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Cardiovascular Research 2007 76(3):442-452; doi:10.1016/j.cardiores.2007.07.013
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Copyright © 2007, European Society of Cardiology

Atrial cardiomyocyte tachycardia alters cardiac fibroblast function: A novel consideration in atrial remodeling*

Brett Bursteina,c, Xiao-Yan Qia, Yung-Hsin Yeha, Angelino Calderoneb and Stanley Nattela,c,*

aDepartment of Medicine, Montreal Heart Institute and Université de Montréal, Montreal, Canada
bDepartment of Physiology, Montreal Heart Institute and Université de Montréal, Montreal, Canada
cDepartment of Pharmacology and Therapeutics, McGill University, Montreal, Canada

*Corresponding author. 5000 Belanger St. E., Montreal H1T 1C8, Quebec, Canada. Tel.: +1 514 376-3330; fax: +1 514 376 1355. stanley.nattel{at}icm-mhi.org

Received 19 March 2007; revised 18 July 2007; accepted 20 July 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective Atrial fibrillation (AF) causes tachycardia-induced atrial electrical remodeling, contributing to the progressive nature of the arrhythmia. Ventricular dysfunction due to a rapid response to AF can cause structural remodeling, but whether AF itself directly promotes atrial fibrosis is controversial. This study investigated the hypothesis that rapid atrial cardiomyocyte activation produces factors that influence atrial fibroblast proliferation and secretory functions.

Methods Cultured canine atrial fibroblasts were treated with medium from rapidly-paced atrial cardiomyocytes, non-paced cardiomyocytes and cardiomyocyte-pacing medium only, and analyzed by [3H]thymidine incorporation, Western blot and real-time RT-PCR.

Results Rapidly-paced cardiomyocyte-conditioned medium reduced [3H]thymidine uptake compared to non-paced cardiomyocyte-conditioned medium and medium alone (~85%, P<0.01). Rapidly-paced cardiomyocyte medium increased {alpha}SMA protein (~55%, p<0.001), collagen-1 (~85%, P<0.05) and fibronectin-1 (~205%, P<0.05) mRNA expression vs. controls. The angiotensin-1 receptor blocker valsartan attenuated pacing-induced {alpha}SMA changes but did not affect fibroblast proliferation. Suppression of contraction with blebbistatin did not prevent tachypacing-induced changes in [3H]thymidine uptake or {alpha}SMA upregulation, pointing to a primary role of electrical over mechanical cardiomyocyte activity. Atrial tissue from 1-week atrial-tachypaced dogs with ventricular rate control similarly showed upregulation of {alpha}SMA protein (~40%, P<0.05), collagen-1 (~380%, P<0.01) and fibronectin-1 (~430%, P<0.001) mRNA versus shams.

Conclusions Rapidly-paced cardiomyocytes release substances that profoundly alter cardiac fibroblast function, inducing an activated myofibroblast phenotype that is reflected by increased ECM-gene expression in vivo. These findings are consistent with recent observations that AF per se may cause ECM remodeling, and have potentially important consequences for understanding and preventing the mechanisms underlying AF progression.

KEYWORDS Fibrosis; Remodeling; Extracellular matrix; Angiotensin


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Atrial fibrillation (AF) changes atrial electrophysiological function, providing a possible explanation for the progressive nature of this arrhythmia. There exists a clearly-established link between atrial extracellular matrix (ECM) remodelling and AF maintenance, likely related to uncoupling of cardiomyocytes by intervening fibrotic deposits [1,2]. Clinical AF is commonly associated with underlying heart disease [3]; however, data about structural remodeling as a potential direct consequence of AF are limited [4,5]. Rapid electrical stimulation of cultured atrial cardiomyocytes produces electrical remodeling, recapitulating the principle phenotypic features of atrial-tachycardia remodeling in vivo [6]. It remains unclear whether rapid cardiomyocyte activation influences neighbouring non-cardiomyocyte cells, an issue of particular importance in the context of cardiomyocyte/fibroblast crosstalk during atrial remodeling [7,8]. We tested the hypothesis that tachypaced atrial cardiomyocytes generate factors which influence fibroblast proliferation and ECM-secreting functions. We also sought evidence for consequences of this potential paracrine mechanism in a clinically-relevant experimental model of atrial-tachypacing (ATP)-induced AF [9].


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1 Cardiomyocyte culture and pacing
HL-1 atrial-derived cardiomyocytes [10] were obtained from William Claycomb (Louisiana State University) and grown to confluence on gelatin-coated culture dishes (Fisher) in culture medium consisting of Complete Claycomb Medium (JRH Biosciences) supplemented with 10%-FBS, 100 µM norepinephrine, 4 mM L-glutamine and 1% penicillin-streptomycin, as previously-described [11]. Confluent HL-1 cells contracted spontaneously at ~0.5 Hz, and were maintained in cardiomyocyte-pacing medium (culture medium with 2% FBS-content) throughout pacing. Cells were subjected to rapid electrical stimulation or cultured in parallel without stimulation. Stimulated cells were paced as previously-described [12] with a C-Pace100-culture pacer and C-Dish100-culture dishes (IonOptix), with 5-ms, 5-Hz square-wave pulses (45±3 V pulse-voltage, ~7 V/cm) for 24 h. Capture efficiency >90% was confirmed by microscopic examination of cell shortening at the beginning and end of stimulation, and pacing resulted in <10% cell death [6]. Conditioned media from rapidly-stimulated myocytes (group 5-Hz), non-paced myocytes (group NP) and cardiomyocyte-pacing medium alone containing no cells (basal, group 0) were transferred to atrial fibroblast cultures isolated from single dogs for parallel study. To exclude non-specific effects of electrical stimulation, experiments were conducted applying electrical stimulation to cardiomyocyte-pacing medium (group 0P). Additional experiments were performed with the excitation–contraction uncoupler (±)-blebbistatin (5 µM, Calbiochem) in the cardiomyocyte-pacing medium throughout electrical stimulation, to evaluate the importance of cardiomyocyte contraction, and with the AT1-receptor blocker, valsartan (10 µM, Novartis), added to the paced myocyte-conditioned medium to assess the role of locally-elaborated angiotensin II. Both blebbistatin and angiotensin were studied in parallel with vehicle-treated controls.

2.2 Fibroblast isolation and culture
Atrial fibroblasts were obtained from right atria of adult mongrel dogs (n=17) weighing 23–31 kg, as described previously [13]. The investigation conforms with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, revised 1996). Only first and second-passage fibroblasts were used, and for all experiments cells from a single dog were plated in culture medium at 200 cells/mm2. Cells were allowed to adhere, then rendered quiescent by 24 h in serum-free medium, followed by 24-h treatment with conditioned cardiomyocyte medium.

2.3 [3H]Thymidine incorporation assay
Fibroblast proliferation was assayed by [3H]thymidine incorporation as described previously [13]. Fibroblasts were plated in 24-well plates at 50 cells/mm2 and, following serum starvation, treated for 24 h. Disintegrations per minute (DPM) for each treatment were measured in triplicate.

2.4 Immunohistochemistry and confocal imaging
Following treatment, fibroblasts plated on glass coverslips in p100 culture dishes were washed with phosphate-buffered saline (PBS) and fixed with 2%-paraformaldehyde. Fixed cells were washed, permeabilized with 0.1% Triton X-100 and blocked with 2%-normal donkey serum (NDS, Jackson ImmunoResearch Laboratories). The cells were then incubated with mouse anti-{alpha}-smooth muscle actin ({alpha}SMA, Sigma, 1/200) primary antibodies, followed by donkey anti-mouse Alexa Fluor-555 secondary antibodies (Invitrogen, 1/600). Excess antibody was removed by three washes and the coverslip-mounted cells applied to glass slides in Dabco mounting medium (Sigma). Cells were imaged with a Zeiss LSM-510 confocal microscope. Parallel negative-control studies with cells incubated with only secondary antibodies revealed no non-specific staining.

2.5 Ca2+-transients and cell contraction
Intracellular Ca2+-transients and cell shortening were recorded as previously-described [14] with Indo-1 AM microfuorometry and video edge-detection, from 5-Hz paced cultured cardiomyocytes in pacing medium before and after supplementation with (±)-blebbistatin (5 µM, n=4).

2.6 Atrial-tachypacing animal model
Adult mongrel dogs (n=10) were subjected to 1-week ATP [9,15,16]. Right-atrial pacing was performed at 400 bpm. Ventricular rate was controlled by producing AV-block (radiofrequency-ablation), and a ventricular lead attached to a demand-pacemaker set to 80 bpm. ATP dogs were compared with similarly-instrumented control dogs (n=8) with atrial pacemaker inactivated. At sacrifice, right-atrial free-wall tissue samples were collected, snap-frozen in liquid-N2 and stored at –80 °C.

2.7 Western-blot analysis
Proteins were extracted from treated fibroblasts and whole right-atrial free-wall tissue as previously-described [13]. Protein was quantified by Bradford assay. Proteins (10 µg/lane) were separated with 10%-polyacrylamide gel-SDS electrophoresis and transferred to PVDF membranes. Membranes were blocked, then incubated with anti-{alpha}SMA (1/40,000) and anti-GAPDH (1/5000, RDI) primary antibodies, followed by anti-mouse (1/20,000, Santa Cruz) HRP-conjugated secondary antibodies, and revealed with Western-Lightning chemiluminescence (PerkinElmer). {alpha}SMA-band intensities were quantified by video-densitometry and expressed relative to GAPDH-intensity from the same sample.

2.8 RNA extraction and TaqMan real-time RT-PCR
Total RNA was isolated from treated fibroblasts and whole right-atrial free-wall tissue by guanidine thiocyanate–phenol–chloroform extraction [17], then DNase-treated (RNeasy mini kit, Qiagen), quantified and quality-controlled by microelectrophoresis on polyacrylamide gels (Agilent 2100 Bioanalyzer). DNA-contamination was excluded by reverse transcription (RT)-negative polymerase chain reaction (PCR). First-strand cDNA was synthesized from 2-µg total RNA with High Capacity cDNA Archive Kits (Applied Biosystems). Real-time RT-PCR was performed with 6-carboxy-fluorescein (FAM)-labeled fluorogenic collagen-1 and fibronectin-1 TaqMan primers and probes (Assay-by-design, sequences in Table 1) and TaqMan universal master mix (Applied Biosystems) with the Stratagene Mx3000P sequence-detection system [15]. Fluorescence-signals were detected in duplicate, normalized to 18S-ribosomal RNA (Applied Biosystems) and quantified using MxPro QPCR software (Stratagene).


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  		Table 1 Gene-specific primers and TaqMan probe sequences used in real-time RT-PCR analysis

 
2.9 Statistical analysis
Data are presented as mean±SEM. [3H]Thymidine incorporation, protein and mRNA expression were studied in parallel in different groups and analyzed by repeated-measures analysis of variance (ANOVA). Bonferroni-corrected paired t-tests were applied to evaluate individual mean differences when ANOVA revealed significant group effects. For mRNA and protein expression in whole-tissue samples, a non-paired Student's t-test was applied. A two-tailed P-value<0.05 was considered statistically-significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Conditioned medium-induced fibroblast proliferation
Following 24-h serum starvation, [3H]thymidine uptake was not different between fibroblasts treated with conditioned medium from non-paced myocytes compared to basal (Fig. 1). Conversely, treatment with conditioned medium from rapidly-paced myocytes had a strong anti-proliferative effect, reducing [3H]thymidine uptake by ~85% (n=5, P<0.01).


Figure 1
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  		Fig. 1 [3H]Thymidine incorporation (mean±SEM) in atrial fibroblasts treated in parallel with cardiomyocyte-pacing medium alone (basal, 0), non-paced cardiomyocyte-conditioned medium (NP) and rapidly-paced cardiomyocyte-conditioned medium (5Hz); n=5, **p<0.01 vs. basal. NS=no significant difference vs. basal.

 
3.2 Myofibroblast differentiation in response to conditioned medium
Fibroblasts stimulated to differentiate acquire a myofibroblast phenotype, resembling the activated secretory cell type involved in myocardial remodeling and fibrosis, characterized by expression of the contractile protein {alpha}SMA [18]. A significantly greater percentage of large cells with strong-staining, organized {alpha}SMA stress-fibers were observed in fibroblast cultures treated with rapidly-paced cardiomyocyte medium compared to control groups (Fig. 2A–C). Increased {alpha}SMA expression was confirmed quantitatively by Western-blot analysis. Immunoblots for fibroblasts subjected to each treatment showed distinct bands (Fig. 2D) corresponding to {alpha}SMA (~42 kD) and GAPDH (~36 kD). Mean {alpha}SMA expression (Fig. 2E) was greater in fibroblasts treated with rapidly-paced cardiomyocyte medium compared to treatment with either non-paced cardiomyocyte medium or pacing medium alone (1.62±0.20 vs. 1.08±0.26 and 1.01±0.24, n=7, p<0.001). GAPDH expression was unchanged by any treatment (Table 2A).


Figure 2
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  		Fig. 2 A–C: Immunofluorescent confocal images with {alpha}SMA staining in red from 1 representative plate of treated fibroblasts per group (original magnificationx63, bar=50 µm, n=3). Arrowheads indicate large cells with strong-staining, organized {alpha}SMA stress fibers, characteristic of a myofibroblast phenotype. D: Representative {alpha}SMA (top) and GAPDH (bottom) bands from two sets of treated fibroblasts. E: Mean±SEM {alpha}SMA-band intensities relative to corresponding GAPDH signals; n=7/observation, ***p<0.001 vs. basal. NS=no significant difference vs. basal.

 

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  		Table 2 GAPDH Western-blot band intensity in arbitrary units*

 
3.3 ECM-gene expression in treated fibroblasts
We next elected to study gene expression changes in collagen-1 (Col1) and fibronectin-1 (FN1), the two most abundant collagen and non-collagen ECM proteins respectively [19], synthesis and secretion of which are both greatly enhanced in activated myofibroblasts during structural remodeling and fibrosis [20–22]. Treatment of fibroblasts did not alter the expression of the normalizing reference gene, 18S-rRNA (Table 3A). Collagen-1 (Col1, Fig. 3A) and fibronectin-1 (FN1, Fig. 3B) mRNA expression was not different between fibroblasts treated with conditioned medium from non-paced myocytes compared to basal. Collagen-1 (2.41±0.22 vs. 1.49±0.33 and 1.10±0.19) and fibronectin-1 (3.05±0.28 vs. 1.80±0.45 and 1.20±0.19) mRNA expression was significantly increased by conditioned medium from rapidly-paced cardiomyocytes. Taken together with the reduced proliferative rate and increased {alpha}SMA expression, results are consistent with the induction of myofibroblast differentiation [23].


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  		Table 3 18S-ribosomal RNA expression by real-time RT-PCR*

 

Figure 3
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  		Fig. 3 Mean±SEM relative quantity of Col1 (A) and FN1 (B) mRNA expression in treated fibroblasts; n=7/observation, **p<0.01, *** p<0.001 vs. basal. NS=no significant difference vs. basal.

 
3.4 Effects of rapid electrical stimulation of cardiomyocyte-pacing medium
Fibroblasts were treated with cardiomyocyte-pacing medium subjected to 5-Hz pacing in the absence of cardiomyocytes (group 0P) or to non-paced cardiomyocyte-pacing medium (group 0) and analyzed by [3H]thymidine incorporation (Fig. 4A), Western-blot (Fig. 4B) and real-time RT-PCR (Fig. 4C). No differences in proliferation, {alpha}SMA expression, collagen-1 or fibronectin-1 mRNA levels were seen between treatment groups. Expression of GAPDH (Table 2B) and 18S-rRNA was also unaltered (Table 3B).


Figure 4
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  		Fig. 4 Control experiments demonstrating no difference in effect of cardiomyocyte-pacing medium alone (basal, 0) versus electrically stimulated cardiomyocyte-pacing medium (0P) on fibroblast function as determined by [3H]Thymidine incorporation (A), {alpha}SMA expression (B), or Col1 and FN1 mRNA expression (C); mean±SEM, n=4/observation.

 
3.5 Effect of excitation–contraction uncoupling in rapidly-paced cardiomyocytes
To dissociate the role of cardiomyocyte excitation and contraction in tachypaced cardiomyocyte effects, cardiomyocytes were paced in the presence and absence of the myosin II inhibitor, blebbistatin [24,25]. Blebbistatin has been shown to be an effective excitation–contraction uncoupler, suppressing cardiomyocyte mechanical function at a concentration of 5 µM without altering electrical properties [26]. Activity of the drug was confirmed by measuring intracellular Ca2+ transients and cell shortening in paced cardiomyocytes before and after addition of blebbistatin to the medium (Fig. 5A). The drug abolished contraction in treated cells, without affecting Ca2+ transients, confirming cell capture and separating excitation from contraction (n=4). Conditioned medium from myocytes tachypaced in the presence of blebbistatin significantly reduced [3H]thymidine uptake (Fig. 5B) and increased {alpha}SMA expression (Fig. 5C, GAPDH values in Table 2C) in treated fibroblasts compared to fibroblasts treated with medium from blebbistatin-exposed non-paced cells and cell-free medium. Furthermore, the effects of blebbistatin-incubated tachypaced cardiomyocyte medium were indistinguishable from those of vehicle-incubated cell medium (Fig. 5B and C), confirming that tachypacing effects were unaffected by the dissociation of excitation and contraction in the rapidly-paced cardiomyocytes (n=6/observation).


Figure 5
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  		Fig. 5 A: Calcium transients and cell shortening in cultured cardiomyocytes, before and after treatment with blebbistatin (5 µM, n=4). Mean±SEM [3H]Thymidine incorporation (B) and normalized {alpha}SMA expression (C, with representative {alpha}SMA/GAPDH immunoblot in upper panel) in fibroblasts treated with conditioned medium from cardiomyocytes paced in the presence or absence of blebbistatin; Bleb=blebbistatin, n=6/observation ***p<0.001 vs. basal and NP, {dagger}{dagger}{dagger}p<0.001 vs. basal+bleb and NP+bleb.

 
3.6 Effects of angiotensin II type 1 (AT1) receptor inhibition on the responses to rapidly-paced cardiomyocyte-conditioned medium
Rapid pacing of cultured cardiomyocytes induces expression and secretion of the pro-fibrotic agent angiotensin II [27]. To elucidate the potential role of angiotensin II signaling, the AT1 receptor blocker valsartan was added to the rapidly-paced cardiomyocyte-conditioned medium before fibroblast exposure, at a concentration (10 µM) demonstrated to completely inhibit the action of up to 100 nM angiotensin II in several cell types [28–30]. The anti-proliferative effect of the conditioned medium was unaffected by valsartan (Fig. 6A). The conditioned medium effect to increase {alpha}SMA expression (Fig. 6B) was attenuated compared to vehicle-treated controls (1.00±0.10 vs. 1.52±0.14, n=6, P<0.001), without altering GAPDH (Table 2D). However, even in the presence of valsartan medium from tachypaced cardiomyocytes continued to enhance {alpha}SMA expression relative to non-paced cardiomyocyte medium (0.49±0.07, n=6) and cell-free medium (0.50±0.09, n=6, P<0.001 vs. tachypaced myocyte medium for each).


Figure 6
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  		Fig. 6 Mean±SEM [3H]Thymidine incorporation (B) and normalized {alpha}SMA expression (C, with representative {alpha}SMA/GAPDH immunoblot in upper panel) in fibroblasts treated with conditioned medium in the presence or absence of valsartan; Val=valsartan, n=6/observation ***p<0.001 vs. basal and NP, {dagger}{dagger}{dagger}p<0.001 vs. basal+Val and NP+Val.

 
3.7 In vivo ECM-gene expression in response to atrial-tachypacing
One week of experimental ATP produces a clinically-relevant model of AF-related remodeling [9,16,31]. To determine whether similar changes in ECM genes occur in vivo, collagen-1 and fibronectin-1 mRNA expression was compared in ATP dogs (n=10) and unpaced controls (n=8). ATP significantly increased both collagen-1 (2.59±0.48 vs. 0.68±0.30, P<0.01) and fibronectin-1 (4.87±0.64 vs. 1.13±0.39, P<0.001) mRNA expression (Fig. 7A and B) without altering 18S-rRNA expression (Table 3C). {alpha}SMA protein expression (Fig. 7C) was also increased by ATP (1.35±0.07 vs. 0.98±0.13, n=7/group, P<0.05), without change in GAPDH expression (Table 2E). The in vivo increases in collagen-1 (~3.8-fold) and fibronectin-1 (~4.3-fold) support the in vitro observation that rapid rates of cardiomyocyte activation modulate cardiac fibroblast behavior, increasing ECM-protein secretory function.


Figure 7
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  		Fig. 7 Mean±SEM relative quantity of Col1 (A) and FN1 (B) mRNA in 1-week atrial-tachypaced dogs (ATP, n=10) and unpaced controls (CTL, n=8). C: Normalized {alpha}SMA expression (lower panel, n=7/group) and representative {alpha}SMA/GAPDH immunoblot (upper panel, with molecular weight markers in center lane separating groups; corresponding MWs indicated with arrows at left)- {alpha}SMA is upper band, GAPDH lower band; *p<0.05, **p<0.01, ***p<0.001.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
4.1 Main findings
We have demonstrated that rapid electrical stimulation of cardiomyocytes produces factors that induce a differentiated myofibroblast phenotype, characterized by reduced proliferation and increased secretory activity. This finding suggests that rapid atrial activation may influence structural remodeling by altering fibroblast function, and may contribute to the upregulation of ECM genes in response to atrial tachycardia.

4.2 Potential mechanisms
Our findings are consistent with previous studies showing that cardiomyocyte–fibroblast interactions can directly promote fibroblast secretory function [7,8], and relate to the central paracrine role of myofibroblasts [32]. Interestingly, the importance of the interaction between cardiomyocytes and fibroblasts in fibrillating atria has been suggested recently, based on observations on atrial myocytes from AF patients [33]. Tachypacing causes cardiomyocyte Ca2+-loading, which can threaten cellular viability [6,34,35]. Conditions of impaired cell-viability are associated with fibrosis in various animal models [36,37]. The results of experiments with the excitation–contraction uncoupler blebbistatin reinforce the notion of an electrically-mediated process, and indicate that contraction per se is not needed to stimulate production of fibroblast-regulating substances from rapidly-firing cardiomyocytes. Various pro-fibrotic stimuli are known to induce a myofibroblast phenotype with enhanced secretory activity and reduced proliferation [23,38], resembling the activated cell type involved in ECM remodeling and fibrosis [39,40]. This phenotype is difficult to detect in vivo, due to fibroblast proliferation related at least in part to active recruitment and chemotaxis [41], but has been observed in cardiac fibroblasts of TGFβ1-overexpressing transgenic mice [42]. Similarly, intravenous TGFβ1 produces myocardial fibrosis with reduced fibroblast DNA synthesis [43].

Locally produced angiotensin II has been well established to exacerbate structural remodeling, and angiotensin II converting enzyme (ACE) inhibition attenuates the appearance of atrial fibrosis [44]. Cardiomyocytes have been shown to possess the machinery to synthesize angiotensin II [45], which is stimulated in cardiomyocytes when paced in vivo [46], and stretched in vitro [47]. Rapid pacing of cultured cardiomyocytes has been shown to result in significantly increased levels of expression and secretion of angiotensin II [27]. AT1-receptor blockade experiments with valsartan indicated that local production of angiotensin II plays a relatively minor role in the paced cardiomyocyte-induced myofibroblast differentiation, with valsartan only partially attenuating changes in secretory indices and showing no effect on proliferation. Other mediators including TGFβ1, endothelin-1, TNF{alpha} and natriuretic peptides, acting alone or in combination, are potential candidates to contribute to the effects of tachypaced cardiomyocytes on fibroblast function. Further studies of the potential factor(s) involved would be of great interest but are beyond the scope of the present study.

4.3 Atrial structural remodeling as a potential consequence of AF
Electrical and structural remodeling are important contributors to the AF substrate [1,31,48]. Atrial fibrosis is common in clinical AF and in AF-promoting animal models of heart failure [1,48]. AF causes atrial cardiomyocyte cell-ultrastructural remodeling [48–50]; however, it is presently unknown whether atrial tachycardia/AF can promote fibrosis in the absence of ventricular dysfunction. Frustaci et al. have documented inflammatory infiltrates, myocyte hypertrophy, myocyte degeneration and fibrosis of unknown etiology in patients with lone AF [51]. Boldt et al. have shown increased ECM deposition in lone-AF patients, as well as in mitral valve disease patients with AF versus those without [52]. Furthermore, among patients with end-stage heart failure, collagen volume fraction is positively correlated with permanence of AF [53]. These findings are consistent with the notion that AF may promote fibrosis, although it is also possible that the association is due to a greater likelihood of AF with underlying fibrosis-inducing conditions.

Ausma et al. have extensively studied structural remodeling in experimental lone AF [48–50,54,55], and have described cellular changes resembling those seen in hibernating ventricular myocardium, characterized by cardiomyocyte volume increase, myolysis, glycogen accumulation, mitochondrial changes and chromatin redistribution. They have shown increased {alpha}SMA expression and ECM accumulation after 4 months of ATP [54], and clear fibrosis after 18 months [55]. Similarly, Chiu et al. have reported greater ECM content throughout the atria of chronic ATP dogs [56], and elevated collagen-1 [57] and fibronectin-1 [58] protein deposition has been documented in a porcine model of ATP. We have previously published a genomic analysis of canine ATP-remodeling revealing qualitatively-similar but statistically non-significant ECM-gene expression changes at 24 h and 1 week [15, on-line supplement].

4.4 Novelty and potential significance
To our knowledge, this is the first reported study of the effects of rapid cardiomyocyte activation on fibroblast function. The changes we observed would be expected to have offsetting effects on atrial pro-fibrotic tendencies: reduced fibroblast proliferation would tend to reduce fibrosis, whereas increased ECM-protein synthesis tends to enhance fibrosis. These offsetting changes may explain why overt atrial fibrosis is not observed after 1-week ATP with a controlled ventricular rate [1], despite the increased ECM-protein synthetic function we show here for 1-week ATP tissue. On the other hand, under conditions that promote the migration of fibroblast progenitors into rapidly activating atria (e.g. CHF, atrial stretch, diastolic dysfunction, etc), the decreased proliferation of atrial fibroblasts caused by ATP may be less relevant than the associated increase in ECM-protein secretion, and enhanced fibrosis could result. Since AF can cause impaired myocardial function, as well as atrial dilation, prolonged AF in itself could lead to atrial fibrosis. Our findings underscore the potential importance of normalizing cardiac rhythm to prevent irreversible AF-associated arrhythmogenic structural remodeling. Conversely, if long-standing sustained AF does not by itself ultimately produce fibrosis despite the observed ECM-gene upregulation, this would imply that the myocardium can adapt to counteract this signal (possibly by inhibiting fibroblast proliferation). An understanding of the underlying mechanisms may provide clues to novel therapeutic targets for the prevention of maladaptive cardiac fibrosis. Our results thus provide new insights into cardiomyocyte-fibroblast interactions in the fibrillating atrium that may have substantial clinical relevance and importance.

4.5 Limitations of the study
The in vitro experiments analyzed fibroblast response after only 24 h of treatment, from myocytes rapidly paced at one frequency, and may not apply to other frequencies or tachypacing durations. The culture conditions do not entirely replicate the in vivo environment, in which the development of fibrosis is regulated by the complex interplay among multiple resident myocyte and non-myocyte cell types, each subject to increased rate, mechanical and electrical influences beyond those of the in vitro system. Native canine cardiomyocytes were not used in the present study, as preliminary experiments revealed that variable cell survival, adhesion and capture rates did not allow reproducible conditioning of the medium. Instead, we elected to use HL-1 cardiomyocytes, a well characterized atrial-derived murine cell-line, shown to recapitulate the primary characteristics of ATP-induced remodeling in vivo [6]. We have not isolated an individual factor through which rapidly-activated cardiomyocytes influence fibroblast function, and given the possibility of multiple secreted agents acting in concert, this will likely be an important and challenging objective for future studies. Finally, factors other than rapid atrial rates, such as atrial-ventricular dyssynchrony, may contribute to AF effects on the ECM, but are beyond the scope of the present study.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
Valsartan was kindly provided by Novartis, Montreal, Canada. BB received a Canadian Institutes of Health Research (CIHR) MD/PhD studentship. AC is a Chercheur-boursier senior of the «Fonds de la recherche en santé du Québec». The authors thank Denis Chartier, Sabrina Le Bouter, Nathalie L'Heureux, Chantal St-Cyr, and Louis Villeneuve for technical assistance, and France Thériault for secretarial support.


   Notes
 
* Supported by the Canadian Institutes of Health Research. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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