C-反應蛋白基因啟動子區域核?˙霰亄妝M心房顫動的關係-遺傳相關及功能性研究

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行政院國家科學委員會專題研究計畫成果報告

C-反應蛋白基因啟動子區域核酸變異和心房顫動的關係-遺傳相關及功能性研究

研究成果報告(精簡版)

計畫類別：個別型計畫編號： NSC 95-2314-B-002-134- 執行期間： 95 年 08 月 01 日至 96 年 07 月 31 日執行單位：國立臺灣大學醫學院內科計畫主持人：黃瑞仁計畫參與人員：學士級-專任助理：江怡萱處理方式：本計畫可公開查詢

中華民國 96 年 10 月 30 日

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C-reactive Protein Gene G1059C Polymorphism and Atrial Fibrillation- Genetic and Functional Studies

Chia-Ti Tsai, MD, PhD,1＊

Juey-Jen Hwang, MD, PhD,1, 2, 3＊

Sheng-Nan Chang, MD,2 Ling-Ping Lai, MD, PhD,1, 2, 3 Li-Ying Huang, MS,1 Chuen-Den Tseng, MD, PhD,1 Jiunn-Lee Lin, MD, PhD,1 Fu-Tien Chiang, MD, PhD,1,4

＊

Chia-Ti Tsai and Juey-Jen Hwang contributed equally to the manuscript

From the 1_{Division of Cardiology, Department of Internal Medicine, National Taiwan}

University Hospital, Taipei, Taiwan, 2_{Cardiovascular Center, National Taiwan}

University Hospital Yun-Lin Branch, Douliou City, Taiwan, 3_{Institute of}

Pharmacology, National Taiwan University, Taipei, Taiwan and 4_{Department of}

Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan

Running title: CRP gene and AF Total word count: 5313

No conflict of interest

This work was supported by two grants from the National Science Council, Taiwan, ROC (95-2314-B-002-087-MY3 and NSC-95-2314-B-002-134).

Corresponding author:

Fu-Tien Chiang, MD, PhD

Department of Laboratory Medicine National Taiwan University Hospital

No. 1, Section 1, Jen-Ai Road, Taipei 100, TAIWAN Tel: 886-2-23123456 ext. 5002

Fax: 886-2-82317099

(3)

Abstract

Background Inflammation plays an important role in AF pathogenesis. Whether genetic variant in the C-reactive protein (CRP) gene predisposes to AF, and molecular

effects of CRP on atrial myocytes and fibroblasts are unknown.

Objective To evaluate association between CRP gene G1059C polymorphism and non-familial atrial fibrillation (AF), and effect of CRP on atrial myocytes and

fibroblasts.

Methods We performed genetic association study to investigate association between CRP gene G1059C polymorphism and non-familial AF. We used whole cell

patch-clamp and reverse-transcription polymerase chain reaction to investigate the

effect of CRP on the transmembranic ionic currents and expression of procollagens in

atrial myocytes and fibroblasts, respectively.

Results Two-hundred and forty-three patients with non-familial AF and 348 controls were recruited. AF patients had a higher plasma CRP level (3.87±3.59 vs 2.91±3.30

mg/L, P=0.0008). The 1059C variant was associated with a lower CRP level, and a

lower risk of AF (OR 0.54 [0.30-0.97] and 0.55 [0.32-0.94] for dominant and additive

models, respectively, after multivariate adjustment). CRP significantly increased the

inward L-type calcium channel, without significant change on T-type calcium current

(4)

2 (COL1A2) procollagens in atrial fibroblasts.

Conclusions CRP gene G1059C polymorphism determines the basal level of CRP and the risk of common AF. The mechanism may be through an augmented inward

calcium current by CRP in atrial myocytes, but not through atrial fibrosis.

Key words: C-reactive protein; Atrial fibrillation; Genetics; Atrial myocytes; Atrial fibroblasts

(5)

Abbreviation List AF = Atrial fibrillation

COL1A1 = type I alpha 1 procollagen,

COL3A1 = type III alpha 1 procollagen

COL1A2 = type 1 alpha 2 procollagen

CRP = C-reactive protein

ICaL = L-type calcium current

ICaT = T-type calcium current

IKr = rapidly-activating delayed rectifier potassium current

IK1 = Inward rectifier potassium current

Ito = transient outward current

LAD = left atrial dimension

LVEF = left ventricular ejection fraction

LVESD = left ventricular end-systolic dimension

LVEDD = left ventricular end-diastolic dimensions

Reverse transcription–polymerase chain reaction = RT-PCR

(6)

Introduction

There is increasing evidence suggesting the role of inflammation and oxidative

stress in the pathogenesis of AF (1-6). It has also been speculated that these processes

contribute to the atrial remodeling (1)(6). Inflammatory markers, mainly C-reactive

protein (CRP), an acute-phase reactant indicating active inflammation, have been used

to predict the risk of AF (4) and AF recurrences after cardioversion (7-9). It has also

been shown that CRP was elevated in AF patients, and the CRP level correlatedwith

the AF burden (10). Furthermore, several pharmacological approaches with

non-channel blocking agents with anti-inflammatory properties show favorable effects

on AF, which include inhibitors of the renin-angiotensin system, statins, dietary

antioxidants, corticosteroids, and others (4)(6)(11-13). It has been shown that

corticosteroid treatment significantly reduced the CRP levels and there was a strong

correlation between CRP level and the risk of AF recurrence (14).

There have been several reports addressing the genetic control of familial AF

(15-17). However, the genetic study for non-familial AF is scarce in the literature

(18-20). Based upon the aforementioned studies on the relationship between

inflammatory state, CRP and AF, we hypothesized that the CRP gene might be one of

the susceptible genes of non-familial AF. There are several single nucleotide

(7)

intronic T to A transversion polymorphisms (21)(22). Prior studies have shown that

G1059C was associated with baseline CRP level (21). There are also reports

investigating the association between CRP G1059C polymorphism with

cardiovascular diseases, such as myocardial infarction, stroke, and post-angioplasty

restenosis (21)(23).

In the present study, we conducted a case-control study to investigate the

association between the CRP gene G1059C polymorphism and non-familial AF. The

effect of CRP on atrial myocytes and cardiac fibroblasts were also investigated to

elucidate the possible molecular mechanism.

Methods

Study population

The study included 243 consecutive patients at the Cardiovascular Clinic and

Adult cardiology ward of the National Taiwan University Hospital with a documented

history of AF, either by surface 12-lead ECG or Holter monitoring. Patients with

hyperthyroidism were excluded. Patients with familial AF were also excluded. The

patient was defined as having familial AF if any of his or herdirectly-related family

(8)

of 343 consecutive patients and were recruited from the same clinic and ward. The

control patients had no documented history of AF and no symptoms suggesting the

attack of AF, such as palpitation, chest discomfort, shortness or breath or dizziness.

Hypertension was defined as the presence of elevated systolic (>140 mm Hg)

and/or diastolic (>90 mm Hg) blood pressure and/or the current use of

antihypertensive drugs. Non- insulin-dependent diabetes mellitus was defined as

history of hypoglycemic treatment and/or a fasting blood glucose of >126 mg/dl.

All of the patients underwent the echocardiographic examination. All standard

measurements were obtained from parasternal long- and short-axis views and the

apical 4-chamber view according to American Society of Echocardiography

guidelines (24).M-mode measurements of left atrial anteroposterior dimension (LAD),

left ventricular end-systolic and end-diastolic dimensions (LVESD and LVEDD) and

left ventricular ejection fraction (LVEF) were used for the analyses. Left ventricular

mass was calculatedwith echocardiographic parameters and the Devereux formula

(25).Doppler and color Doppler studies were performed for detection of valvular

heart disease (VHD). Significant VHD was defined as at least moderate aortic or

mitral stenosis/regurgitation. The study was approved by the local institutional review

(9)

Genotyping of CRP gene G1059C polymorphism and measurement of plasma CRP

concentration

Blood was withdrawn, centrifuged soon, then DNA extraction was performed

with standard phenol–chloroform method and plasma was stored in 80°C freezer

before analysis. Genotyping of CRP gene G1059C polymorphism was performed by

polymerase chain reaction (PCR) and further digestion by MaeIII restriction enzyme,

as previously described (26). Briefly, PCR was performed by using the following

primers: Forward-5′GATCTGTGTGATCTGAGAAACCTCT3′

and Reverse- 5′GAGGTACCAGAGACAGAGACGTG3′. Target DNA was amplified

using 94°C for 5 minutes, then 30 cycles of 94°C for 30 seconds, 57°C for 30 seconds

and 72°C for 30 seconds, followed by extension step with 72°C for 10 minutes. Two

percent agarose gel was used for visualization. Quality control for the experiments

was evaluated by re-genotyping of 20 random samples blinded to the technician. The

plasma CRP concentration was measured by a high sensitivity Immunonephelometry

(Nephelometry, Behring Nephelometer II, Dade Behring Marburg GmbH, Germany)

with the lowest detection limit of 0.16 mg/L. All laboratory analyses were performed

blinded with respect to the diagnosis and patients’ characteristics. Subjects with CRP

level more than 20 mg/L were excluded from the study due to the possibility of the

(10)

Transmembraneous ionic current measurement

Transmembrane currents were measured by using a patch-clamp amplifier (8900;

Dagan Corporation, Minneapolis, Minn) by applying a whole-cell recording technique

as previously described (27). The extracellular solution contained NaCl 137 mM, KCl

5.4 mM, MgSO4 1.2 mM, CaCl2 1.8 mM, KH2PO4 1.2 mM, dextrose 22 mM, and

HEPES buffer 10 mM (pH 7.4). The internal pipette solution contained K-aspartate

120 mM, KCl 20.0 mM, Mg adenosine 5'-triphosphate 5.0 mM, K2 ethylene glycol

tetraacetic acid 11.0, HEPES buffer 10.0 mM, MgCl2 2.0 mM, CaCl2 1 mM, Na

creatine phosphate 5, mM Na guanosine 5'-triphosphate. 0.2 mM, and adenosine

3',5'-cyclic monophosphate 0.2 (pH 7.2).

The L-type (ICaL) and T-type (ICaT) calcium currents were recorded as

previously described (27). The contamination of INa and IK was abolished by replacing

the sodium ion with NMDG (137 mM) and adding Cs+_{(12 mM) in the bath solution.}

The pipette internal solution also contained TEA (30 mM) and Cs+ (130 mM). Ba2+

(20 mM) was employed as the charge carrier in the bath solution. Total calcium

currents, including ICaL and ICaT currents, were obtained by using a series of

depolarization steps to +60 mV from the holding potential –80 mV. ICaL was

(11)

mV. ICaT was obtained after subtracting the ICaL total calcium current. HL-1

myocytes exhibit either dominant L (calcium currents obtained from holding potential

-80 mV were identical to those obtained from holding potential -50 mV ) or dominant

T type (no current obtained from holding potential -50 mV) calcium currents, which

were sensitive to nifedipine 3 µmol/L and nickel 1 µmol/L, respectively (27).

The rapidly-activating delayed rectifier potassium current, or IKr, which is

sensitive to specific blockers such as E4031 or dofetilide, is a prominent component

of the outward rectifier potassium currents of HL-1 cells (28)(29). We also measured

the effect of CRP on IKr. IKr was obtained by applying a series of depolarization

steps (500 ms) from -80 mV and was measured as the tail currents at the

repolarization step to -40 mV, which was sensitive to E4031 (1 µmol/L). We also

measured the effect of CRP on other outward currents. The transient outward current

(Ito) was measured as the 4-aminopyridine-sensitive peak current (10 mM). The

inward rectifier potassium current (IK1) was obtained by using a series of

hyperpolarization steps (200 ms) to -140 mV after a pre-pulse to -20 mV (200 ms)

from a holding potential –80 mV.

(12)

The HL-1 atrial cell line was derived from adult mouse atria, which were

obtained from Louisiana State University in New Orleans, LA, USA. The culture and

maintenance of HL-1 atrial myocytes were as previously described (27)(28). HL-1

myocytes were serum starved for 24 hours on the 3rd_{day after subculture and then}

used for experiments (CRP stimulation).

Isolation and Culture of Neonatal Rat Atrial Fibroblasts

Both atria from week-old neonatal Wistar rats were cut into chunks and were

subjected to trypsin (0.125%) digestion in a balanced salt solution. The disaggregated

cells were collected by centrifugation at 300 g for 10 min. The cell pellet was

re-suspended in serum-containing medium (Ham’s F-12:DMEM;1:1 with 20% fetal

bovine serum and 1% penicillin-streptomycin), plated onto a Petri dish and kept for

2.5h in a 5% CO2 atmosphere at 37°C to allow fibroblasts to attach to the bottom of

the dish. The enriched cells weresubsequently incubated with DMEM supplemented

with 10% fetal calfserum and 1% penicillin-streptomycin for an additional 3 days.

After 3 days, the cells were transferred to a serum-free medium (Ham’s

F-12:DMEM;1:1 for myocytes and DMEM for nonmyocytes) overnight and then used

(13)

Extraction of RNA and Reverse Transcription–Polymerase Chain Reaction

Accumulation of extracellular matrix is an important structural change in AF

(30-32). Therefore, we used the expression of type I alpha 1 (COL1A1), type III alpha 1 (COL3A1) and type 1 alpha 2 (COL1A2) procollagens to evaluate the change of

collagen synthesis by atrial fibroblasts after CRP stimulation.

The extraction and quantification of mRNA by means of reverse

transcription–polymerase chain reaction (RT-PCR) were performed as reported

(27)(33). In brief, 5 µg total RNA was isolated andreverse transcribed.

Single-stranded cDNA was amplified with PCR. PCR with each specific primer pair

yielded only one DNA band of predicted size. The PCR products were confirmed by

means of direct sequencing. The glyceraldehyde 3-phosphate dehydrogenase

(GAPDH) gene was used as the internal control for equal loading. The reaction

products were analyzed with agarose gel electrophoresis. Optic densitometry was

performed after the gel was stained with ethidium bromide to measures its DNA

amount. Expression of mRNA was represented by its ratio to the mRNA for GAPDH.

The primer sequences were as follows:

Rat COL1A1 (250 bp):

Forward: 5’TCTCCACTCTTCTAGTTCCT3’

(14)

Rat COL3A1 (557 bp): Forward: 5’AGCTGGTCAGCCTGGAGATA3’ Reverse: 5’GACCTCGTGCTCCAGTTAGC3’ Rat COL1A2 (352 bp): Forward: 5’CACCTGGTCCTGTTGGAAGT3’ Reverse: 5’ATACCTGGCAGACCACGTTC3’ Rat GAPDH (408 bp): Forward: 5’TTGCCATCAACGACCCCTTC3’ Reverse: TTGTCATGGATGACCTTGGC Statistic method

For comparisons of the baseline characteristics, the between-group data were

compared with Student’s unpaired t test for continuous data and with the χ2 or

Fisher’s exact test for categorical data (degree of freedom [df] =1). Hardy-Weinberg

equilibrium of thegenotype distribution of polymorphisms was tested using a χ2 test

(df=2).

We compared the allele and genotype frequencies between the cases and controls

with the χ2 test or Fisher’s exact test (df=1 for the two by two allele frequencies χ2

(15)

genotype-phenotypecorrelation was examined with additive, dominant, and recessive

models with logistic regression. The dominant geneticmodel compares individuals

with one or two C alleles (GC+CC)with the baseline group with no C allele (GG

genotype). The recessive genetic model compares the CC genotypewith the combined

GG+GC genotypes, which form the baseline group.The additive genetic model

assumes that there is a linear gradientin risk between the GG, GC, and CC genotypes

(GG genotype as the baseline). Odds ratios (ORs) and their 95% confidence intervals

(CIs) werecalculated. Because LAD, LVEF and the percentage of patients with

significant VHD were not balanced in the casesand controls (Table 1), we also

performed the analyses with adjustmentfor LAD, LVEF and the percentage of

patients with significant VHD. The STATA software was used for analyses and

P<0.05 was considered statistically significant.

Results

Basline characteristics

The baseline characteristics of the entire study population according to the

presence (cases) or absence (controls) of AF are shown in Table 1. The AF patients

had a lower mean LVEF, higher mean LAD, and a greater incidence of significant

(16)

G1059C polymorphism and AF

CRP gene G1059C polymorphism significantly affected the basal CRP level.

Subjects with GC and CC genotypes had significantly lower basal plasma CRP

concentration than those with GG genotype (GG: 3.02 ± 3.33; GC: 2.50 ± 3.21;CC:

1.85 ± 1.76 mg/L, all p <0.01 compared to the those with GG genotype).

No deviation from the expected population genotype proportions predicted by

Hardy–Weinberg equilibrium was detected (Table 2). A significant difference in

genotype distribution and allele frequencies between the AF patients and controls

were found for G1059C polymorphisms (Table 2). The allele frequency of 1059C

variant was significantly lower in the AF patients. The genotype frequencies of GC

and CC were also significantly lower in the AF patients.

In the univariate analysis, C allele of the CRP gene G1059C polymorphism was

significantly associated with a protecting effect on AF under dominant and additive

models (Table 3). Because the mean LAD, LVEF and the percentage of patients with

significant VHD were significantly different in the AF patientsand controls, we also

performed the multivariate analysis to adjustfor the possible confounding effects from

LAD, LVEF and the presence of significant VHD. In the multivariate analysis, the

(17)

adjustment for LAD, LVEF and the presence of significant VHD (Table 3). In other

words, G allele of the CRP gene G1059C polymorphism was associated with a higher

basal CRP level, and a higher risk of AF.

Effect of CRP on the transmembranic ionic currents of HL-1 atrial myocytes

The Effect of CRP on the transmembranic ionic currents of HL-1 atrial myocytes

was evaluated by using a tight-sealed whole-cell recording technique. The CRP was

applied and the currents were recorded after a steady-state of current amplitude

(around 3 min after membrane rupture). The cells could sustain a tight-sealed

condition without leak for around 20 min. Cells with leak were not used for study.

CRP significantly increased ICaL current density (Figure 1A and 1B), without a

significant change in ICaT (data not shown). HL-1 myocytes also had typical IKr

currents (27). CRP (1 mg/L) did not affect IKr current density (data now shown). We

also observed no significant changes in IK1 and Ito after CRP stimulation (data not

shown).

Effect of CRP on cardiac fibroblasts

Accumulation of extracellular matrix is an important structural change in AF

(18)

amount of collagen synthesis by atrial fibroblasts. We found that CRP did not increase

or decrease COL1A1, COL1A3 and COL1A2 expressions in atrial fibroblasts with

either different concentrations or different stimulation times (Figure 2).

Discussion

Main findings

To the best of our knowledge, this is the first report to demonstrate the

association between the CRP gene variant with non-familial AF. The 1059G allele was

associated with a higher basal CRP level, and predisposition to AF. The mechanism of

the association may be through an augmented inward calcium current induced by CRP

in atrial myocytes, which may play an important role in the genesis of calcium

overload and initiation of AF.

Mechanisms of the Association

Our results are similar to those of other studies that CRP gene G1059C

polymorphism affects the basal CRP level, with the C allele associated with a lower

plasma CRP level (21). So far the mechanism of this association has never been

established. The CRP G1059C polymorphism locates in exon 2 of the CRP gene,

(19)

mechanism to explain the association between G1059C polymorphism and plasma

CRP level is that it may be in tight linkage disequilibrium with polymorphisms in the

promoter region of the CRP gene (34).

In the present study, we also demonstrated that CRP increases the ICaL current in

atrial myocytes, which has never been reported in the literature. The effect could be

observed with a physiological range of CRP concentration (0.5-1 mg/L). The

CRP-induced increase in inward calcium current may increase calcium influx during

rapid depolarization and thus exacerbate the condition of calcium overload in AF.

However, the change of ICaL current induced by CRP (up-regulation) is the reverse of

those observed in animal models of AF (35) or human AF (36)(down-regulation).

The possible explanation is that increased ICaL may induce calcium overload

and thus initiation of AF. Using a computer simulation model, it has been

demonstrated that increased ICaL plays a critical role in induction of dynamic spatial

dispersions of repolarization, which caused conduction block, reentry, and thus

initiation of AF (37). In humans, it has also been found that increased ICaL is a strong

predictor of post-operative AF (38). Therefore, it is possible that CRP increases the

risk of AF through augmenting ICaL. This hypothesis is in accordance with the

finding that baseline CRP predicted risk for developing future AF in the general

(20)

AF (36) may be an adaptive process (38) to prevent further damage from calcium

overload after a longer term of AF-induced calcium overload, and is important for the

mechanism of shortening of atrial refractory period and maintenance of AF.

In addition to the electrophysiological mechanisms, atrial fibrosis also plays a

very important role in the promotion of AF (30-32). However, in the present study, we

found that CRP did not affect the expression of procollagens in atrial fibroblasts.

Therefore, it is relatively unlikely that CRP promotes AF through atrial fibrosis.

Limitations

First, we only studied a single polymorphism in the CRP gene. However, this

polymorphism has been reported to affect the basal plasma CRP level in many studies

(21) (40)(41), and thus has functional significance. Second, we only investigated the effect of CRP in the cellular level, and did not provide a mechanic link between CRP

and AF by using an animal model. There are several gene over-expression models of

AF (42)(43). However, the animal model with cardiac-specific CRP gene

over-expression has been lacking so far. Finally, we also did not provide the

mechanism by which CRP augments ICaL. The CRP receptor and the detailed

signaling pathway of CRP in cardiomyocytes has been unknown so far, and have

(21)

In conclusion, the evaluation of CRP gene G1059C polymorphism has

demonstrated a possible role forCRP gene as a predisposing factor to AF. Future

studies to substantiate our results in other ethnic population are warranted.

Furthermore, from a pharmacogenetic point of view, whether this polymorphism

could also predict the efficiency of non-channel blocking drugs with

(22)

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Acknowledgement

This work was supported by two grants from the National Science Council,

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Figure Legends

Figure 1. C-reactive protein (CRP) increases L-type calcium current (ICaL) in HL-1 atrial myocytes. A, Representative recordings of whole-cell ICaL currents are shown

for the control cells and CRP-treated (1 mg/L, 10 min). ICaL was obtained by a

family of depolarization steps to +70 mV from the holding potential –50 mV. Inset,

Voltage protocol. B, Representative density-voltage relationships of ICaL in control

cells and CRP-treated cells (all n=6).

Figure 2. No effect of C-reactive protein (CRP) on expressions of procollagens in atrial fibroblasts. A, Total RNA was isolated from atrial fibroblasts left untreated

(controls) or treated for indicated times with CRP (1 mg/L). Reverse

Transcription–Polymerase Chain Reaction (RT-PCR) with specific primers for type I

alpha 1 (COL1A1), type III alpha 1 (COL3A1) and type 1 alpha 2 (COL1A2)

procollagens were performed, and the PCR products were visualized by

electrophoresis and ethidium bromide staining. B, Total RNA was isolated from atrial

fibroblasts left untreated (controls) or treated with indicated concentrations (1 denotes

1 mg/L) of CRP for 24h. RT-PCR for COL1A1, COL1A3 and COL1A2 were

performed and the PCR products were visualized by electrophoresis and ethidium

bromide staining. GAPDH was used for internal control to show equal loading of the

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