Atrial Fibrillation Induces Myocardial Fibrosis Through Angiotensin II Type 1 Receptor–Specific Arkadia-Mediated Downregulation of Smad7Novelty and Significance
Rationale: Tachycardia-induced atrial fibrosis is a hallmark of structural remodeling of atrial fibrillation (AF). The molecular mechanisms underlying the AF-induced atrial fibrosis remain unclear.
Objective: To determine the role of angiotensin II (Ang II)/Ang II type 1 (AT1) receptor–coupled transforming growth factor (TGF)-β1/Smad signaling pathway in the AF-induced atrial fibrosis.
Methods and Results: Rapid atrial pacing (1000 ppm) was applied to the left atrium of rabbit heart to induce atrial fibrillation and fibrosis. Quantitative PCR and Western blot analysis revealed that rapid atrial pacing caused a marked increase in the expression of Ang II, TGF-β1, phosphorylated Smad2/3 (P-Smad2/3), Arkadia, and hydroxyproline synthesis. However, the expression of Smad7, a key endogenous antagonist of the TGF-β1/Smad-mediated fibrosis, was significantly decreased. These changes were dose-dependently reversed by AT1 receptor antagonist losartan, implicating the involvement of AF-induced release of Ang II and activation of AT1 receptor–specific pathway. In the adult rabbit cardiac fibroblasts, Ang II increased the expression of TGF-β1, P-Smad2/3, Smad4, Arkadia, and collagen I synthesis and significantly reduced Smad7 expression. These effects of Ang II were reversed by losartan but not by the AT2 antagonist (PD123319). In addition, extracellular signal-regulated kinase inhibitor and anti–TGF-β1 antibody also blocked the Ang II–induced downregulation of Smad7. Silencing of Smad7 gene by small interfering RNA abolished the antagonism of losartan on the fibrogenic effects of Ang II on cardiac fibroblasts, whereas overexpression of Smad7 blocked Ang II–induced increase in collagen I synthesis.
Conclusions: Ang II/AT1 receptor–specific activation of Arkadia-mediated poly-ubiquitination and degradation of Smad7 may decrease the inhibitory feedback regulation of TGF-β1/Smad signaling and serves as a key mechanism for AF-induced atrial fibrosis.
Atrial fibrillation (AF) is the most common arrhythmia encountered in clinical practice1,–,3 and remains a major morbidity and mortality.4 Tachycardia-induced atrial fibrosis is a hallmark of AF-induced structural remodeling.1,2 Experimental studies in animal models have shown that atrial fibrosis plays an important role in the induction and perpetuation of AF.2,5,6 Atrial fibrosis causes intra- and interatrial inhomogeneity in conduction, thus creating a substrate for local reentry and contributing to the progressive nature of AF.6 Among the plethora of identified fibrogenic factors, the rennin–angiotensin system, especially angiotensin II (Ang II), has been implicated to play an important role in the development of cardiac remodeling during AF. However, the precise downstream molecules important in the genesis of AF-induced atrial fibrosis are currently unclear.7
Ang II activates multiple intracellular second messenger molecules.8 Ang II may mediate the profibrotic responses including cell growth, inflammation, fibroblast proliferation, and transformation and extracellular matrix (ECM) deposition primarily through transforming growth factor (TGF)-β1.9,–,11 TGF-β1 is expressed in the adult heart, where it is secreted by cardiomyocytes and myofibroblasts and retains in significant amounts in ECM as a latent cytokine. TGF-β1 signal transduction pathways are initiated by binding of TGF-β1 to membrane-bound heteromeric receptor kinases (TβRI and TβRII) that transduce intracellular signals via both Smad and non-Smad pathways.12,–,14 The primary TGF-β1 signal transduction pathway is the highly conserved Smad pathway. Activated TβRI and TβRII receptors phosphorylate receptor-regulated Smads (R-Smads) (such as Smad2/3), which form homomeric complexes and heteromeric complexes with comediator Smad (co-Smad, such as Smad4).12,–,14 These active Smad complexes translocate into the nucleus, where they accumulate and bind to target genes to directly regulate their transcription. The inhibitory Smad proteins (I-Smads) (such as Smad6/7) are capable of inhibiting TGF-β1 signaling.15 Smad7 binds to activated TβRI and prevents phosphorylation of Smad2/3 or recruits the ubiquitin ligases Smurf1 and Smurf2 to induce proteasomal degradation of the receptor complexes.15 Therefore, Smad7 may act as a major negative regulator forming autoinhibitory feedback loops and mediating crosstalk with other signal pathways.
In addition, Ang II can also activate the Smad pathways by a TGF-β1–independent activation of Ang II type 1 (AT1) receptors and mitogen-activated protein kinases (MAPKs).16 It has been reported that Ang II–induced left ventricular remodeling and fibrosis are dependent on both extracellular signal-regulated kinase (ERK) and Smad activation and that inhibition of either pathway is equally efficacious in restoring left ventricular function and architecture.17 However, it is unknown whether ERK is also involved in Smad signaling in the AF-induced atrial fibrosis. It remains to be elucidated how the Smad pathways are involved in AF-induced fibrosis.
In the present study, we investigated the role of Smad pathways in AF-induced atrial fibrosis by examining the expression of Ang II, TGF-β1, Smad, and collagen synthesis in rabbit atria subjected to rapid atrial pacing (RAP) and in the adult rabbit cardiac fibroblasts in the absence or presence of antagonists of AT1 receptors. Our results suggest a key causal role of AT1 receptor–specific downregulation of the inhibitory Smad7 in AF-induced atrial fibrosis.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
All experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals (NIH Publication No.85-23, revised 1996) and were in compliance with the guidelines specified by the Chinese Heart Association policy on research animal use and the Public Health Service policy on the use of laboratory animals.
Male New Zealand rabbits (2.0 to 2.5 kg) were randomly divided into 6 groups (n=8 for each group): normal control (N), sham control (S), RAP alone (P), RAP+losartan 10 mg·kg−1 daily (D1), RAP+losartan 20 mg·kg−1 daily (D2), and RAP+losartan 30 mg·kg−1 daily (D3).
Left Atrial Rapid Pacing
Rabbits were anesthetized with 30 mg·kg−1 pentobarbital sodium (IV) and then intubated and ventilated with a volume-cycled ventilator (Model HX-200, TAIMENG, Chengdu, China). The left thoracic cavity was opened via the sternum from the second intercostals to the fourth intercostals, and then the heart was exposed by a dilator. A custom-designed set of electrodes, comprising a pair of electrodes with a distal hook for pacing and a pair of electrodes with an interelectrode distance of 15 mm aligned proximally for recording, was sutured to the epicardial surface of the left atrium. The reason for choosing the left atrium for RAP was because of its higher inducibility of AF than right atrium.18 The distal ends of these electrodes leads were tunneled subcutaneously and exposed on the back and connected to a pacemaker (output of 6V with 1.0 ms pulse duration, Guangzhou Academy of Sciences, China) in the jacket. The pacemaker was programmed to provide RAP at 1000 ppm, and this pacing rate was maintained continuously for 4 weeks with a brief period of break for measurement of electrophysiological and mechanical parameters. Rabbits in normal control (group N) were not subjected to surgery, whereas those in sham control (group S) were operated with the identical surgery procedure but RAP. When surgery was completed, the rabbits were given antibiotics and then allowed to recover for 5 days. Postoperative care included the administration of antibiotics and analgesics. In groups D1, D2, and D3, oral administration of losartan (Merck) started at the same time as RAP and continued for 4 weeks during the pacing period. The same amount of normal saline was given to rabbits in groups N, S, and P.
ECG was recorded before and after the pacing. During the 4-week period of pacing, ECG was measured every day to ensure that the pacemakers were working properly. Atrial effective refractory period (AERP) of the left atrial appendage was measured at basic cycle lengths (BCLs) of either 120 or 200 ms. Five basic drive stimuli were followed by 1 single premature stimulus, and all stimuli were twice the diastolic threshold. The interval between S1 and S2 was decreased in steps of 2 ms, and AERP was determined to be the shortest S1 to S2 interval resulting in a propagated atrial response.
At the end of the experiment, all rabbits were euthanized, and the hearts were removed and weighed immediately. Left atria were then quickly removed and cut into 3 (upper, meddle, lower) sections. Each section was divided equally into 4 pieces. Three pieces from each section were randomly chosen to form a new part for radioimmunity assay of Ang II accumulation and hydroxyproline content analysis. One part was paraffin-embedded for Masson trichrome staining. The remaining parts were quickly frozen in liquid nitrogen and maintained at −80°C until use for mRNA and protein analysis. The entire procedure was performed in cold conditions.
Masson Trichrome Staining for Collagen
Masson trichrome staining of the paraffin section prepared from Bouin-fixed samples was performed as previously described.19 To quantitate atrial collagen content, images were captured with a digital camera and the red pixel content of the myocardium was measured using Adobe Photoshop 5.5 and Scion Images for Windows Beta 4.0.2 software as described.19 The analyses were performed by at least 2 independent investigators on coded specimens in a blinded fashion.
Left Atrial Collagen Content
Hydroxyproline content was measured as an index of the amount of collagen, which reflects the degree of myocardial fibrosis. The specimens were minced and then homogenized for 2 minutes at 4°C in sufficient deionized water to yield a 10% mixture (wt/vol). The hydroxyproline content of homogenates was assayed as described by Jamall et al.20
Radioimmunity Assay of Ang II Accumulation
Tissues (50 mg) from each sample was homogenated in cool acetic acid, centrifuged, and torrefied per the instructions accompanying the kit (125ICAMP RIA kit, Medical College of SUN Yet-San University, Guangzhou China) and assayed with a γ-immunity indicator (FM2000, Xi'an, China).
Cardiac fibroblasts were isolated from left atria of adult New Zealand rabbit heart as described previously.21,22 Cells were subcultured and passed as they reached 80% to 90% confluence. The purity of fibroblasts used in these experiments was 90% using routine phenotyping methods as described previously.21,22
Small Interfering RNA Transfection
Silencer small interfering (si)RNAs targeting Smad2, Smad3, or Smad7 were synthesized by Ambion based on the sequences of rabbit Smad2 (GenBank accession no. AAGW02032355), Smad3 (GenBank accession no. AAGW02025187), and Smad7 (GenBank accession no. AAGW02032373) and were used in knockdown experiments. To demonstrate that the transfection does not induce nonspecific effect on gene expression, a control siRNA (GenBank no. NM001082253) that has no homology to known sequences from rabbit or humans was used. The adult rabbit cardiac fibroblasts were cultured to approximately 80% to 90% confluence and then transfected with 400 pmol of siRNA using Lipofectamine 2000 in a 6-well plate according to the instructions of the manufacturer. Clones of Smad2, Smad3, or Smad7 siRNA that presented at least 90% inhibition of target genes were chosen for further analysis. For experimental procedures, second to third passages of each clone were used. The morphology of knockdown cells was monitored during culture under an inverted microscope. After 24 hours of transfection, cells were treated with or without Ang II (1 μmol/L)/losartan (10 μmol/L) for 48 hours. Levels of Smad2/3, Smad7, and collagen I expression were determined by Western blot.
Smad7 Expression Plasmid and Transfection
The Smad7 expression plasmid pcDNA3-FLAG-Smad7 was constructed as described previously.23 Fibroblasts were grown to 50% confluence in 100-mm dishes. After 4 hours of incubation with serum-free medium, cultures were transfected with 1.0 μg of pcDNA3-FLAG-Smad7 or 1.0 μg of empty pcDNA3 vector in 6-well plates by using Lipofectin (Invitrogen, Carlsbad, Calif) according to the instructions of the manufacturer. After transfection, cells were treated with Ang II (1 μmol/L) for 48 hours in media. In this experiment, total DNA in each well was adjusted to the same amount using vector DNA. All assays were performed in triplicate, and the results are presented as the means(±SE) of 3 independent transfections.
Quantitative Real-Time Polymerase Chain Reaction
Total RNA was extracted with TRIzol reagent (Gibco-BRL Life Technologies). cDNA was synthesized with SYBR ExScript RT-PCR kit (TOYOBO, Japan) according to the protocol provided by the manufacturer. PCR primers for TGF-β1 (forward: 5′-ACA TTG ACT TCC GCA AGG AC-3′; reverse: 5′-TAG TAC ACG ATG GGC AGT GG-3′) and Smad7 (forward: 5′-GTG GCA TAC TGG GAG GAG AA-3′; reverse: 5′-GAT GGA GAA ACC AGG GAA CA-3′) were designed with Primer Express software (Applied Biosystems). GAPDH (forward: 5′-GCA CCG TCA AGG CTG AGA AC-3′; reverse: 5′-ATG GTG GTG AAG ACG CCA GT-3′) was used as reference to normalize input amounts of RNA for all samples. Real-time PCR was performed using an ABI7300 Real-Time PCR system (Applied Biosystems) with SYBR green fluorophore.24,25 All reactions were performed in at least duplicate for every sample. Threshold cycle (Ct) data were collected using the Sequence Detection Software version 1.2.3 (Applied Biosystems). GAPDH was used as an internal control. mRNA fold change relative to GAPDH was calculated with the comparative Ct method of 2−ΔΔCt.25
Immunohistochemistry was performed using a microwave-based antigen retrieval technique as described previously.26 Briefly, cells after culturing in 6-chamber glass slides were stained with antibodies against Smad7 (Santa Cruz Biotechnology) using a 3-layer peroxidase antiperoxidase method. For analysis of Smad7 in cultured cardiac fibroblasts, positive stain for Smad7 was counted in 500 cells and expressed as percentage. All examinations were performed on coded slides in a blinded fashion.
Low-molecular-weight marker (Cell Signaling Technology) and 50 μg of protein from samples were separated on 10% or 12% SDS gels by SDS-PAGE. Separated protein was transferred to a poly(vinylidene difluoride) membrane that was blocked at room temperature for 1 hour in Tris-buffered saline with 0.2% Tween 20 (TBS-T) containing 5% skim milk and probed with primary antibodies overnight at 4°C. The diluted concentrations of the primary antibodies (Santa Cruz Biotechnology) were as follows: TGF-β1, 1:200; phosphorylated Smad2/3, 1:250; Smad4, 1:200; Smad7, 1:200; collagen I, 1:250; β-actin, 1:500. Secondary antibodies (Cell Signaling Technology) included horseradish peroxidase–labeled and were diluted 1:1000/2000 with 0.2% TBS-T and 1% skim milk and incubated for 1 hour at room temperature. Protein bands on Western blots were visualized using ECL Plus (Amersham, Arlington Heights, Ill). Relative band densities of proteins in Western blots were normalized against β-actin.
Data are expressed as means±SE. ANOVA and Student t test were used to determine statistical significance. A 2-tailed probability value of ≤0.05 was considered statistically significant.
RAP-Induced Atrial Fibrosis
Conventional ECGs were recorded in anesthetized intact rabbits. Figure 1 depicts representative ECG recordings before (Figure 1A, a) and after (Figure 1A, b) 4 weeks of RAP. It is clear from the ECG recordings that RAP caused disappearance of P waves and absolutely irregular RR intervals, typical signs of sustained AF. RAP induced sustained (>24 hours) AF in 6 of 8 (75%) animals in group P but none in groups D1, D2, and D3. Epicardium stimulation induced sustained AF in 2 of 8 (25%) animals in group P and group D1 but none in groups D2 and D3. The pacemaker was not turned off automatically during AF, except for a brief period of interruption for measurement of electrophysiological and mechanical parameters. Before RAP (group N), the baseline AERP was 107±3 and 93±2 (n=8) at BCLs of 200 and 120 ms, respectively. In response to 4 weeks of RAP (group P), AERP decreased significantly to 81±2 (n=8; P<0.05 versus group N) at BCL 200 ms and 71±3 (n=8; P<0.05 versus group N) at BCL 120 ms. In the 3 groups with different doses of losartan, AERP at BCL 200 ms was prolonged from 81±2 to 91±4 in group D1 (n=8; P<0.05 versus group N or group P), 94±2 in group D2 (n=8; P<0.05 versus group N or group P), and 96±2 in group D3 (n=8; P<0.05 versus group N or group P). Similarly, AERP at BCL 120 ms was prolonged from 71±3 to 83±2 ms in group D1 (n=8; P<0.05 versus group N or group P), 86±2 ms in group D2 (n=8; P<0.05 versus group N or group P), and 87±2 ms in group D3 (n=8; P<0.05 versus group N or group P) (Table 1). No significant difference of left ventricular ejection fraction in rabbits between pacing groups and no-pacing groups was observed (data not shown). There was no evidence of heart failure in any groups of the rabbits studied. Therefore, it is highly unlikely that the results we observed in our RAP model can be attributed to an indirect effect of RAP-induced heart failure.
We then examined whether RAP effectively caused fibrosis in the left atria. Hydroxyproline content was measured as an index of the amount of collagen, which reflects the degree of myocardial fibrosis. As shown in Figure 1B and 1C and Table 2, RAP caused a marked deposition of collagens as estimated by Masson trichrome staining (Figure 1B and 1C). The hydroxyproline content in the heart of group P was significantly higher than that in the heart of group N or group S. Left atrial weight and left atrial mass index in group P were significantly higher than those in group N. These results indicate that RAP at 1000 ppm for 4 weeks caused significant atrial fibrosis.
RAP-Induced Changes in Ang II Levels
Because Ang II has been implicated an important role in cardiac fibrosis, we tested whether RAP-induced atrial fibrosis is associated with any changes in Ang II level in the left atria. As shown in Table 2, the accumulation of Ang II, as estimated by radioimmunity in the left atrium, was significantly increased in group P, D1, D2, and D3 when compared with that in the nonpaced group N (P<0.05), whereas it is comparable among the groups subjected to RAP (P>0.05). Therefore, RAP-induced atrial fibrosis was accompanied with a significant increase in Ang II accumulation in the atrial tissues.
Effects of AT1 Receptor Antagonist on the RAP-Induced Atrial Fibrosis
As shown in Table 2 and Figure 1B and 1C, treatment of the animals with AT1 receptor antagonist losartan caused a significant decrease in hydroxyproline content, collagen I and collagen III deposition, left atrial weight, and left atrial mass index and attenuated the progression of RAP-induced atrial fibrosis. These data suggest that the RAP-induced atrial fibrosis may be mediated through an activation of AT1 receptors in the heart.
RAP-Induced Changes in the Expression of TGF-β1 and Smads in the Left Atrium
Previous studies have shown that activation of the AT1 receptors by Ang II upregulates TGF-β1 expression in cardiac myocytes and fibroblasts.27,28 Therefore, we next examined whether RAP causes changes in the expression level of TGF-β1 and Smads and whether the changes are AT1 receptor–specific.
As shown in Figure 2, real-time PCR and quantitative Western blot analysis found that the expression level of TGF-β1 mRNA (Figure 2A, a) and protein (Figure 2B, a and b) was significantly increased in the left atrium of group P compared with the nonpaced group N and group S. In addition, the phosphorylated Smad2/3 (P-Smad2/3; Figure 2B, a and d) and the E3 ubiquitin ligase Arkadia (Figure 2B, a and e) were also increased in the left atrium among the pacing groups compared with the nonpaced groups. However, the expression of the inhibitory Smad, Smad7, was significantly decreased in group P (Figure 2B, a and c). These RAP-induced changes in TGF-β1, P-Smad2/3, Arkadia, and Smad7 expression were antagonized by AT1 receptor agonist losartan in a dose-dependent manner.
The above results from the whole-animal model strongly suggest that RAP may cause atrial fibrosis through an Ang II/AT1 receptor–specific activation of TGF-β1/Smad pathway. The downregulation of Smad7 may be a key for the increased activation of P-Smad2/3 that is responsible for the increased atrial fibrosis during AF. To further confirm our observations in the in vivo study and gain more information about how Smad7 is downregulated during RAP, we examined whether stimulation of the growth-arrested adult rabbit cardiac fibroblast with Ang II (10−6 mol/L) for 48 hours would cause similar changes in the Smad signaling.
Ang II–Induced TGF-β1 and Smad Expression in Cultured Cardiac Fibroblasts
The primary cardiac fibroblasts (passage 2) were serum starved for 24 hours, followed by treatments with the following for 48 hours: (1) DMSO (0.005%) as control group; (2) Ang II (1 μmol/L); (3) Ang II (1 μmol/L)+losartan (10 μmol/L); and (4) Ang II (1 μmol/L)+PD123319 (AT2 antagonist, 100 μmol/L with 0.005% DMSO). Cells were treated with losartan or PD123319 at 1 hour before Ang II stimulation. Either losartan (10 μmol/L) alone or PD123319 (100 μmol/L) alone failed to significantly affect Smad7 expression. The protein expression of TGF-β1, P-Smad2/3, Smad4, Arkadia, and collagen I were determined by Western blot analysis. Continuous stimulation of cardiac fibroblasts with Ang II for 48 hours caused a significant increase in the expression of TGF-β1, P-Smad2/3, Smad4, Arkadia, and collagen I (Figure 3). AT1 antagonist losartan (10−5 mol/L) inhibited the effects of Ang II on TGF-β1 protein expression, Smad2/3 phosphorylation, and collagen I expression. The AT2 receptor antagonist PD123319, however, had no effect on the Ang II–induced changes in TGF-β1 and Smad expression, indicating an AT1 receptor–specific mechanism in the activation of the TGF-β1/Smad signaling pathway.
Ang II–Induced Downregulation of Smad7 and Increased Ubiquitin-Dependent Protein Degradation
As shown in Figure 4A, the Smad7 protein expression in cardiac fibroblasts was significantly reduced by Ang II stimulation, which could be antagonized by the AT1 receptor antagonist losartan but not by the AT2 receptor antagonist PD123319. These findings are in agreement with the findings in the in vivo model (see Figure 2). Because Ang II also caused an increase in Arkadia (see Figures 2 and 3) and previous evidence indicated that Arkadia may act as an E3 ubiquitin ligase and play a key role in the regulation of TGF-β/Smad signaling through degradation of signal molecules,29 we investigated whether the ubiquitin-dependent protein degradation through Arkadia is involved in the AT1 receptor–specific downregulation of Smad7 expression. As shown in Figure 4A, pretreatment of the cardiac fibroblasts with a specific proteasome inhibitor, lactacystin (10−4 mmol/L), for 3 hours before Ang II stimulation reversed Ang II–induced Smad7 downregulation, suggesting that Arkadia-mediated ubiquitin–proteasome protein degradation of Smad7 may be responsible for the AT1 receptor–specific downregulation of Smad7.
AT1 Receptor–Specific Downregulation of Smad7 Through Both TGF-β1 and ERK Pathways
Because Ang II/AT1 receptors activate multiple intracellular signaling pathways, including the TGF-β1 and ERK1/2 pathways that participate in the regulation of Smads, we further tested whether both TGF-β1 and ERK1/2 pathways are involved in the regulation of Smad7 expression. As shown in Figure 4B and 4C, treatment of adult rabbit cardiac fibroblasts with anti–TGF-β1 antibody or the ERK inhibitor PD98059 both blocked the Ang II–induced downregulation of Smad7 expression. Smad7 expression was increased by blockade of endogenous TGF-β1 to an almost identical level as that by ERK inhibitor. Blockage of AT1 receptor by losartan caused nearly 1-fold increase in Smad7 expression (Figure 4B) compared with blockade of endogenous TGF-β1 or inhibition of ERK. Blockade of AT2 receptor by PD123319, however, had no effect on the expression of Smad7 in these cells. Similar results were found in immunohistochemistry staining of Smad7 in the primary cardiac fibroblasts (Figure 4C).
Effects of Smad7 Gene Silencing by siRNA on Ang II–Induced Collagen Synthesis in Cardiac Fibroblasts
To further determine the relative role of Smad2/3 and Smad7 in the AF/Ang II–induced fibrosis, silencer siRNAs targeting Smad2, Smad3, or Smad7 were transfected into the primary cardiac fibroblasts (passage 2 to 3). Cells were serum starved for 24 hours and then transfected with 400 pmol of control siRNA, Smad2 siRNA, Smad3 siRNA, or Smad7 siRNA under the same conditions for 24 hours. The cells were then treated with DMSO (0.005%) as control group, Ang II (10−6 mmol/L), losartan (10−5 mmol/L), or Ang II (10−6 mmol/L)+losartan (10−5 mmol/L. Cells were treated with losartan at 1 hour before Ang II stimulation) for 48 hours.
Transfection with siRNAs targeting Smad2, Smad3, or siSmad7 significantly downregulated the expression level of Smad2, Smad3, or Smad7 (>90%), as measured by quantitative real-time PCR (data not shown) and Western blotting (Figure 5A [a and b] and Figure 6A [a], respectively).
As shown in Figure 5A through 5C, Ang II caused a significant increase in collagen I expression (0.369±0.017 versus 0.193±0.012; n=10; P<0.001) and a decrease in Smad7 expression (0.184±0.003 versus 0.084±0.002; n=10; P<0.001) in cardiac fibroblasts transfected with control siRNA. When Smad2 and Smad3 genes were silenced by siRNA the Ang II– induced increase in collagen I expression in the fibroblasts was significantly less than that in the fibroblasts transfected with control siRNA (0.237±0.017 for Smad2 siRNA and 0.236±0.016 for Smad3 siRNA versus 0.369±0.017 for control siRNA; n=10; P<0.001, respectively). However, knockdown of Smad2 or Smad3 did not completely block the Ang II–induced increase in collagen I expression (0.237±0.017 and 0.236±0.016 for Smad2 siRNA and Smad3 siRNA, respectively, versus 0.193±0.012 for nontransfected; n=10; P<0.05) and had no effect on the Ang II–induced decrease in Smad7 expression. Losartan alone had no effects on collagen I and Smad7 expression in these cells. However, the Ang II–induced increase in collagen I expression was completely blocked by losartan. In the presence of losartan, Ang II caused a significant increase (instead of decrease) in Smad7 expression.
In the cells transfected with control Smad7 siRNA, as shown in Figure 6A (b and c), Ang II induced a significant increase in collagen I (0.35±0.02 versus 0.18±0.01 in nontransfected cells; n=10; P<0.001) and decrease in Smad7 expression (0.094±0.001 versus 0.189±0.004 in the nontransfected cells; n=10; P<0.001), which was comparable to that in the fibroblasts under control conditions (see Figure 3F and Figure 4). When Smad7 gene were silenced by siRNA, Smad7 expression was significantly decreased in the cells (0.009±0.001; n=7; P<0.001 versus control siRNA and nontransfection). Under basal conditions without Ang II stimulation siRNA knockdown of Smad7 slightly increased the collagen I synthesis but it was not statistically significant (0.176±0.004 versus 0.192±0.002, respectively; n=5; P>0.05). Losartan alone had no effects on collagen I expression in these cells but partially blocked the Ang II–induced increase in collagen I expression (0.27±0.01; n=7; P<0.001 versus control siRNA and nontransfection, respectively).
These results suggest that although upregulation of Smad2/3 may contribute to the Ang II–induced increase in the collagen I synthesis, the downregulation of Smad7 may play a major role in the AF/Ang II–induced atrial fibrosis.
Effects of Overexpression of Smad7 on Ang II–Induced Collagen Synthesis in Cardiac Fibroblasts
To further confirm whether downregulation of Smad7 plays a causal role in the AF/Ang II–induced atrial fibrosis, we examined the effects of overexpression of Smad7 on the Ang II–stimulated expression of collagen I in the isolated cardiac fibroblasts.
As shown in Figure 6B, overexpression of Smad7 caused a significant decrease in collagen I expression under basal conditions (in the absence of Ang II) compared with the control cells (without transfection) and the cells transfected with empty vectors (n=7; P<0.05 versus nontransfection or empty vector). Overexpression of Smad7 also antagonized the Ang II–induced increase in collagen I expression (0.169±0.002 [n=7] versus 0.345±0.033 [n=4]; P<0.001; see Figure 3F) and masked the antagonism of losartan on the fibrogenic effects of Ang II (0.169±0.001 [n=7] versus 0.184±0.014 [n=4]; P=0.176; see Figure 3F). In the presence of losartan, the expression level of collagen I (0.150±0.001; n=7) is comparable to that under basal conditions (0.151±0.001; n=7; P=0.493). These results strongly suggest that downregulation of Smad7 is required for the Ang II–induced increase in collagen I synthesis and that the reduced expression of inhibitory Smad7 may play a causal role in the AF-induced atrial fibrosis.
In this study, we examined the role of the Ang II/AT1 receptor/Smad signaling pathway in the AF-induced atrial fibrosis. The major novel finding of this study include the following: (1) our real-time PCR and quantitative Western blot analyses revealed that RAP-induced AF caused a significant downregulation of Smad7 expression in rabbit atria and increased P-Smad2/3 activity and atrial fibrosis; (2) the AF-induced downregulation of Smad7 and upregulation of P-Smad2/3 and collagen production were dose-dependently reversed by the AT1 receptor antagonist losartan but not by the AT2 receptor antagonist PD123319; (3) in isolated adult rabbit cardiac fibroblasts, losartan also caused an upregulation of Smad7 expression and decreased the level of P-Smad2/3, Smad4, and collagen I; (4) blockade of TGF-β1 or inhibition of ERK both upregulated Smad7 expression, indicating that both TGF-β1 and ERK signaling pathways may be involved in the AT1 receptor–specific regulation of Smad7 expression; (5) both RAF and Ang II increased Arkadia protein expression and degradation of Smad7, which could be reversed by AT1 receptor antagonist but not by AT2 receptor antagonist; (6) blockage of proteasome could diminish Ang II–induced degradation of Smad7; and (7) silencing of Smad7 gene by siRNA abolished the antagonism of losartan on the fibrogenic effects of Ang II on cardiac fibroblasts, whereas overexpression of Smad7 blocked Ang II–induced increase in collagen I synthesis. These results strongly suggest that AT1 receptor–specific downregulation of Smad7 through increased Arkadia-medicated protein degradation may be a novel mechanism for the AF-induced atrial fibrosis.
AF and Atrial Fibrosis
A significant feature of AF-induced structural remodeling is tachycardia-induced atrial fibrosis,1,2 which plays an important role in the induction and perpetuation of AF.2,5,6 Biopsy and autopsy from patients and animal models with AF have displayed the presence of atrial fibrosis.30 There exist, however, controversial opinions about whether structural changes in the atria are attributable to tachycardia or related to underlying diseases.31,32 In this study, we demonstrated that RAP alone induced profound changes in gene expression of collagens and fibrogenic factors in the heart, strongly suggesting that tachycardia during AF may cause atrial remodeling resulting from atrial fibrosis. In addition to the increase in the hydroxyproline content and left atrial weight, a significant deposition of cardiac collagen I and III was observed in the atria subjected to rapid pacing. These results also support previous observations that atrial fibrosis consisting of the collagen type I and III is a typical feature of AF, especially in the permanent form of AF.33 Atrial fibrosis causes intra- and interatrial inhomogeneity in conduction, thus creating a substrate for local reentry and contributing to the progressive nature of AF.6
Several animal models of chronic sustained AF have been developed to assess the atrial electric remodeling and arrhythmias of AF. In canine models of pacing-induced AF,34 AERP was decreased, and the animals became more vulnerable to AF. It has been reported that rapid pacing induces AF with much higher incidence at the left atrial site than at the right atrium or any other sites, partly because of the inhomogeneous dispersion of AERP.18 In our rabbit model of left atrial RAP, the AERP of group P was statistically shortened compared with the baseline. After 4 weeks of treatment with AT1 receptor antagonist, AERPs of groups D1, D2, and D3 were shortened to a significantly less extent than group P in a dose-dependent manner. The shortening of AERP correlated with atrial fibrosis. The structural abnormalities of the atria, especially their increased size, likely contributed to their continued vulnerability to electric stimulation, suggesting that atrial electric remodeling has a close relationship with atrial fibrosis.
Ang II/TGF-β1/Smad Pathways in AF-Induced Atrial Fibrosis
Both Ang II and TGF-β1 have been found to stimulate the progression of cardiac fibrosis during cardiac hypertrophy and heart failure.27,35,36 Ang II participates in the development of AF-induced myocardial fibrosis through activation of AT1 and AT2 receptors.9,–,11 AT1 receptor antagonism significantly attenuates fibrosis process of atrial fibrillation in dogs.37 In the present study, we found that application of losartan decreased the deposition of cardiac collagens in a dose-dependent manner, suggesting that activation of AT1 receptors may be an important mechanism for AF-induced atrial fibrosis. Recent studies indicate that Ang II and TGF-β1 do not act independently from one another but rather act as part of a network that promotes cardiac remodeling.27 Ang II mediates the expression of TGF-β1 in vitro38 and in vivo39 in various cell types, including cardiac fibroblasts. Serum level of TGF-β1 was increased in patients with AF, and it was downregulated after defibrillation therapy.40 Changes in genes regulating TGF-β1 function and signaling were observed in patients with permanent AF41 and in canine AF models.42 TGF-β1 is a known profibrotic agent, and its enhanced expression has been shown to increase myocardial fibrosis.12,43 Overexpression of constitutively active TGF-β1 in mouse caused only atrial interstitial fibrosis but not the ventricles.36 It seems that atria are more susceptible to TGF-β1 influences than ventricles. In the present study, we found that TGF-β1 was significantly upregulated in left atria of the pacing group (group P) as compared with the nonpacing controls (group N and group S in Figure 2). This corresponds well with an excessive atrial collagen synthesis observed in group P with AF (Table 1 and Figure 1). Consistent with our in vivo observations, we found that AT1 receptor antagonist, but not AT2 receptor antagonist, inhibited the Ang II–induced increase in TGF-β1 expression in isolated adult rabbit cardiac fibroblasts (Figure 3), implicating an AT1 receptor–specific mechanism for the Ang II activation of TGF-β1 signaling pathway.
Although it has been demonstrated that TGF-β1/Smad pathway is involved in the cardiac fibrosis in myocardial infarction, it is not clear whether and how TGF-β1/Smad pathway is involved in the AF-induced atrial fibrosis. Binding of TGF-β1 to its 2 membrane-bound receptor kinases, TβRI and TβRII, initiates a series intracellular signals via both non-Smad pathways and Smad-mediated transcriptional regulation.12,–,14 The signaling of TGF-β1 is finely regulated at different levels. Inhibitory Smads, including Smad6 and Smad7, are key regulators of TGF-β1 signaling through negative-feedback loops. They can form stable complexes with activated TβRI and block the phosphorylation of regulatory Smads (R-Smads) (Smad2/3) or recruit ubiquitin E3 ligases, such as Smurf1/2, resulting in the ubiquitination and degradation of the activated TβRI. Smad6/7 can also inhibit TGF-β1 signaling in the nucleus by interacting with transcriptional repressors, such as histone deacetylases, or disrupting the formation of the TGF-β1–induced functional Smad-DNA complexes.15 Smad7, in turn, is regulated by various mechanisms such as Arkadia-mediated ubiquitination and degradation (see below). Therefore, Smad7 may act as a major negative regulator in the autoinhibitory feedback loops and mediate the crosstalk with other signal pathway. Deregulation of Smad7 expression has been associated with various human diseases, such as inflammatory disease and carcinogenesis. Overexpression of Smad7 has been shown to antagonize TGF-β–mediated fibrosis, carcinogenesis, and inflammation, suggesting a therapeutic potential of Smad7 to treat these diseases.44 In the present study, we found that the RAP-induced stimulation of Ang II/AT1/TGF-β1 pathway caused a significant downregulation of Smad7 expression, which might result in a marked increase in P-Smad 2/3 and collagen I and III synthesis in the atria subjected to RAP.15 Therefore, a reduction in Smad7 may be the major mechanism for the AF-induced atrial fibrosis.
Mechanisms Underlying AF-Induced Downregulation of Smad7 Expression
In the present study, we found that the RAP-induced Ang II release and AT1 receptor activation caused a significant reduction of Smad7 expression in the atria. We also demonstrated that Smad7 expression was significantly upregulated by the AT1 receptor antagonist losartan in a dose-dependent manner. Because both anti–TGF-β1 antibody and ERK inhibitor PD98059 were almost equally efficacious in increasing Smad7 expression in adult rabbit cardiac fibroblasts, the AT1 receptor–specific downregulation of Smad7 expression may involve both TGF-β1 and ERK signaling pathways.16 In addition, we found that RAP-induced stimulation of Ang II/AT1 receptor increased Arkadia expression in the left atria, and AT1 receptor antagonist diminished Arkadia expression in rabbit cardiac fibroblasts. Proteasome inhibitor prevented the Ang II–induced Smad7 downregulation (Figure 4A). These results indicated that activation of the Arkadia–ubiquitin–proteasome pathway may be responsible for the AT1 receptor–specific downregulation of Smad7 during AF. Furthermore, overexpression of Smad7 blocked Ang II–induced increase in collagen I synthesis, and silencing of Smad7 gene by siRNA abolished the antagonism of losartan on the fibrogenic effects of Ang II in cardiac fibroblasts, whereas knockdown of Smad2 or Smad3 did not completely inhibit the Ang II–induced increase in collagen I synthesis, implicating a causal role for Smad7 downregulation, but not upregulation of Smad2/3, in AF/Ang II–induced atrial fibrosis.
In summary, the present study provides compelling in vivo and in vitro experimental evidence that tachycardia during AF may activate the Ang II/AT1 receptor/TGF-β1 and ERK/Smad signaling pathways and AT1 receptor–specific downregulation of the inhibitory Smad proteins (I-Smads) (Smad7) may serve as a key mechanism for the AF-induced atrial fibrosis. These results may provide new insights into the understanding of the mechanisms for AF-induced atrial fibrosis and myocardial remodeling and valuable information for novel therapeutic targets of AF.
Sources of Funding
This study was supported by the Science Fund Committee of Guangdong Province of China grant 06021342 (to X.G.); National Center for Research Resources grant P-20 RR-15581 (to D.D.D.); National Heart, Lung, and Blood Institute grant HL63914 (to D.D.D.); American Diabetes Association Innovation Award 7-08-IN-08 (to D.D.D.); and National Basic Research Program of China grant 2009CB521903 (to D.D.D.).
Non-standard Abbreviations and Acronyms
- atrial effective refractory period
- atrial fibrillation
- Ang II
- angiotensin II
- angiotensin II type 1
- angiotensin II type 2
- basic cycle length
- extracellular matrix
- extracellular signal-regulated kinase
- mitogen activated protein kinase
- phosphorylated Smad
- rapid-atrial pacing
- rennin–angiotensin system
- small interfering RNA
- transforming growth factor
- Received June 6, 2009.
- Revision received March 6, 2010.
- Revision received October 11, 2010.
- Revision received November 12, 2010.
- Accepted November 19, 2010.
- © 2011 American Heart Association, Inc.
- Wyse DG,
- Gersh BJ
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- Koutsopoulos AV,
- Mavrakis HE,
- Chlouverakis GI,
- Vardas PE
- Buxton IL,
- Duan D
- Rodriguez-Vita J,
- Sanchez-Lopez E,
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Novelty and Significance
What Is Known?
The renin–angiotensin system and, more specifically, angiotensin II (Ang II), is involved in the genesis of the atrial fibrosis induced by excessively rapid heartbeat during atrial fibrillation (AF).
What New Information Does This Article Contribute?
Rapid atrial pacing induces atrial fibrosis in adult rabbit heart through release of Ang II.
Rapid atrial pacing–induced stimulation of Ang II type 1 (AT1) receptor increases expression of TGF-β1, ERK, Smad2/3, Smad4, and collagen I but significantly decreases Smad7 through activation of the Arkadia-mediated protein degradation.
Ang II/AT1 receptor–specific downregulation of the inhibitory Smad7 plays a key causal role in AF/Ang II–induced atrial fibrosis.
Although many fibrogenic factors have been implicated in the development of cardiac remodeling during AF, the precise downstream molecules important in the genesis of AF-induced atrial fibrosis are currently unclear. Here, we provide in vivo and in vitro evidence that tachycardia during AF activates the Ang II/AT1 receptor/TGF-β1 and ERK/Smad signaling pathways and increases the Arkadia–mediated degradation of Smad7. Downregulation of Smad7 may play a causal role in the AF/Ang II–induced atrial fibrosis. These results may provide new insights into the understanding of the mechanisms for AF-induced atrial fibrosis and myocardial remodeling and valuable information for novel therapeutic targets of AF.