Pirfenidone Prevents the Development of a Vulnerable Substrate for Atrial Fibrillation in a Canine Model of Heart Failure
Background— Atrial fibrosis is an important substrate in atrial fibrillation (AF), particularly in the setting of structural heart disease. In a canine model, congestive heart failure (CHF) produces significant atrial fibrosis and the substrate for sustained AF. This atrial remodeling is a potential therapeutic target. The objective of the present study is to evaluate the effects of the antifibrotic drug pirfenidone (PFD) on arrhythmogenic atrial remodeling in a canine CHF model.
Methods and Results— We studied 15 canines, divided equally into 3 groups: control, CHF canines not treated with PFD, and CHF canines treated with PFD. CHF was induced by ventricular tachypacing (220 bpm for 3 weeks), and oral PFD was administered for the 3-week pacing period. We performed electrophysiology and AF vulnerability studies, atrial fibrosis measurements, and atrial cytokine expression studies. Only canines in the untreated CHF group developed sustained AF (>30 minutes, 4 of 5 canines; P<0.05). Treatment of CHF canines with PFD resulted in an attenuation of arrhythmogenic left atrial remodeling, with a significant reduction in left atrial conduction heterogeneity index (median [25% to 75% interquartile range] 4.96 [3.53 to 5.64] versus 2.52 [2.11 to 2.82], P<0.01; pacing cycle length 300 ms), left atrial fibrosis (16.0% [13.0% to 17.5%] versus 8.7% [5.7% to 10.6%], P<0.01), and AF duration (1800 [1020 to 1800] seconds versus 6 [5 to 22] seconds, P<0.01). Immunoblotting studies demonstrated the drug’s effects on multiple cytokines, including a reduction in transforming growth factor-β1 expression.
Conclusions— Treatment of CHF canines with PFD results in significantly reduced arrhythmogenic atrial remodeling and AF vulnerability. Pharmacological therapy targeted at the fibrotic substrate itself may play an important role in the management of AF.
Received March 2, 2006; revision received August 16, 2006; accepted August 18, 2006.
Atrial fibrillation (AF) is a common clinical arrhythmia, and its management remains a challenge. Correlative data in biopsy and autopsy specimens from patients with AF have uncovered the presence of atrial fibrosis.1,2 Animal studies have shown atrial fibrosis to be an important substrate for AF, especially in the setting of structural heart disease.3–6 Pharmacological therapy targeted at the substrate may reduce AF vulnerability.
Clinical Perspective p 1712
Pirfenidone (PFD) is an orally active, antifibrotic drug shown to significantly attenuate and potentially reverse collagen deposition in various experimental animal models.7–12 We hypothesize that PFD will attenuate atrial fibrosis that is characteristic of canines with congestive heart failure (CHF). The objective of the present study was to evaluate the effects of PFD on arrhythmogenic atrial remodeling and AF vulnerability in canines with ventricular tachypacing (VTP)–induced CHF.
This study was approved and monitored by the Laboratory Animal Resource Center at the University of California, San Francisco, and conformed to the “Guide for the Care and Use of Laboratory Animals” (National Institutes of Health publication No. 85-23, revised 1996). Fifteen adult mongrel canines (weight 25 to 32 kg) divided into 3 groups (n=5 per group) were studied: (1) control (normal animals neither paced nor treated with PFD); (2) HF (animals with VTP-induced CHF not treated with PFD); and (3) HF+PFD (animals with VTP-induced CHF treated with PFD). Canines in the paced groups underwent placement of a single-chamber pacemaker with the pacing lead positioned in the right ventricular apex, followed by radiofrequency ablation of the AV junction to create complete heart block. Canines in the HF and HF+PFD groups underwent 3 weeks of VTP at 220 bpm. For the animals in the HF+PFD group, oral PFD (800 mg 3 times per day; InterMune, Brisbane, Calif) was started 2 days before the initiation of pacing and was discontinued >6 half-lives (24 hours) before the open-chest study. On follow-up, the animals underwent electrophysiological study (EPS), as described previously.13,14 Atrial tissues were processed for immunoblotting and histology studies.
Monitoring of the Congestive Heart Failure Model
The paced groups underwent transesophageal echocardiography at the time of pacemaker implantation and at follow-up. Follow-up studies included transthoracic echocardiography, ECG monitoring, and physical examination on a weekly basis. CHF was established by clinical signs (eg, oral mucous membrane color, lethargy, and edema) and the presence of left ventricular (LV) dysfunction on echocardiography. Left atrial (LA) size was determined by measuring the LA area by planimetry from 2D echocardiographic images (2-chamber view during diastole). LV systolic function was determined by measuring LV fractional shortening (LVFS) at the level of the papillary muscle. Two repeated measurements were made for LA area and LVFS, and the mean was used for analysis.
During EPS, animals were anesthetized with isoflurane and mechanically ventilated. The pacemaker rate was set at 80 bpm for the EPS. The chest was opened with a midline sternotomy. A pericardial cradle was created, and 4 high-density plaques were placed on the epicardial surface of the atria (LA free wall, LA Bachmann’s bundle, right atrial [RA] free wall, and RA Bachmann’s bundle; a total of 512 unipolar electrodes with an interelectrode distance of 3.5 mm), similar to that described previously.13 Signals were sampled at 2 kHz and stored with the UnEmap system (University of Auckland, New Zealand). Neighboring plaque electrode pairs were used for bipolar stimulation at twice the diastolic threshold. Effective refractory periods (ERPs) were measured at 12 atrial sites (6 in the LA, 6 in the RA) with the single extrastimulus protocol at an 8-beat drive train with basic cycle lengths (BCLs) of 200, 300, and 400 ms. ERPs were determined in 2-ms increments. Dispersion of repolarization was taken as the coefficient of variation (SD/mean×100%) of the ERP at each BCL. During stimulation of the contralateral atrium, conduction velocity (CV) was calculated between pairs of plaque electrodes perpendicular to the activation wavefront with a customized software program.15 As described previously,15 conduction heterogeneity was quantified as the phase difference (milliseconds/millimeters), defined as the average difference in activation time between a plaque electrode and that of its neighbors normalized by the interelectrode distance. Frequency histograms were constructed for the phase differences within an atrial region. The histograms were summarized as (1) median phase (P50) and (2) 5th and 95th percentile phase, or P5 and P95 of the distribution, respectively. To quantify conduction heterogeneity, we calculated the conduction heterogeneity index, defined as the absolute conduction heterogeneity normalized by the median phase, or P95−5/P50. The same pacing signals were used to calculate CV and phase.
AF vulnerability was assessed by single-extrastimulus pacing and an atrial burst pacing protocol that entailed pacing at 1 LA site and 1 RA site. A total of 16 burst stimulations were performed for each animal, with each atrial site receiving 8 burst pacings (4 for 6 seconds, 4 for 12 seconds) at a cycle length of 50 ms and a stimulus output of 0.5 V plus twice the diastolic threshold. AF was considered sustained if the induced episode lasted >30 minutes. If sustained AF was achieved at any point in the protocol, further testing was not performed, and the longest AF duration was taken as 1800 seconds.
Histological, Immunoblotting, and Immunohistochemistry Studies
Atrial tissue samples were fixed in 10% neutral buffered formalin. The samples were embedded in paraffin and sectioned (4- to 5-μm thick) and were stained with hematoxylin and eosin, Masson’s trichrome, or Sirius red. Fibrosis was quantified from the digital photomicrographs of the Sirius red–stained sections as described previously.6 Areas containing blood vessels and perivascular interstitial cells were excluded from fibrosis quantification.
Atrial tissue samples were also frozen in liquid nitrogen for immunoblotting studies of cytokines using standard techniques. Cytokines of interest included those that have been reported to potentially play an important role in the development of AF substrate (ie, mediators of inflammation or fibrosis development).5,16–24 After homogenization in solubilizing buffer, atrial tissue that contained an equal amount of total protein (determined by DC Protein Assay, Bio-Rad, Hercules, Calif) was electrophoresed on a 4% to 20% Tris-glycine gel and then transferred onto a nitrocellulose filter. Nonspecific binding sites were blocked with 4% BSA, and the filter was incubated with diluted antibody and matched secondary antibody (Chemicon, Temecula, Calif). Protein bands were analyzed with a chemiluminescence detection method (NEN Life Science, Boston, Mass). Densitometry was used to quantify protein band signals from all animals under investigation. Quantitative protein levels were normalized to β-actin levels in each specimen run concomitantly in each lane.
Atrial distribution of the gap junction proteins connexin 43 (Cx43) and 40 (Cx40) was studied with immunohistochemistry techniques, as described previously.13,25 Specimens were incubated with mouse monoclonal antibody against Cx40 and rabbit polyclonal antibody against Cx43 (Dako, Glostrup, Denmark) overnight at 4°C. Incubation with FITC-labeled goat anti-rabbit (for Cx43) and Texas Red–labeled donkey anti-mouse (for Cx40) antibodies (Jackson ImmunoResearch Laboratories, West Grove, Pa) was performed. The specimens were processed and analyzed with fluorescent microscopy.
Intragroup data variables were compared with the Friedman test. Data variables among the 3 groups (intergroup) were compared with the Kruskal-Wallis test, and if P was <0.05, follow-up comparisons of the different groups were done with the Mann-Whitney test. Proportion comparisons were performed with the Fischer exact test. Results were presented as median (25% to 75% interquartile range), and P<0.05 was deemed statistically significant.
The authors had full access to the data and take full responsibility for their integrity. All authors have read and agree to the manuscript as written.
LV Function and LA Dilatation
At the time of follow-up, there was no significant difference in arterial blood pressure (BP) between the paced groups (systolic BP 95 [92–101] mm Hg for HF versus 96 [93–99] mm Hg for HF+PFD, P=NS; diastolic BP 59 [52–64] mm Hg for HF versus 61 [55–63] mm Hg for HF+PFD, P=NS).
LVFS was similar in all 3 groups at baseline (41% [38% to 43%] in control, 39% [36% to 42%] in HF, and 40% [36% to 44%] in HF+PFD, P=NS). After 3 weeks of pacing, LVFS was markedly reduced for the HF (ΔLVFS=−63% [−58% to −73%], P<0.01) and HF+PFD (ΔLVFS=−65% [−57% to −75%], P<0.01) groups compared with baseline. The LVFS at each weekly time point was similar in the paced groups.
At baseline, LA area was similar in all groups (8.6 cm2 [8.0 to 9.0 cm2] in control, 8.7 cm2 [7.9 to 9.2 cm2] in HF, and 8.9 cm2 [8.2 to 9.1 cm2] in HF+PFD, P=NS). For the paced groups, LA area (Figure 1) increased significantly after 1 week of VTP, and this increase was progressive. At each weekly time point, the increase in LA area was slightly greater in the HF+PFD group than in the HF group, although the difference between the 2 paced groups was not statistically significant.
Clinical signs of CHF were similar between the 2 paced groups. All animals in the drug-treated group received all scheduled doses of the drug. There were no overt signs of intolerance to the drug.
Atrial ERP findings are shown in Figures 2A and 2B. In the LA, CHF resulted in a trend toward longer ERPs at all BCLs compared with controls. ERPs in the HF+PFD group were significantly longer than those in controls (P<0.04 for all BCLs) but did not reach statistical significance compared with those in the HF group. In the RA, ERPs were similar in all groups.
Atrial dispersion of repolarization (DOR) measurements are shown in Figures 2C and 2D. In the LA, CHF resulted in a trend toward an increase in DOR compared with controls. LA DOR in the HF+PFD group was not significantly different from that in the HF group at all BCLs but was significantly greater than that in controls at BCLs of 200 and 300 ms (P<0.02). In the RA, CHF resulted in an increase in DOR at all BCLs compared with controls, and this increase reached statistical significance at BCLs of 200 and 400 ms (P<0.05). RA DOR in the HF+PFD group was similar to that in the HF group and was significantly greater than that in controls at the BCL of 300 ms (P<0.02).
Atrial CV findings are shown in Figures 2E and 2F, respectively. Compared with those in controls, LA CVs in the HF group were decreased at all BCLs but only reached statistical significance at the shortest BCL of 200 ms (P<0.05). LA CV in the HF+PFD group was generally greater than that in the HF group and was similar to that in controls (P=NS). In the RA, CVs were similar in all 3 groups.
Representative isochronal activation maps generated from each of the 4 individual atrial plaques (BCL 300 ms) are shown in Figure 3. Atrial conduction was more heterogeneous (ie, more discrete areas of slow conduction) in the HF group than in controls, and this was reduced with PFD.
Atrial conduction heterogeneity was analyzed quantitatively with phase maps and derivation of the conduction heterogeneity index (CHI; Figures 4A and 4B). In the LA, CHF resulted in significantly increased CHI at all BCLs compared with controls (P<0.01). Treatment with PFD resulted in a significantly reduced CHI at all BCLs (P<0.01). The CHI in the HF+PFD group was not significantly different from that in the control group. In the RA, there was an increase in CHI in the HF group compared with controls, and this increase reached statistical significance at BCLs of 300 and 400 ms (P<0.05). This increase was less than that seen in the LA. Drug treatment resulted in a decrease in RA CHI, and this decrease reached statistical significance at a BCL of 300 ms (P<0.05). RA CHI in the control and HF+PFD groups was similar.
Atrial Fibrillation Vulnerability
Only canines in the HF group developed sustained AF (4/5, P<0.05 versus HF+PFD group). AF duration in the remaining untreated canine with nonsustained AF was 240 seconds (induced with 6-second RA burst pacing). CHF resulted in a significant increase in mean AF duration (Figure 5), from 7 (2 to 35) seconds in controls to 1800 [1020 to 1800] seconds in the HF group (P<0.01); PFD treatment resulted in a significant reduction in mean AF duration to 6 (5 to 22) seconds (P<0.01 versus HF), which was similar to that in controls.
Representative LA sections stained with Sirius red are shown in Figure 6. Histological evaluations of all control atria were normal. However, LA sections of HF canines had extensive interstitial fibrosis, with prominent myocyte hypertrophy and cell loss. PFD treatment resulted in significant attenuation of interstitial fibrosis. Histological alterations were also seen in the RA (not shown) of the HF group, although they were much less extensive than those in the LA.
There was a significant increase in percentage LA fibrosis (Figure 7) in the HF group compared with controls (16.0% [13.0% to 17.5%] versus 3.1% [2.5% to 4.0%], P<0.01). PFD treatment resulted in a significant reduction in LA fibrosis (8.7% [5.7% to 10.6%], P<0.01 versus HF group), although it was still greater than that in controls (P<0.02).
Shown in Figure 8 are LA cytokine immunoblotting results with corresponding protein band density measurements for transforming growth factor (TGF)-β1, total extracellular signal-regulated protein kinase-1 and -2 (ERK[1/2]), total c-Jun N-terminal kinase (JNK), total p38, tissue inhibitor of metalloproteinase (TIMP)-4, active matrix metalloproteinase (MMP)-9, and tumor necrosis factor (TNF)-α. CHF resulted in an increase in TGF-β1, JNK, MMP-9, and TNF-α (P=0.05), whereas PFD resulted in a decrease in each of these cytokines (P=0.05). CHF also resulted in a trend toward a small increase in ERK1/2 and p38 and resulted in a large decrease in TIMP-4 (P=0.05), whereas PFD resulted in a trend toward a small decrease in ERK1/2 and p38 and a large increase in TIMP-4 (P=0.05). Spatial distribution of Cx43 and Cx40 in the LA was similar in the 3 groups (Data Supplement).
In this study, we investigated the effects of a unique antifibrotic agent on CHF-induced atrial fibrosis and vulnerable substrate for AF. Although CHF produced marked arrhythmogenic atrial remodeling with increased AF vulnerability, PFD treatment resulted in a significant reduction in atrial fibrosis, conduction heterogeneity, and AF vulnerability.
Atrial Remodeling and Atrial Fibrillation
Atrial interstitial fibrosis can lead to increased nonuniform anisotropy in conduction26,27 and has been found to be an important substrate for AF.3,5 Canines in the present study developed significant LA fibrosis, LV dysfunction, and LA dilatation after 3 weeks of VTP, an outcome that was similar to results reported by others.3,4 These animals had a significant increase in atrial conduction heterogeneity and AF vulnerability. Although animals that were treated with PFD had similar severity of CHF and degree of LA dilatation as their untreated counterparts, the drug-treated group had a significant reduction in arrhythmogenic atrial remodeling, with reduced AF vulnerability.
In a similar canine model of pacing-induced CHF, Li et al3 observed an increase in atrial ERPs. Here, we observed a trend toward an increase in LA ERP in canines with CHF. This initial increase in LA ERP may be caused in part by an electrophysiological effect from atrial dilatation (from CHF), as has been reported.28,29 A progressive lengthening in LA ERP was observed with PFD treatment, and this lengthening may be caused by a further increase in atrial dilatation because less interstitial fibrosis is present. It is unlikely that these effects are a primary effect of PFD, because the drug was stopped >6 half-lives before the EPS, and because no alteration in RA ERP was observed.
Although CHF resulted in an increase in conduction heterogeneity and a decrease in CV, treatment with PFD prevented these changes. These effects are consistent with the histological finding of increased atrial fibrosis in the HF group that was markedly attenuated with PFD, with elimination of AF vulnerability. Li et al5 found an almost 10-fold increase in atrial fibrosis in their CHF canines (5 weeks of VTP) compared with controls. They also found that the increased atrial fibrosis was associated with increased conduction heterogeneity and increased AF vulnerability. In the present study, CHF resulted in an almost 5-fold increase in atrial fibrosis, and this was associated with increased conduction heterogeneity and AF vulnerability. Although Li et al5 found that treatment of CHF canines with enalapril resulted in a marked reduction in atrial fibrosis, with reduced conduction heterogeneity and AF vulnerability, the AF vulnerability was still significantly greater than that in controls. In contrast, we found that PFD treatment of CHF canines significantly reduced not only conduction heterogeneity but also AF vulnerability to levels similar to those in controls, although the reduced level of atrial fibrosis was still significantly greater than that in controls. This reduction in AF vulnerability was mostly accounted for by a marked reduction in the number of sustained AF episodes. We speculate that a certain threshold amount of atrial fibrosis is required to maintain AF, and when this threshold is not met, conduction heterogeneity will be insufficient, and AF episodes will not be sustained.
Effects of Pirfenidone
Mitogen-activated protein kinases (MAPKs) mediate the effects of angiotensin II on the tissue level.16 Li et al5 reported that in a canine model, VTP-induced CHF resulted in increased expression of the major MAPK subfamilies, including ERK, JNK, and p38 (total and phosphorylated). They found that treatment with enalapril resulted in a reduction in ERK and JNK (both total and phosphorylated forms). Interestingly, they also found that treatment with enalapril resulted in no effect on phosphorylated p38 and a small increase in total p38. In the present study, CHF resulted in an increase in all 3 MAPKs: a significant increase in total JNK and a trend toward a small increase in total ERK and total p38. PFD treatment resulted in a large decrease in total JNK and a trend toward a small decrease in total ERK and total p38. It has been reported that PFD decreases TGF-β1 expression,30 which we have also found in our model, and this decrease in TGF-β1 may influence MAPK expression or downstream signaling.
Atrial extracellular matrix homeostasis is regulated by a balance of MMPs and their endogenous inhibitors (TIMPs), with TIMP-4 being the most cardiospecific.31 The roles of AF and CHF in MMP and TIMP expression are complex. AF itself may differentially modulate MMP and TIMP expression.32 Furthermore, AF often occurs in the setting of CHF, which also affects MMP and TIMP expression.33 In the present study, MMP-9 was increased with CHF (with a concomitant increase in LA size) and was decreased with PFD treatment (with a concomitant decrease in AF vulnerability). Anné et al34 found no difference in MMP-9 expression in mitral valve disease patients with and without AF. Recently, Mukherjee et al32 found that LA MMP-9 was greater in CHF patients with AF than in those without AF. Nakano et al20 reported that MMP-9 was significantly increased in patients with paroxysmal or chronic AF. In that study, interestingly, LA diameter in patients with paroxysmal AF was similar to that in patients with sinus rhythm, and the authors speculated that MMP-9 itself may promote arrhythmogenic atrial remodeling, with increased AF vulnerability. Results from the present study implicate MMP-9 as a potential contributor to arrhythmogenic atrial remodeling.
The role of TIMPs in atrial remodeling and AF vulnerability has also been studied. Boixel et al21 have found that progressive CHF and LA fibrosis are not associated with an upregulation of TIMP-4. Anné et al34 found no difference in TIMP-4 expression in mitral valve disease patients with or without AF. In contrast, we found a change in TIMP-4 with CHF. Part of the discrepancy in the present findings may be accounted for by the differences in the experimental model under study. Although we observed an increase in MMP-9 with CHF, surprisingly, it was not associated with an increase in TIMP-4, and instead, we found a reduction in TIMP-4. This reduction would favor increased collagen deposition. This dysregulation in MMP-9 and TIMP-4 expression may have played a significant role in the development of marked atrial fibrosis. The effects of PFD included an attenuation of MMP-9 expression and an increase in TIMP-4 expression. These effects blunted the alteration in MMP/TIMP stoichiometry, which helped to restore atrial extracellular matrix homeostasis, resulting in less atrial fibrosis.
Recent studies suggest that inflammation may play an important role in promoting AF.35,36 However, the role of specific inflammatory cytokines in AF is not clear. Goette et al22 and Anné et al34 have found no significant increase in TNF-α in humans with AF. More recently, in a transgenic mouse model of cardiomyopathy that overexpressed TNF-α, Saba et al36 found both alterations in atrial morphology (increased fibrosis) and abnormalities in action potential propagation and calcium handling in atrial myocytes with an increased susceptibility to atrial arrhythmias. However, it is unclear from this particular study how much of the proarrhythmia risk is accounted for by TNF- α itself versus how much is accounted for by severe ventricular failure and the associated alterations in other important cytokines. In the present study, we observed an increase in TNF-α with CHF, and this increase may have played a role in elevating MMP-9 levels.24 PFD has been found to reduce TNF-α expression,37 and we observed a similar effect. The reduction in TNF-α may have contributed to the reduction in MMP-9.
Previous work from our laboratory has shown that overexpression of TGF-β1 in transgenic mice resulted in increased atrial fibrosis, conduction heterogeneity, and AF vulnerability.6 PFD has been shown to reduce expression of TGF-β1, with a significant reduction in fibrosis in multiple experimental animal models, including those of lung,30 hepatic,38 and renal8,9,39 fibrosis. We observed similar results: CHF resulted in an increase in TGF-β1 and atrial fibrosis, and PFD treatment resulted in a marked reduction in both.
Alterations in expression and distribution of gap junction proteins have been reported to play a role in promoting AF.40,41 Although PFD attenuated arrhythmogenic atrial remodeling and reduced AF vulnerability, it did not appear to alter the atrial spatial distribution of Cx40 and Cx43.
PFD has been reported to have broad antifibrotic and anti-inflammatory effects.7,10,30,42–44 The present results suggest that PFD attenuates CHF-related arrhythmogenic atrial remodeling via a complex set of actions, affecting multiple levels and components of the fibrosis formation and degradation signaling cascade.
In most patients, AF has traditionally been treated with antiarrhythmic drugs, with their accompanying proarrhythmia risks. Pharmacological therapy targeted at the underlying substrate has been investigated.5,45 Although angiotensin-converting enzyme inhibitors and angiotensin II type 1 receptor antagonists have been shown to be effective in attenuating arrhythmogenic atrial remodeling, these studies have only shown a blunting of AF vulnerability, perhaps because of the complex, redundant pathways for atrial fibrosis formation. Because it has the potential to attenuate increases in all 3 major cardiac MAPKs, PFD may be a more potent antifibrotic drug. We investigated this drug with no known hemodynamic effects and found it to have striking beneficial effects on arrhythmogenic atrial remodeling.
Serum drug levels were not monitored during the present study. Doses were extrapolated from presumed optimal doses based on known pharmacokinetics of the compound. It is not known whether higher doses would be more effective or lower doses equally effective. The present study did not investigate whether concomitant use of other agents used to treat CHF (eg, angiotensin II type 1 receptor antagonists, angiotensin-converting enzyme inhibitors, endothelin-1 receptor antagonists, β-adrenoceptor antagonists, or aldosterone antagonists) would have additive effects against atrial fibrosis formation. It is possible that agents used to treat CHF directly (ie, those that affect hemodynamics) may augment or complement the action of PFD, because this drug has no known vasoactive (afterload reduction) properties.
Although the effects of PFD on various important cytokines are shown here, its specific actions on these proteins (ie, whether it is selective for the MAPKs or acts on other pathways responsible for fibrosis) remain to be elucidated. It is not known, and not likely, that this drug intervention would affect the substrate of primary electrical remodeling due to AF itself (“AF begets AF”), because PFD has no known direct electrophysiological effects. However, this was not studied as part of the present investigation.
Atrial fibrosis and CHF are but one substrate for AF. Other mechanisms of increased AF vulnerability (eg, delayed afterdepolarization in nonischemic CHF) warrant consideration, but they were not part of the present investigation. PFD has broad antifibrotic and antiinflammatory effects, and additional effects beyond those studied here also warrant further investigation.
PFD treatment of canines with VTP-induced CHF results in a marked attenuation of arrhythmogenic atrial remodeling, with a reduction in AF vulnerability. This appears to be due to the drug’s broad effects on potent profibrotic mediators. Pharmacological therapy targeted at the underlying fibrotic substrate may play an important role in the management of AF.
Sources of Funding
This work was supported by National Heart, Lung, and Blood Institute grant RO1-HL072854 (Dr Olgin) and American Heart Association Western States Affiliate Postdoctoral Fellowship Award (Dr Lee).
Kostin S, Klein G, Szalay Z, Hein S, Bauer EP, Schaper J. Structural correlate of atrial fibrillation in human patients. Cardiovasc Res. 2002; 54: 361–379.
Boldt A, Wetzel U, Lauschke J, Weigl J, Gummert J, Hindricks G, Kottkamp H, Dhein S. Fibrosis in left atrial tissue of patients with atrial fibrillation with and without underlying mitral valve disease. Heart. 2004; 90: 400–405.
Li D, Fareh S, Leung TK, Nattel S. Promotion of atrial fibrillation by heart failure in dogs: atrial remodeling of a different sort. Circulation. 1999; 100: 87–95.
Shinagawa K, Shi YF, Tardif JC, Leung TK, Nattel S. Dynamic nature of atrial fibrillation substrate during development and reversal of heart failure in dogs. Circulation. 2002; 105: 2672–2678.
Li D, Shinagawa K, Pang L, Leung TK, Cardin S, Wang Z, Nattel S. Effects of angiotensin-converting enzyme inhibition on the development of the atrial fibrillation substrate in dogs with ventricular tachypacing-induced congestive heart failure. Circulation. 2001; 104: 2608–2614.
Verheule S, Sato T, Everett Tt, Engle SK, Otten D, Rubart-von der Lohe M, Nakajima HO, Nakajima H, Field LJ, Olgin JE. Increased vulnerability to atrial fibrillation in transgenic mice with selective atrial fibrosis caused by overexpression of TGF-β1. Circ Res. 2004; 94: 1458–1465.
Iyer SN, Gurujeyalakshmi G, Giri SN. Effects of pirfenidone on procollagen gene expression at the transcriptional level in bleomycin hamster model of lung fibrosis. J Pharmacol Exp Ther. 1999; 289: 211–218.
Verheule S, Wilson E, Everett Tt, Shanbhag S, Golden C, Olgin J. Alterations in atrial electrophysiology and tissue structure in a canine model of chronic atrial dilatation due to mitral regurgitation. Circulation. 2003; 107: 2615–2622.
Verheule S, Wilson E, Banthia S, Everett THT, Shanbhag S, Sih HJ, Olgin J. Direction-dependent conduction abnormalities in a canine model of atrial fibrillation due to chronic atrial dilatation. Am J Physiol Heart Circ Physiol. 2004; 287: H634–H644.
Yano M, Kim S, Izumi Y, Yamanaka S, Iwao H. Differential activation of cardiac c-jun amino-terminal kinase and extracellular signal-regulated kinase in angiotensin II-mediated hypertension. Circ Res. 1998; 83: 752–760.
Thomas CV, Coker ML, Zellner JL, Handy JR, Crumbley AJ III, Spinale FG. Increased matrix metalloproteinase activity and selective upregulation in LV myocardium from patients with end-stage dilated cardiomyopathy. Circulation. 1998; 97: 1708–1715.
Spinale FG, Coker ML, Krombach SR, Mukherjee R, Hallak H, Houck WV, Clair MJ, Kribbs SB, Johnson LL, Peterson JT, Zile MR. Matrix metalloproteinase inhibition during the development of congestive heart failure: effects on left ventricular dimensions and function. Circ Res. 1999; 85: 364–376.
Spinale FG, Coker ML, Bond BR, Zellner JL. Myocardial matrix degradation and metalloproteinase activation in the failing heart: a potential therapeutic target. Cardiovasc Res. 2000; 46: 225–238.
Goette A, Arndt M, Rocken C, Staack T, Bechtloff R, Reinhold D, Huth C, Ansorge S, Klein HU, Lendeckel U. Calpains and cytokines in fibrillating human atria. Am J Physiol Heart Circ Physiol. 2002; 283: H264–H272.
Bradham WS, Bozkurt B, Gunasinghe H, Mann D, Spinale FG. Tumor necrosis factor-alpha and myocardial remodeling in progression of heart failure: a current perspective. Cardiovasc Res. 2002; 53: 822–830.
Bradham WS, Moe G, Wendt KA, Scott AA, Konig A, Romanova M, Naik G, Spinale FG. TNF-alpha and myocardial matrix metalloproteinases in heart failure: relationship to LV remodeling. Am J Physiol Heart Circ Physiol. 2002; 282: H1288–H1295.
Verheule S, Wilson EE, Arora R, Engle SK, Scott LR, Olgin JE. Tissue structure and connexin expression of canine pulmonary veins. Cardiovasc Res. 2002; 55: 727–738.
Spach MS, Miller WT III, Dolber PC, Kootsey JM, Sommer JR, Mosher CE Jr. The functional role of structural complexities in the propagation of depolarization in the atrium of the dog: cardiac conduction disturbances due to discontinuities of effective axial resistivity. Circ Res. 1982; 50: 175–191.
Boyden PA, Tilley LP, Albala A, Liu SK, Fenoglio JJ Jr, Wit AL. Mechanisms for atrial arrhythmias associated with cardiomyopathy: a study of feline hearts with primary myocardial disease. Circulation. 1984; 69: 1036–1047.
Iyer SN, Gurujeyalakshmi G, Giri SN. Effects of pirfenidone on transforming growth factor-beta gene expression at the transcriptional level in bleomycin hamster model of lung fibrosis. J Pharmacol Exp Ther. 1999; 291: 367–373.
Greene J, Wang M, Liu YE, Raymond LA, Rosen C, Shi YE. Molecular cloning and characterization of human tissue inhibitor of metalloproteinase 4. J Biol Chem. 1996; 271: 30375–30380.
Mukherjee R, Herron AR, Lowry AS, Stroud RE, Stroud MR, Wharton JM, Ikonomidis JS, Crumbley AJ III, Spinale FG, Gold MR. Selective induction of matrix metalloproteinases and tissue inhibitor of metalloproteinases in atrial and ventricular myocardium in patients with atrial fibrillation. Am J Cardiol. 2006; 97: 532–537.
Li YY, Feldman AM, Sun Y, McTiernan CF. Differential expression of tissue inhibitors of metalloproteinases in the failing human heart. Circulation. 1998; 98: 1728–1734.
Chung MK, Martin DO, Sprecher D, Wazni O, Kanderian A, Carnes CA, Bauer JA, Tchou PJ, Niebauer MJ, Natale A, Van Wagoner DR. C-reactive protein elevation in patients with atrial arrhythmias: inflammatory mechanisms and persistence of atrial fibrillation. Circulation. 2001; 104: 2886–2891.
Saba S, Janczewski AM, Baker LC, Shusterman V, Gursoy EC, Feldman AM, Salama G, McTiernan CF, London B. Atrial contractile dysfunction, fibrosis, and arrhythmias in a mouse model of cardiomyopathy secondary to cardiac-specific overexpression of tumor necrosis factor-α. Am J Physiol Heart Circ Physiol. 2005; 289: H1456–H1467.
van der Velden HM, Ausma J, Rook MB, Hellemons AJ, van Veen TA, Allessie MA, Jongsma HJ. Gap junctional remodeling in relation to stabilization of atrial fibrillation in the goat. Cardiovasc Res. 2000; 46: 476–486.
Atrial fibrosis is an important substrate in atrial fibrillation (AF), especially in the setting of structural heart disease. In a canine model of heart failure, there is marked atrial arrhythmogenic remodeling, with increased AF vulnerability. This arrhythmogenic remodeling is a potential therapeutic target. In this investigation, we studied the effects of the antifibrotic drug pirfenidone on arrhythmogenic atrial remodeling. Drug treatment resulted in a reduction in atrial fibrosis, conduction heterogeneity, and AF vulnerability. Pharmacological therapy targeted at the underlying fibrotic substrate may play an important role in the prevention of AF.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.624320/DC1.