged as above. The crude nuclear pellets were washed once with buffer A and resuspended in buffer C (20 mmol/L hydroxyethyl piper-azine-ethanesulfonic acid [pH 7.9], 25% glycerol (volume/vol-ume), 0.42 M NaCl, 1.5 mmol/L MgCl₂, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol, and 1 mmol/L PMSF) for 30 min at 4°C. Nuclear proteins were recovered after centrifugation at 25,000g for 30 min. The resulting supernatants were the nuclear extracts.

Polyacrylamine Gel Electrophoresis and Immunoblotting

Proteins were separated by 8% sodium dodecylsulfate-polyac-rylamine gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore; Bedford, MA). The membranes were blocked for 1 h at room temperature using nonfat dry milk dissolved in Tris-buffer with 0.1% Tween-20. The primary antibodies used in the present study included rabbit polyclonal anti–NFAT-c4 antibody, mouse monoclonal anti–NFAT-c3, and anticalcineurin antibody specific for calcineurin A-␣ (all from Santa Cruz Biotechnology; Santa Cruz, CA). Membranes were incubated with primary antibody in blocking buffer for 12 h at 4°C. Peroxidase-conjugated secondary antibodies were used for detection of primary antibody. Membranes were incubated in blocking buffer containing secondary antibody for 1 h at room temperature. Signals were detected with an enhanced chemilu-minescence kit (Amersham Biosciences; Buckinghamshire, UK) and analyzed using image-editing software (Adobe Photoshop 6.0; Adobe Systems Incorporated; San Jose, CA; and Image Gauge V3.12; Fujifilm; Tokyo, Japan).

Immunohistochemistry

For immunohistochemistry, paraffin-embedded left atrial tis-sue was used. Deparaffinized and rehydrated sections were blocked at room temperature for 20 min with 5% nonimmune goat serum in Tris-buffered saline solution (pH 7.5) after micro-wave treatment (boiled 5 min in citrate buffer, pH 6) and quenching of endogenous peroxidase with 3% hydrogen perox-ide/methanol for 15 min. Anti–NFAT-c3 (1:20 dilution) or

anti–NFAT-c4 (1:50 dilution) were used as primary antibody and incubated at room temperature for 1 h. Staining was performed (VECTASTAIN ABC; Vector Laboratories; Burlingame, CA) as described by the manufacturer, and the color was developed with diaminobenzidine. Hematoxylin was used for counterstaining.

Nonisotopic Electrophoretic Mobility Shift Assays

For nonisotopic electrophoresis mobility shift assay (EMSA), 10␮g of nuclear extracts were incubated with 10 ng of biotin-labeled double-strand oligonucleotide probe in 10-␮L binding buffer containing 10 mmol/L Tris (pH 7.5), 50 mmol/L NaCl, 1 mmol/L dithiothreitol, 0.5 mmol/L EDTA, 5% glycerol, and 1␮g poly-d(I-C) (Panomics; Redwood City, CA). The oligonucleotide was the consensus NFAT binding site from the interleukin-2 promoter, and the base sequence was 5⬘-ACGCCCAAAGAG-GAAAATTTGTTTCATACA-3⬘. Competitive binding assays were conducted under the same condition with the addition of 50-fold molar excess of unlabeled NFAT or nonspecific (scram-bled) probes. Anti-NFAT antibody from Santa Cruz Biotechnol-ogy was used for supershift assay. Complexes were resolved on 6% polyacrylamide gel at 4°C in 0.5⫻ Tris-borate-EDTA buffer and then transferred to a nylon membrane (positive charged, Roche, Mannheim, Germany). Following UV cross-linking (UV-Stratalinker-1800; Stratagene, La Jolla, CA), the membrane was incubated with strepavidin-horse radish peroxidase in blocking buffer (Gel-Shift Kit; Panomics). The membrane was washed and subsequently developed using an enhanced chemiluminescence kit (Amersham) and a chemiluminescence imaging system (Syn-gene; Cambridge, UK).

Statistical Analysis

All data were expressed as mean⫾ SD. Parametric data were compared using Student t test. A p value⬍ 0.05 was considered statistically significant.

Results

Porcine Model of AF

All eight pigs in the active pacing group showed

AF at the end of the study, while all six pigs in the

control group showed sinus rhythm. The two groups

did not differ significantly regarding the left atrial

dimension, left ventricular dimension, and left

ven-tricular ejection fraction at the beginning. However,

the left atrial dimension increased significantly in the

AF group after 6 weeks of rapid atrial depolarization

(Table 1), while there was no significant change of

the left atrium size in the control group. The left

ventricular dimension and left ventricular ejection

fraction were not significantly altered in both groups.

Calcineurin Enzyme Activity and Protein Amount

We found that calcineurin (phosphatase 2B)

en-zyme activity was significantly higher in AF pigs than

in control pigs (0.143

⫾ 0.034 vs 0.038 ⫾ 0.063 nmol

PO

released, p

⬍ 0.01). The increase was greater

than threefold (372

⫾ 87%) [Fig 1]. Calcineurin

protein amount was also measured using antibody

specific for calcineurin A-␣ for immunoblotting

anal-ysis. We found that the protein amount of

cal-cineurin was not significantly different between AF

and control pigs. These results indicated that

cal-cineurin activity increased due to activation of the

protein without significant changes in the protein

amount.

Translocation of NFAT-c3 and NFAT-c4

To investigate the translocation of NFAT-c from

the cytosolic compartment to the nuclear

compart-ment, we performed immunoblotting for NFAT-c3

and NFAT-c4 using cytosolic and nuclear fractions

from atrial tissues (Fig 2, 3). These fractions were

first confirmed using immunoblotting for

␤-actin and

nucleolin to serve as cytosolic and nuclear markers,

respectively. We demonstrated that there was little

␤-actin in the nuclear extract, while there was little

nucleolin in the cytosolic extract (Fig 2). In the

cytosolic fraction, NFAT-c4 decreased significantly

in AF tissue, while NFAT-c3 also decreased but did

not reach statistical significance (p

⫽ 0.06) [Fig 3,

top, A]. In contrast, both NFAT-c3 and NFAT-c4

increased significantly in the nuclear fraction in pigs

with AF (Fig 3, bottom, B).

Immunohistochemistry

Immunohistochemical studies were performed to

investigate the distribution of NFAT-c3 and

NFAT-c4 in atrial tissue. In microscopy, the brown

signals indicate NFAT-c3 or NFAT-c4, and the

nuclei appear blue with hematoxylin counterstain.

On translocation of NFAT into the nuclei, darker

signals were observed when brown and blue signals

overlap. There was more NFAT-c3 and NFAT-c4

translocation into the nuclei in AF pigs than in control

pigs. We also measured the percentage of nuclei

showing overlapping signals. The ratio was significantly

Table 1—Echocardiographic Measurements in AF and

Control Groups*

Variables

AF Group Control Group Baseline 6 wk Baseline 6 wk LAD, mm 26⫾ 4 31⫾ 4†‡ 26⫾ 3 27⫾ 3 LVESD, mm 29⫾ 4 31⫾ 5 29⫾ 5 30⫾ 6 LVEDD, mm 48⫾ 4 52⫾ 6 49⫾ 5 51⫾ 6 LVEF, % 69⫾ 10 67⫾ 10 68⫾ 9 67⫾ 10 *Data are presented as mean⫾ SD.

†p⬍ 0.05 when compared to LAD at baseline in AF group. ‡p⬍ 0.05 when compared to LAD at 6 weeks in control group. LAD⫽ left atrial dimension; LVEDD ⫽ left ventricular end-diastolic dimension; LVEF⫽ left ventricular ejection fraction; LVESD⫽ left ventricular end-systolic dimension.

higher in the AF group than in the control group

(73.3

⫾ 27.8% vs 36.4 ⫾ 21.9%, p ⬍ 0.05).

Nonisotopic EMSA for NFAT

To further investigate the NFAT-c activity in the

nucleus, we performed EMSA using specific NFAT-c–

binding oligonucleotides (Fig 4). We used an unlabeled

NFAT probe and an unlabeled nonspecific probe for

competition to confirm that the band showing the shift

was NFAT specific. Furthermore, a supershift was

observed when anti-NFAT antibody was added. In the

nuclear extracts from AF pigs, the optical density of the

band with the mobility shift was significantly larger than

the control pigs. These results further indicated that

the NFAT-c translocated to the nucleus had binding

affinity with NFAT-c–responsive elements.

Discussion

In the present study, we demonstrated the

activa-tion of calcineurin-NFAT signal transducactiva-tion

path-way in AF tissue after rapid atrial depolarization for

6 weeks. We showed that tissue calcineurin

enzy-matic activity was increased. We also showed that the

downstream effectors of calcineurin, NFAT-c3 and

NFAT-c4, were translocated into the nuclei. Binding

activity to NFAT-c–specific probes was increased in

nuclear extracts as demonstrated using EMSA.

Figure 1. Enhancement of calcineurin enzyme activity but not protein amount in the atria of pigs with AF. The calcineurin activity was measured using a synthetic peptide R-II as the substrate. The calcineurin enzyme activity was significantly increased in pigs with AF than in control pigs. The elevation of calcineurin enzyme activity was greater than threefold (left, A). Immunoblotting of calcineurin revealed no significant change of calcineurin protein amount (right, B). CaN⫽ calcineurin; n⫽ No. of pigs; Sh ⫽ sham; n.s. ⫽ not significant. The error bars represent SEM.

Figure 2. Confirmation of the purity of the cytosolic (Cyto) and nuclear (Nucl) protein extracts. The cytosolic and nuclear fractions were verified using immunoblotting for␤-actin and nucleolin to serve as cytosolic and nuclear markers, respectively. There was little␤-actin in the nuclear protein extract, while there was little nucleolin in the cytosolic protein extract.

NFAT-c and the Heart

NFAT-c has been extensively studied in the

im-mune system. It was named NFAT because of its

essential roles in T-cell activation.

_The

associa-tion between NFAT-c and the heart was found by a

yeast two-hybrid study showing binding affinity

be-tween NFAT-c and the heart-specific GATA4

tran-scription factor.

_{It is therefore hypothesized that}

NFAT-c plays important roles in the regulation of

gene expression in cardiac tissue. Later studies

on NFAT-c in the heart focused on ventricular

hypertrophy. Both a transgenic animal study

_{and a}

pharmacologic study

_{using cyclosporine A and}

FK506 indicate that calcineurin-NFAT pathway is

involved in cardiac hypertrophy.

Past studies on the calcineurin-NFAT pathway in

atrial tissue are few. To the best of our knowledge,

we showed for the first time that the

calcineurin-NFAT pathway was activated in AF. In AF, the

cytosolic calcium level undergoes characteristic

changes. During rapid atrial depolarization, the

dia-stolic period shortens, which results in a decrease of

Figure 3. Translocation of NFAT-c3 and NFAT-c4 from cytosolic fraction to nuclear fraction in atrial tissue in pigs with AF. Immunoblotting studies on both cytosolic and nuclear fractions using anti–NFAT-c3 and anti–NFAT-c4 antibodies were performed respectively (panel A). Panel B shows the summary data. In the cytosolic fraction, NFAT-c4 decreased significantly in pigs with AF, while NFAT-c3 also showed a decrease, although not statistically significant (p⫽ 0.06). In the nuclear fraction, both NFAT-c3 and NFAT-c4 increased significantly in pigs with AF. Error bars represent SEM. See Figure 1 legend for expansion of abbreviations.

calcium re-uptake into the sarcoplasmic reticulum. It

has been demonstrated that the diastolic cytosolic

calcium level increases and the calcium transient

decreases in AF.

_{This exactly fulfills the}

condi-tion needed for activacondi-tion of calcineurin-NFAT

path-way, which depends on a sustained calcium elevation

instead of a transient increase of cytosolic calcium.

The gene expression regulatory effects of NFAT-c

are promiscuous. It has been shown that NFAT-c

regulated the expression of myosin heavy chain,

inflammatory cytokines such as interleukins, tumor

necrosis factors, and inducible cyclooxygenase 2

_;

ion channels such as calcium-activated potassium

channel

_{; and apoptosis-related genes such as Fas}

legend and tumor necrosis factor-related

apoptosis-inducing ligand genes.

_{Brain-type natriuretic}

peptide, endothelin-1, and myocyte-enriched

cal-cineurin interacting protein 1 are also under the

regulation of NFAT-c.

_{It has also been reported}

that overexpression of calcineurin resulted in a

de-crease of Ito potassium channel.

_{Another group of}

researchers

_{reported that expression of Kv4.2}

po-tassium channel was regulated by GATA4

transcrip-tion factor, which is also called NFAT-n, and binds

with NFAT-c. The remodeling processes of atrial

tissue in AF are manifold and include structural

remodeling, electrical remodeling, and contractile

remodeling. The activation of calcineurin-NFAT

ac-tivation may contribute to these changes by altering

the expression of a plethora of genes.

NFAT-c Subtypes

There are five subtypes of NFAT-c identified in

mammalian tissues.

_{In adult cardiac tissue, NFAT-c3}

and NFAT-c4 are the most important ones.

There have been reports showing that these two

subtypes are redundant. The DNA-binding

se-quence was 100% homologous between NFAT-c3

and NFAT-c4.

_{They might regulate the}

transcrip-tion of the same genes, and both can compensate for

the loss of each other. At the ventricular level, it has

been reported that NFAT-c3 is more important than

NFAT-c4 in causing cardiac hypertrophy.

_{In the}

present study, we showed an increase of both

NFAT-c3 and NFAT-c4 in the nuclei.

Limitations

Although we showed that calcineurin-NFAT

path-way is activated in AF and a lot of genes are under

the regulation of NFAT-c, a direct link between

atrial tissue remodeling and calcineurin-NFAT

path-Figure 4. Increased NFAT-specific probe bind capacity in nuclear extracts from pigs with AF. Biotin-labeled NFAT-c–specific probes used EMSA for nuclear extracts from atrial tissue. The lower arrow indicates the mobility shift due to binding of the probe with NFAT-c, while the upper arrow indicated a supershift after adding anti–NFAT-c antibody. Ab⫽ antibody against NFAT-c; CP ⫽ cold probe in 50⫻ excess. Error bars represent SEM.

way activation is lacking. Pharmacologic blockade of

the calcineurin-NFAT pathway using cyclosporine A

or FK506 was not performed in the present study.

The study was performed after rapid atrial

depo-larization for 6 weeks. The changes therefore can

only represent the change at 6 weeks. A time course

study was not performed. We cannot answer how

quick the pathway was activated.

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