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Determinants of atrial fibrillation in an animal model of obesity and acute obstructive sleep apnea
Determinants of atrial fibrillation in an animal model of obesity and acute obstructive sleep apnea Yu-ki Iwasaki, MD,* Yanfen Shi, MD,* Begoña Benito, MD,* Marc-Antoine Gillis, PhD,* Kyoichi Mizuno, MD,† Jean-Claude Tardif, MD,* Stanley Nattel, MD, FHRS* From the *Department of Medicine and Research Center, Montréal Heart Institute, Quebec, Canada, and †Division of Cardiology, Department of Internal Medicine, Nippon Medical School, Tokyo, Japan. BACKGROUND Obesity and obstructive sleep apnea (OSA) are risk factors for atrial fibrillation (AF), but the underlying mechanisms are poorly understood.
83.3% AF prevention (P ⬍.05). Rocuronium also was protective (66.7%), but autonomic blockade had smaller effects (42.9% prevention).
OBJECTIVE The purpose of this study was to assess the mechanisms underlying AF promotion by obesity and OSA in rat models.
CONCLUSION Obesity and acute obstructive apnea interacted to promote AF in this model. Forced inspiration–induced acute LA distension related to diastolic dysfunction may be an important component of the arrhythmogenic substrate for AF during OSA episodes in obese patients.
METHODS Zucker obese rats (ORs) and lean rats (LRs) were intubated and ventilated with air and 2% isoflurane. OSA was mimicked by stopping the ventilator and closing the airway for 40 seconds. For nonobstructive control periods, the protocol was repeated with an open airway. Fifteen seconds after apnea onset, AF susceptibility was tested with 6 atrial burst pacing cycles (25 Hz, 3 seconds, 1-second intercycle pauses). RESULTS AF was not inducible in ORs or LRs at baseline or in nonobstructive control periods. AF was induced in 24 of 28 ORs (85.7%) vs 5 of 18 LRs (27.8%) during obstructive apnea (P ⬍.001). Negative intrathoracic pressure generation (esophageal pressure monitoring) was substantial during obstructive apnea. Echocardiography showed left ventricular hypertrophy with diastolic dysfunction in ORs. Obstructive apnea caused acute left atrial (LA) dilation, increasing LA diameter significantly more in ORs than in LRs. To clarify AF mechanisms, 24 AF-inducible ORs were divided into 4 groups: saline (n ⫽ 5), pharmacologic autonomic blockade (n ⫽ 7), respiratory muscle paralysis with rocuronium (n ⫽ 6), and inferior vena cava (IVC) balloon occlusion to unload the LA (n ⫽ 6). Balloon catheter–induced IVC occlusion prevented LA distension during obstructive apnea, leading to
Introduction A number of studies have demonstrated that obesity increases the risk of atrial fibrillation (AF).1– 4 Obesity commonly clusters with the metabolic syndrome, diabetes, hypertension, and obstructive sleep apnea (OSA), all of which may contribute to the development of AF. OSA is particularly associated with obesity,5 with OSA present in more This work was supported by Canadian Institutes of Health Research (MGP6957), Quebec Heart and Stroke Foundation, European-North American AF-Research Alliance (ENAFRA) award from Fondation Leducq, and MITACS Network of Centers of Excellence. Address reprint requests and correspondence: Dr. Stanley Nattel, University of Montreal, Montreal Heart Institute Research Center, 5000 Belanger St E, Montreal, Quebec, H1T 1C8, Canada. E-mail address: stanley.nattel@icm-mhi. org.
KEYWORDS Atrial fibrillation; Left atrial distension; Negative intrathoracic pressure; Obesity; Obstructive sleep apnea ABBREVIATIONS AF ⫽ atrial fibrillation; AV ⫽ atrioventricular; D ⫽ diastolic; ERP ⫽ effective refractory period; IVC ⫽ inferior vena cava; IVRT ⫽ isovolumic relaxation time; IVRTc ⫽ corrected isovolumic relaxation time; LA ⫽ left atrium; LAD ⫽ left atrial dimension; LV ⫽ left ventricle; LV-AWT ⫽ left ventricular anterior wall thickness; LVDd ⫽ left ventricular dimension at enddiastole; LVDs ⫽ left ventricular dimension at end-systole; LVEDP ⫽ left ventricular end-diastolic pressure; LV-FS ⫽ left ventricular fractional shortening; LV-PWT ⫽ left ventricular posterior wall thickness; OSA ⫽ obstructive sleep apnea; PAC ⫽ premature atrial contraction; PVF ⫽ pulmonary venous flow; RA ⫽ right atrium; RV-AWT ⫽ right ventricular anterior wall thickness; S ⫽ systolic; S/D ratio ⫽ systolic/diastolic flow ratio (Heart Rhythm 2012;9:1409 –1416) © 2012 Heart Rhythm Society. All rights reserved.
than 40% of obese patients.6 Obesity and OSA are associated with multiple abnormalities implicated in the pathogenesis of AF, including hypoxia,7 negative intrathoracic pressure leading to increased atrial wall stress, sympathovagal imbalance,8,9 left ventricular (LV) diastolic dysfunction,10,11 systemic inflammation,12 and increased intravascular volume.13 OSA induces deeply negative intrathoracic pressure,14 increases venous return, impairs LV filling, and diminishes stroke volume. Strongly negative intrathoracic pressures activate intrathoracic baroreceptors, inducing autonomic reflex responses that promote AF.9 AF onset tends to occur during sleep apnea episodes, suggesting that episodes of OSA acutely enhance the risk of AF.15 The pathogenesis of AF in obesity is uncertain. Although studies in animal models mimicking sleep apnea have been
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http://dx.doi.org/10.1016/j.hrthm.2012.03.024
1410 performed,9,16 none have assessed potential interactions with obesity. This study was designed to assess the mechanisms underlying AF promotion in obesity and OSA by utilizing Zucker obese rats, which are widely used in experimental studies of metabolic syndrome and obesity.17
Methods Animal model All experimental protocols were approved by the local animal research ethics committee and conformed with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (Publication No. 85–23, revised 1996). Twenty-week-old male Zucker obese rats (n ⫽ 38) were studied, with age-matched Zucker lean rats (n ⫽ 26) used as controls (all rats from Charles River, Wilmington, MA, USA). Rats were intubated and ventilated with 2.0% isoflurane in room air. Octapolar electrode catheters (1.9F; Scisense FTS-1913A-1018, London, Ontario, Canada) were introduced into the right atrium (RA) through the right internal jugular vein. A surface ECG (lead II) and intracardiac electrograms were recorded and digitized (IOX 2.516 A/D-converter, EMKA Technologies, Paris, France) for monitoring and subsequent offline analysis. A 3F pressure transducer catheter (Scisense P catherter-3F) was inserted into the esophagus for evaluation of intrathoracic pressure.18 Three-way stopcocks were attached to the endotracheal tube to control the airway.
Electrophysiologic study Programmed RA stimulation was performed in subsets of animals (12 rats/group) at a cycle length of 150 ms (pulse width 2 ms, 2⫻ threshold current) to determine the RA and atrioventricular (AV) conducting system effective refrac-
Heart Rhythm, Vol 9, No 9, September 2012 tory period (ERP). Atrial and AV conducting system ERPs were defined as the longest S1-S2 coupling interval that failed to generate a propagated beat. To assess atrial tachyarrhythmia inducibility, 25-Hz burst pacing (pulse width 2 ms, 4⫻ threshold current) was applied for 3 seconds, with six 3-second burst cycles separated by 1-second intervals. AF was defined as a rapid (⬎800 bpm) irregular atrial rhythm, and AF inducibility was defined as AF lasting for at least 5 seconds immediately following the 6-burst cycle protocol. If AF was induced after fewer than 6 burst pacing cycles, burst pacing was suspended so as not to interfere with the evolution of AF. Wenckebach cycle length was defined by failure of 1:1 AV conduction as determined by RA pacing with decremental steps of 5 ms. Sinus node recovery time was determined by 30-second RA pacing with a cycle length of 150 ms.
OSA simulation OSA was mimicked by turning off the ventilator and closing the airway for 40 seconds. Fifteen seconds after ventilator arrest, the same AF induction protocol was applied during apnea (Figure 1A). A nonobstructive control intervention was also studied, in which the same protocol was repeated in each rat during ventilatory arrest with an open airway (Figure 1B).
Hemodynamic study A 2F pressure transducer catheter (Scisense P catheterRAT) was introduced into the LV of subsets of animals (6 rats/group) through the left internal carotid artery. Systolic, diastolic, and LV end-diastolic pressures were determined during apnea. LV catheter position was adjusted to avoid catheter-induced arrhythmia during apneic episodes. In
Figure 1 Schematic of experimental protocol. A: Obstructive apnea was induced by stopping the ventilator at end-expiration and closing the airway for 40 seconds. Forced inspiration against a closed airway generated large negative intrathoracic pressures. B: To obtain a nonobstructive control period, the same protocol was repeated during ventilatory arrest with an open airway. During both apnea paradigms, 25 Hz-burst pacing (2-ms pulse width, 3-second duration) was begun 15 seconds after apnea onset as 6 burst cycles separated by 1-second intervals.
Iwasaki et al Table 1A
AF Mechanism in Obesity and Obstructive Apnea
Results
Electrophysiologic variables Lean (n ⫽ 12) Obese (n ⫽ 12)
Sinus cycle length (ms) 181.3 ⫾ 6.4 RA ERP (ms) 40.0 ⫾ 1.7 AV ERP (ms) 80.3 ⫾ 3.9 Wenckebach cycle length (ms) 95.9 ⫾ 2.3 Sinus node recovery time (ms) 228.9 ⫾ 9.1
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178.5 39.5 84.2 101.1 219.7
⫾ ⫾ ⫾ ⫾ ⫾
7.2 2.0 2.2 3.5 7.8
AV ⫽ atrioventricular; ERP ⫽ effective refractory period; RA ⫽ right atrial.
some animals, the role of LA dilation was tested by preventing LA dilation during obstructive apnea with a balloon catheter (Maveric 2.0 mm ⫻ 2.0 cm, Boston Scientific, MA, USA) inserted in the inferior vena cava (IVC) from the right femoral vein through a 0.014-inch guidewire. The IVC balloon was inflated to 12 atm during obstructive apnea, after AF inducibility during obstructive apnea had been confirmed at baseline.
Echocardiography Transthoracic echocardiographic studies were performed at baseline and during obstructive apnea with a phased-array 10S-probe (4.5–11.5 MHz) in a Vivid 7 Dimension system (GE-Healthcare Ultrasound, Horten, Norway). M-mode echocardiography was used to measure left atrial (LA) dimension (LAD); LV dimension at end-diastole (LVDd) and end-systole (LVDs); and LV anterior wall (LV-AWT), LV posterior wall (LV-PWT), and right ventricular anterior wall (RV-AWT) thickness. LV fractional shortening (LVFS) was calculated as (LVDd – LVDs)/LVDd ⫻ 100%. LV mass was calculated following Reffelmann and Kloner19 and corrected by LVDd. Apical 4-chamber view was recorded to measure LA area and RA diameter. To study LV diastolic properties, pulsed-wave Doppler was used to evaluate transmitral flow and pulmonary venous flow (PVF). Systolic (S) and (D) diastolic PVF were measured and the S/D ratio calculated. LV isovolumic relaxation time (IVRT) was measured with continuous-wave Doppler and corrected isovolumic relaxation time (IVRTc) by the simultaneously recorded R-R interval. LV diastolic filling patterns were classified according to published criteria.20 The average of 3 consecutive cardiac cycles was used for each measurement. Detailed echocardiographic characterization was obtained in subsets of 6 rats/group, with studies performed in additional animals to relate dynamic changes in LA diameter with OSA to AF inducibility.
Statistical analysis Data are expressed as mean ⫾ SEM. Unpaired t tests were used to compare single nonrepeated means from different groups The Fisher exact test with Bonferroni correction was used to analyze frequencies. For time-dependent comparisons, two-way repeated measures ANOVA with time as a repeated factor were used. Two-tailed P ⬍.05 was considered significant.
Electrophysiologic parameters There were no significant differences between obese and lean rats in baseline electrophysiologic (Table 1A) or hemodynamic (Table 1B) parameters.
Negative intrathoracic pressure generation during obstructive apnea Time from ventilatory arrest to spontaneous breathing onset was not different between obese and lean rats (obese 7.1 ⫾ 0.8 second vs lean 8.2 ⫾ 1.0 second, P ⫽ NS). Negative intrathoracic pressure gradually increased during obstructive apnea (Figure 1A). During nonobstructive control interventions, the same rats showed only very small intrathoracic pressure changes (Figure 1B). Maximum negative intrathoracic pressure increased similarly during obstructive apnea in both groups (obese ⫺39.6 ⫾ 2.5 mm Hg vs ⫺18.2 ⫾ 1.0 mm Hg, P ⬍.001; lean ⫺44.1 ⫾ 3.4 mm Hg vs ⫺15.0 ⫾ 0.7 mm Hg, P ⬍.001). Spontaneous premature atrial contractions (PACs) were observed during obstructive apnea in both groups (Online Figure 1); however, there were no differences in the numbers of PACs between groups (obese 3.3 ⫾ 0.7 beats/40 seconds [n ⫽ 9] vs lean 2.6 ⫾ 0.7 beats/40 seconds [n ⫽ 9], P ⫽ NS). PACs were rare in nonobstructive control periods (obese 0.2 ⫾ 0.2 beats/40 seconds vs lean 0.1 ⫾ 0.1 beats/40 seconds).
AF vulnerability Figure 2 shows recordings during typical burst pacing cycles during obstructive apnea and the nonobstructive control period in one obese rat. Under obstructive apnea, a longlasting AF episode immediately followed the burst cycle (left panel). In contrast, no arrhythmia was induced by burst pacing in the same rat during open airway apnea (right panel). Overall, no AF was induced by burst pacing under baseline conditions or nonobstructive control periods in either the obese or the lean group. Obstructive apnea significantly increased AF inducibility in both lean and obese rats (Online Figure 2), but the AF-promoting effects of obstructive apnea were greater in obese animals. Under obstructive apnea, AF was induced in 24 of 28 obese rats (86%) vs only 5 of 18 lean rats (28%) (P ⬍.001). Once AF was induced, there were no significant differences in AF duration between obese (91 ⫾ 35 seconds) and lean (100 ⫾ 35 seconds) rats.
Cardiac remodeling Echocardiography (Table 2) showed significant increases in LV-PWT, LV mass, and LV mass/LVDd, indicating LV Table 1B
Basic hemodynamic parameters
SBP (mm Hg) DBP (mm Hg) LVEDP (mm Hg)
Lean (n ⫽ 6)
Obese (n ⫽ 6)
121.3 ⫾ 2.1 84.2 ⫾ 3.5 7.3 ⫾ 0.6
118.0 ⫾ 2.0 87.1 ⫾ 3.3 6.0 ⫾ 0.4
DBP ⫽ diastolic arterial pressure; LVEDP ⫽ left ventricular end-diastolic pressure; SBP ⫽ systolic arterial pressure.
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Figure 2 Examples of burst pacing-induced atrial fibrillation (AF) during obstructive apnea (left) and failure to induce AF during a nonobstructive control period (right) in an obese rat.
hypertrophy, in obese rats. LV diastolic filling patterns indicated impaired diastolic function in obese rats, with decreased systolic PVF and S/D ratio for PVF, as well as prolonged IVRT and IVRTc. No appreciable chamber size differences were observed between obese and lean rats.
Table 2
LAD (mm) LA area (mm2) RA (mm) LV-AWT (mm) LV-PWT (mm) LVDd (mm) LVDs (mm) LV FS (%) LV mass (mg) LV mass/LVDd (mg/mm) RV-AWT (mm) PVF left upper S (cm/s) PVF left upper D (cm/s) S/D ratio PVF left lower S (cm/s) PVF left lower D (cm/s) S/D IVRT (ms) IVRTc
5.4 30.6 4.69 1.8 1.70 7.6 3.5 54.9 915 120 0.49 33.1 41.8 0.80 53.1 31.7 1.69 16.6 1.28
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.2 1.3 0.2 0.04 0.06 0.3 0.4 3.4 50 3 0.04 2.4 1.9 0.06 2.0 1.5 0.08 1.5 0.11
Arterial blood samples were obtained from the left internal carotid artery at baseline and after 30 seconds of obstructive apnea. Severe hypoxia and mild hypercapnia occurred with obstructive apnea in both groups of rats (Table 3), with no significant differences in pH, pO2, pCO2, and SaO2 between groups.
Hemodynamic changes
Echocardiographic parameters Lean (n ⫽ 6)
Arterial blood gases during obstructive apnea
Obese (n ⫽ 6) 5.1 27.8 4.94 1.94 1.91 7.8 4.1 47.8 1058 136 0.72 20.3 43.7 0.47 39.0 28.00 1.40 21.9 1.61
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.2 1.8 0.2 0.06 0.06* 0.3 0.2 1.5 22* 4† 0.05* 1.2‡ 1.7 0.04‡ 2.6‡ 1.69 0.09* 1.9* 0.11*
AWT ⫽ anterior wall thickness; D ⫽ diastolic; FS ⫽ fractional shortening; IVRT ⫽ isovolumic relaxation time; IVRTc ⫽ corrected isovolumic relaxation time; LA ⫽ left atrium; LAD ⫽ left atrial dimension; LV ⫽ left ventricle; LVDd ⫽ left ventricular dimension at end-diastole; LVDs ⫽ left ventricular dimension at end-systole; PVF ⫽ pulmonary venous flow; PWT ⫽ posterior wall thickness; RA ⫽ right atrium; RV ⫽ right ventricular; S ⫽ systolic; S/D ratio ⫽ systolic/diastolic PVF ratio. *P ⬍.05, †P ⬍.01, ‡P ⬍.001 vs lean.
There were no significant differences in systolic, diastolic, and LV end-diastolic (LVEDP) pressures at baseline between obese and lean rats (Table 1B). During obstructive apnea, LVEDP increased on a beat-to-beat basis during each expiratory phase and also showed a progressive increase during obstructive apnea cycles (Figure 3A). The maximum value of LVEDP during obstructive apnea was significantly greater in obese than in lean rats (Figure 3B).
LA enlargement during obstructive apnea We suspected that the greater increases in LVEDP during obstructive apnea in obese rats might be associated with acute LA dilation, which could contribute to AF; therefore, we performed echocardiography to study dynamic changes during obstructive apnea conditions. Figure 4A shows representative M-mode images across the LA. LA diameter was significantly increased vs baseline after 30 seconds of obstructive apnea in both obese and lean rats, but LA enlargement was more prominent in obese rats (Figure 4B). The RA was also significantly enlarged after 30 seconds of obstructive apnea, with no significant difference between lean and obese rats (obese 5.4 ⫾ 0.2 mm vs 4.9 ⫾ 0.2 mm at baseline [n ⫽ 6], P ⬍.01; lean 5.3 ⫾ 0.2 mm vs 4.7 ⫾ 0.2 mm [n ⫽ 6], P ⬍.05).
Iwasaki et al Table 3
AF Mechanism in Obesity and Obstructive Apnea
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Arterial blood gases and electrolytes during obstructive apnea Lean (n ⫽ 7) Baseline
pH pCO2 (mm Hg) pO2 (mm Hg) SaO2 (%) Na⫹ (mEq/L) K⫹ (mEq/L) Base excess (mmol/L)
7.42 37.7 80.9 94.7 136.7 4.1 0.7
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
Obese (n ⫽ 6) Apnea 30 seconds
0.01 2.8 3.7 0.7 0.6 0.1 0.5
7.36 49.0 24.3 36.0 137.7 4.7 2.3
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.01† 2.2† 1.8‡ 2.3‡ 0.7 0.1* 0.5*
Baseline 7.43 38.2 81.7 93.7 134.2 4.4 1.5
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
Apnea 30 seconds 0.02 4.3 8.9 1.4 0.8 0.2# 0.9
7.32 54.5 26.3 37.2 135.2 4.9 1.3
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.04† 6.7 2.3‡ 15‡ 0.5# 0.2 1
SaO2 ⫽ oxygen saturation. *P ⬍.05, †P ⬍.01, ‡P ⬍.001 vs baseline. # P ⬍.05 vs lean.
Response of AF vulnerability to various interventions To investigate the potential mechanisms underlying increased AF susceptibility with OSA and obesity in our model, intervention studies were performed. Twenty-four AF-susceptible obese rats with reproducibly inducible AF during obstructive apnea were randomly assigned to 1 of 4 intervention groups: saline injection (control intervention), pharmacologic autonomic blockade, rocuronium (to induce ventilatory muscle paralysis), and IVC balloon occlusion (to reduce LA volume). Results are shown in Figure 5. After saline injection (1 mL), AF remained inducible in all 5 rats tested, demonstrating the stability of the model over the evaluation study period. Pharmacologic autonomic block with intravenous propranolol (1 mg/kg) and atropine (1
mg/kg) prevented AF induction in 3 of 7 rats (43%), indicating that although autonomic changes may contribute to AF inducibility, AF still can be induced during OSA despite the absence of autonomic tone in many rats. To clarify whether forced respiration against a closed airway contributes to increased AF inducibility during obstructive apnea, rocuronium (1 mg/kg) was administered intravenously immediately before the obstructive apnea cycle. Pretreatment with rocuronium prevented spontaneous respiration (Online Figure 3), and AF inducibility occurred during obstructive apnea in only 2 of 6 rats after drug administration. Based on the greater LA dilation during obstructive apnea in obese vs lean rats as well as the recognized effect of LA dilation to promote AF,21 we considered the possibility that LA dilation is a key factor in AF promotion. Therefore, we pre-
Figure 3 Left ventricular pressure changes during obstructive apnea. A: Obstructive apnea progressively increased left ventricular end-diastolic pressure (LVEDP), with a gradual increase (dashed arrow) superimposed on periodic changes associated with respiration (open arrows). B: Maximum LVEDP during obstructive apnea was higher in obese vs lean rats.
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Figure 4 Acute left atrial (LA) distension during obstructive sleep apnea (OSA). A: M-mode images showing changes in LA dimensions with OSA in a lean rat (LR), an obese rat (OR), and the obese rat after inferior vena cava (IVC) balloon occlusion. B: LA diameter changes (mean ⫾ SEM).
vented LA dilation via IVC blood flow occlusion with IVC balloon catheter inflation. IVC balloon occlusion completely prevented LA distension during obstructive apnea in AF-susceptible obese rats (Figure 4). Prevention of LA dilation was associated with substantial suppression of AF inducibility, with AF induction totally prevented by balloon inflation in 5 of 6 rats (83.3%) that had been inducible during obstructive apnea at baseline.
Discussion In this study, we examined the effects on AF vulnerability of obesity and acute obstructive apnea episodes, alone and
Figure 5 Atrial fibrillation (AF) inducibility after various interventions in obese rats with AF inducibility at baseline. After AF inducibility was confirmed, interventions were performed in distinct sets of rats to assess the role of autonomic function, forced inspiration, and left atrial dilation. SR ⫽ sinus rhythm.
in combination. In our model, obesity greatly enhanced the capacity of obstructive apnea to promote AF. Forced respiration–induced acute LA stretch superimposed on LV diastolic dysfunction proved to be central to AF promotion.
Comparison with previous studies of AF mechanisms in obstructive apnea and obesity Few experimental analyses of AF mechanisms related to OSA have been reported. Ghias et al16 studied obstructive apnea in dogs, with apneic periods up to 2.5 minutes. Autonomic neural activity was enhanced, and ganglion plexus ablation prevented AF induction. Linz et al9 applied negative intrathoracic pressure during 2-minute tracheal occlusions in pigs and noted enhanced AF inducibility that was prevented by atropine. Similar to the results of those studies, we observed a reduction in AF inducibility after autonomic blockade. However, unlike the results of the prior studies, protection was incomplete. Our data suggest a role for acute LA dilation in AF inducibility. To our knowledge, this is the first such demonstration in an animal model of OSA. We were unable to find any previous experimental studies of AF promotion associated with obesity. In our Zucker obese rats, basic electrophysiologic properties were unchanged, and AF inducibility was not enhanced in the absence of obstructive apnea. However, in the presence of obstructive apnea, obesity clearly enhanced AF vulnerability. The increase in AF vulnerability that we observed was related to increased LA dilation in response to obstructive apnea and was suppressed by precluding negative intrathoracic pressure rises with rocuronium or abrogating obstructive apnea-induced LA dilation by IVC occlusion. Obese rats showed significantly greater LA dilation under obstructive apnea conditions than did lean rats (Figure 4), apparently because of LV diastolic dysfunction (Table 2) that
Iwasaki et al
AF Mechanism in Obesity and Obstructive Apnea
augmented LVEDP rises (Figure 3) during obstructive apnea. Thus, obesity and obstructive apnea interacted to promote AF.
Potential role of acute LA enlargement Negative intrathoracic pressure draws blood into the thorax (increasing venous return), enhancing atrial filling and volume. The AF promoting properties of LA dilation are well recognized.22 A variety of mechanisms have been implicated in these effects, including altered atrial refractoriness, conduction, and (with long-term stretch) fibrosis.22 Acute atrial distension increases refractoriness heterogeneity,23 an important determinant of atrial vulnerability to AF induction.24 A variety of ion channels modulated by stretch are known to affect electrophysiologic properties and arrhythmia susceptibility.25 Modest increases in atrial pressure can cause appreciable AF promotion in man.26 Of note, although obesity greatly enhanced the AF-promoting effect of obstructive apnea and augmented the associated LA enlargement, even without concomitant obesity (i.e., in lean rats) obstructive apnea caused significant LA dilation (Figure 4B) and increased AF inducibility.
Cardiac remodeling in obesity In this study, obesity caused diastolic dysfunction that conspired with increased venous return during obstructive apnea to enhance LA dilation and increase AF inducibility. We compared Zucker obese rats, which have a leptin-signaling deficiency, with Zucker lean control rats, which lack the leptin defect.17,27 Our findings of LV dysfunction associated with cardiac hypertrophy in Zucker obese rats are consistent with recent reports.28 It has been suggested that lipotoxicity due to lipid deposits29 and/or oxidative stress28 might contribute to adverse remodeling in this model.
Role of hypoxia Hypoxia has been considered an important factor in AF promotion by OSA.9 In the present study, hypoxia itself was insufficient to promote AF because there were no significant differences in blood gas abnormalities during obstructive apnea between lean and obese rats (Table 3), yet AF inducibility was clearly much greater during obstructive apnea in obese vs lean rats. However, this observation does not exclude a contribution of hypoxia to apnea-related AF promotion; it merely indicates that hypoxia alone was insufficient to substantially increase AF inducibility in our rats. Indeed, 2 of 6 rocuronium-treated rats still had inducible AF during obstructive apnea. Hypoxia is known to impair LV contractile and diastolic function,30 which are important factors in AF susceptibility. Repeated exposure to intermittent hypoxia causes structural remodeling in Zucker obese rats.31
Novelty and potential clinical relevance To our knowledge, our study is the first experimental work to address mechanisms of AF promotion associated with obesity and the first to examine the potential interactions
1415 between obesity and OSA in an animal model. Obesity and OSA are increasingly recognized to be important but poorly understood contributors to population risk of AF.32 Our findings may help in appreciating why the risk of paroxysmal atrial tachyarrhythmia is increased 18-fold immediately after a disturbed breathing episode.15 By providing mechanistic insights, our work may contribute to improved understanding and ultimately to new therapeutic approaches to AF associated with these conditions.33 Our work also could lead to mechanistic hypotheses for testing in future clinical studies.
Study limitations In this study, we examined only acute effects of obstructive apnea related to the short-term increase in atrial arrhythmia risk immediately following an OSA episode.15 A recent study reported atrial enlargement and conduction abnormalities in AF patients with repetitive nocturnal OSA.34 Longterm repeated obstructive apnea episodes might cause structural changes leading to conduction disturbances and electrophysiologic abnormalities that further contribute to the AF substrate. Further work is needed to examine the long-term effects of repeated nocturnal OSA episodes on cardiac structure and AF susceptibility. Our work was performed in a rat model, and, as with all studies of animal models of human disease, extrapolation to clinical AF should be cautious, ultimately requiring confirmation by clinical investigation. Although considered an excellent model for clinical obesity and the metabolic syndrome,27 the Zucker obese rat is only one experimental obesity paradigm among many, and further work in other animal models and human subjects is needed. Although our observations point to LA dilation as an important contributor to AF promotion in the presence of combined obesity and OSA, in no way do they exclude contributions from other factors. Indeed, our finding that autonomic blockade prevented AF induction in 3 rats that had been AF-susceptible prior to autonomic blockade suggests a role for autonomic regulation, albeit less significant than the contribution of LA dilation in the present model. The detailed electrophysiologic mechanisms of AF in our model are still uncertain. ERP shortening associated with acute LA distension might play an important role in AF promotion; however, accurate measurement of ERP during obstructive apnea was difficult because of unstable catheter contact due to important chest wall motion and because of the dynamic and short-lasting nature of electrophysiologic changes during apneic episodes. Conduction abnormalities during acute LA dilation also might play an important role. However, accurate measurement of conduction would require optical or dense multielectrode atrial mapping methods that are challenging to apply in the closed-chest rats needed to study obstructive apnea. Obesity increased AF inducibility but not AF duration. Although AF was much more likely to be induced during obstructive apnea in obese than lean rats, once it was induced its duration did not differ between groups. Obstruc-
1416 tive apnea episodes were relatively short (40 seconds total), with rapid resolution of LA dilation upon breathing restoration. Therefore, any effects of obstructive apnea/LA dilation on AF maintenance would have been quite self-limited, making it difficult to detect underlying differences in the AF-maintaining substrate between groups.
Heart Rhythm, Vol 9, No 9, September 2012
15. 16. 17. 18.
Acknowledgements We thank Nathalie L’Heureux, Chantal St-Cyr, and Audrey Bernard for technical help, Ange Maguy for help in establishing experimental methods, and France Thériault for secretarial assistance.
References 1. Krahn AD, Manfreda J, Tate RB, Mathewson FA, Cuddy TE. The natural history of atrial fibrillation: incidence, risk factors, and prognosis in the Manitoba Follow-Up Study. Am J Med 1995;98:476 – 484. 2. Stewart S, Hart CL, Hole DJ, McMurray JJ. Population prevalence, incidence, and predictors of atrial fibrillation in the Renfrew/Paisley study. Heart 2001;86: 516 –521. 3. Wilhelmsen L, Rosengren A, Lappas G. Hospitalizations for atrial fibrillation in the general male population: morbidity and risk factors. J Intern Med 2001;250: 382–389. 4. Wanahita N, Messerli FH, Bangalore S, Gami AS, Somers VK, Steinberg JS. Atrial fibrillation and obesity—results of a meta-analysis. Am Heart J 2008;155: 310 –315. 5. Coromilas J. Obesity and atrial fibrillation: is one epidemic feeding the other? JAMA 2004;292:2519 –2520. 6. Young T, Palta M, Dempsey J, Skatrud J, Weber S, Badr S. The occurrence of sleep-disordered breathing among middle-aged adults. N Engl J Med 1993;328: 1230 –1235. 7. Tkacova R, Rankin F, Fitzgerald FS, Floras JS, Bradley TD. Effects of continuous positive airway pressure on obstructive sleep apnea and left ventricular afterload in patients with heart failure. Circulation 1998;98:2269 –2275. 8. Roche F, Xuong AN, Court-Fortune, et al. Relationship among the severity of sleep apnea syndrome, cardiac arrhythmias, and autonomic imbalance. Pacing Clin Electrophysiol 2003;26:669 – 677. 9. Linz D, Schotten U, Neuberger HR, Bohm M, Wirth K. Negative tracheal pressure during obstructive respiratory events promotes atrial fibrillation by vagal activation. Heart Rhythm 2011;8:1436 –1443. 10. Alchanatis M, Paradellis G, Pini H, Tourkohoriti G, Jordanoglou J. Left ventricular function in patients with obstructive sleep apnoea syndrome before and after treatment with nasal continuous positive airway pressure. Respiration 2000;67:367–371. 11. Fung JW, Li TS, Choy DK, et al. Severe obstructive sleep apnea is associated with left ventricular diastolic dysfunction. Chest 2002;121:422– 429. 12. Shamsuzzaman AS, Winnicki M, Lanfranchi P, et al. Elevated C-reactive protein in patients with obstructive sleep apnea. Circulation 2002;105:2462–2464. 13. Messerli FH, Christie B, DeCarvalho JG, et al. Obesity and essential hypertension. Hemodynamics, intravascular volume, sodium excretion, and plasma renin activity. Arch Intern Med 1981;141:81– 85. 14. Walsh KP, Williams TD, Spiteri C, Pitts E, Lightman SL, Sutton R. Role of
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atrial pressure and rate in release of atrial natriuretic peptide. Am J Physiol 1988;254:R607–R610. Monahan K, Storfer-Isser A, Mehra R, et al. Triggering of nocturnal arrhythmias by sleep-disordered breathing events. J Am Coll Cardiol 2009;54:1797–1804. Ghias M, Scherlag BJ, Lu Z, et al. The role of ganglionated plexi in apnearelated atrial fibrillation. J Am Coll Cardiol 2009;54:2075–2083. Boudina S, Abel ED. Diabetic cardiomyopathy revisited. Circulation 2007;115: 3213–3223. Hall MJ, Ando S, Floras JS, Bradley TD. Magnitude and time course of hemodynamic responses to Mueller maneuvers in patients with congestive heart failure. J Appl Physiol 1998;85:1476 –1484. Reffelmann T, Kloner RA. Transthoracic echocardiography in rats. Evaluation of commonly used indices of left ventricular dimensions, contractile performance, and hypertrophy in a genetic model of hypertrophic heart failure (SHHFMcc-facp-Rats) in comparison with Wistar rats during aging. Basic Res Cardiol 2003;98:275–284. Shi Y, Denault AY, Couture P, Butnaru A, Carrier M, Tardif JC. Biventricular diastolic filling patterns after coronary artery bypass graft surgery. J Thorac Cardiovasc Surg 2006;131:1080 –1086. Kalifa J, Jalife J, Zaitsev AV, et al. Intra-atrial pressure increases rate and organization of waves emanating from the superior pulmonary veins during atrial fibrillation. Circulation 2003;108:668 – 671. De Jong AM, Maass AH, Oberdorf-Maass SU, Van Veldhuisen DJ, Van Gilst WH, Van Gelder IC. Mechanisms of atrial structural changes caused by stretch occurring before and during early atrial fibrillation. Cardiovasc Res 2011;89: 754 –765. Satoh T, Zipes DP. Unequal atrial stretch in dogs increases dispersion of refractoriness conducive to developing atrial fibrillation. J Cardiovasc Electrophysiol 1996;7:833– 842. Fareh S, Villemaire C, Nattel S. Importance of refractoriness heterogeneity in the enhanced vulnerability to atrial fibrillation induction caused by tachycardiainduced atrial electrical remodeling. Circulation 1998;98:2202–2209. Isenberg G, Kazanski V, Kondratev D, Gallitelli MF, Kiseleva I, Kamkin A. Differential effects of stretch and compression on membrane currents and [Na⫹]c in ventricular myocytes. Prog Biophys Mol Biol 2003;82:43–56. Antoniou A, Milonas D, Kanakakis J, Rokas S, Sideris DA. Contractionexcitation feedback in human atrial fibrillation. Clin Cardiol 1997;20:473– 476. Aleixandre de Artiñano A, Miguel Castro M. Experimental rat models to study the metabolic syndrome. Br J Nutr 2009;102:1246 –1253. Zhou X, Ma L, Habibi J, et al. Nebivolol improves diastolic dysfunction and myocardial remodeling through reductions in oxidative stress in the Zucker obese rat. Hypertension 2010;55:880 – 888. Zhou YT, Grayburn P, Karim A, et al. Lipotoxic heart disease in obese rats: implications for human obesity. Proc Natl Acad Sci U S A 2000;97:1784 –1789. Cargill RI, Kiely DG, Lipworth BJ. Adverse effects of hypoxaemia on diastolic filling in humans. Clin Sci (Lond) 1995;89:165–169. Lee SD, Kuo WW, Bau DT, et al. The coexistence of nocturnal sustained hypoxia and obesity additively increases cardiac apoptosis. J Appl Physiol 2008;104:1144 –1153. Needleman M, Calkins H. The role of obesity and sleep apnea in atrial fibrillation. Curr Opin Cardiol 2011;26:40 – 45. Nattel S. From guidelines to bench: implications of unresolved clinical issues for basic investigations of atrial fibrillation mechanisms. Can J Cardiol 2011 JanFeb;27(1):19 –26. Dimitri H, Ng M, Brooks AG, et al. Atrial remodeling in obstructive sleep apnea: Implications for atrial fibrillation. Heart Rhythm 2012;9:321–327.
Iwasaki et al
AF Mechanism in Obesity and Obstructive Apnea
1416.e1
Online Figure 1 PACs during OSA. Very few PACs occurred with open-airway apnea.
Online Figure 2 AF-induction rates. No rats could be induced into AF under baseline and open-airway conditions. During obstructive apnea, AF was inducible in 24/28 obese rats versus 4/18 lean rats (P⬍0.001).
Online Figure 3 Examples of AF-induction during obstructive apnea before rocuronium (left) and failure to induce AF after rocuronium (right) in an obese rat. Rocuronium suppressed forced respiration against the closed airway during obstructive apnea, preventing intra-thoracic pressure changes and AF-induction.
83.3% AF prevention (P ⬍.05). Rocuronium also was protective (66.7%), but autonomic blockade had smaller effects (42.9% prevention).
OBJECTIVE The purpose of this study was to assess the mechanisms underlying AF promotion by obesity and OSA in rat models.
CONCLUSION Obesity and acute obstructive apnea interacted to promote AF in this model. Forced inspiration–induced acute LA distension related to diastolic dysfunction may be an important component of the arrhythmogenic substrate for AF during OSA episodes in obese patients.
METHODS Zucker obese rats (ORs) and lean rats (LRs) were intubated and ventilated with air and 2% isoflurane. OSA was mimicked by stopping the ventilator and closing the airway for 40 seconds. For nonobstructive control periods, the protocol was repeated with an open airway. Fifteen seconds after apnea onset, AF susceptibility was tested with 6 atrial burst pacing cycles (25 Hz, 3 seconds, 1-second intercycle pauses). RESULTS AF was not inducible in ORs or LRs at baseline or in nonobstructive control periods. AF was induced in 24 of 28 ORs (85.7%) vs 5 of 18 LRs (27.8%) during obstructive apnea (P ⬍.001). Negative intrathoracic pressure generation (esophageal pressure monitoring) was substantial during obstructive apnea. Echocardiography showed left ventricular hypertrophy with diastolic dysfunction in ORs. Obstructive apnea caused acute left atrial (LA) dilation, increasing LA diameter significantly more in ORs than in LRs. To clarify AF mechanisms, 24 AF-inducible ORs were divided into 4 groups: saline (n ⫽ 5), pharmacologic autonomic blockade (n ⫽ 7), respiratory muscle paralysis with rocuronium (n ⫽ 6), and inferior vena cava (IVC) balloon occlusion to unload the LA (n ⫽ 6). Balloon catheter–induced IVC occlusion prevented LA distension during obstructive apnea, leading to
Introduction A number of studies have demonstrated that obesity increases the risk of atrial fibrillation (AF).1– 4 Obesity commonly clusters with the metabolic syndrome, diabetes, hypertension, and obstructive sleep apnea (OSA), all of which may contribute to the development of AF. OSA is particularly associated with obesity,5 with OSA present in more This work was supported by Canadian Institutes of Health Research (MGP6957), Quebec Heart and Stroke Foundation, European-North American AF-Research Alliance (ENAFRA) award from Fondation Leducq, and MITACS Network of Centers of Excellence. Address reprint requests and correspondence: Dr. Stanley Nattel, University of Montreal, Montreal Heart Institute Research Center, 5000 Belanger St E, Montreal, Quebec, H1T 1C8, Canada. E-mail address: stanley.nattel@icm-mhi. org.
KEYWORDS Atrial fibrillation; Left atrial distension; Negative intrathoracic pressure; Obesity; Obstructive sleep apnea ABBREVIATIONS AF ⫽ atrial fibrillation; AV ⫽ atrioventricular; D ⫽ diastolic; ERP ⫽ effective refractory period; IVC ⫽ inferior vena cava; IVRT ⫽ isovolumic relaxation time; IVRTc ⫽ corrected isovolumic relaxation time; LA ⫽ left atrium; LAD ⫽ left atrial dimension; LV ⫽ left ventricle; LV-AWT ⫽ left ventricular anterior wall thickness; LVDd ⫽ left ventricular dimension at enddiastole; LVDs ⫽ left ventricular dimension at end-systole; LVEDP ⫽ left ventricular end-diastolic pressure; LV-FS ⫽ left ventricular fractional shortening; LV-PWT ⫽ left ventricular posterior wall thickness; OSA ⫽ obstructive sleep apnea; PAC ⫽ premature atrial contraction; PVF ⫽ pulmonary venous flow; RA ⫽ right atrium; RV-AWT ⫽ right ventricular anterior wall thickness; S ⫽ systolic; S/D ratio ⫽ systolic/diastolic flow ratio (Heart Rhythm 2012;9:1409 –1416) © 2012 Heart Rhythm Society. All rights reserved.
than 40% of obese patients.6 Obesity and OSA are associated with multiple abnormalities implicated in the pathogenesis of AF, including hypoxia,7 negative intrathoracic pressure leading to increased atrial wall stress, sympathovagal imbalance,8,9 left ventricular (LV) diastolic dysfunction,10,11 systemic inflammation,12 and increased intravascular volume.13 OSA induces deeply negative intrathoracic pressure,14 increases venous return, impairs LV filling, and diminishes stroke volume. Strongly negative intrathoracic pressures activate intrathoracic baroreceptors, inducing autonomic reflex responses that promote AF.9 AF onset tends to occur during sleep apnea episodes, suggesting that episodes of OSA acutely enhance the risk of AF.15 The pathogenesis of AF in obesity is uncertain. Although studies in animal models mimicking sleep apnea have been
1547-5271/$ -see front matter © 2012 Heart Rhythm Society. All rights reserved.
http://dx.doi.org/10.1016/j.hrthm.2012.03.024
1410 performed,9,16 none have assessed potential interactions with obesity. This study was designed to assess the mechanisms underlying AF promotion in obesity and OSA by utilizing Zucker obese rats, which are widely used in experimental studies of metabolic syndrome and obesity.17
Methods Animal model All experimental protocols were approved by the local animal research ethics committee and conformed with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (Publication No. 85–23, revised 1996). Twenty-week-old male Zucker obese rats (n ⫽ 38) were studied, with age-matched Zucker lean rats (n ⫽ 26) used as controls (all rats from Charles River, Wilmington, MA, USA). Rats were intubated and ventilated with 2.0% isoflurane in room air. Octapolar electrode catheters (1.9F; Scisense FTS-1913A-1018, London, Ontario, Canada) were introduced into the right atrium (RA) through the right internal jugular vein. A surface ECG (lead II) and intracardiac electrograms were recorded and digitized (IOX 2.516 A/D-converter, EMKA Technologies, Paris, France) for monitoring and subsequent offline analysis. A 3F pressure transducer catheter (Scisense P catherter-3F) was inserted into the esophagus for evaluation of intrathoracic pressure.18 Three-way stopcocks were attached to the endotracheal tube to control the airway.
Electrophysiologic study Programmed RA stimulation was performed in subsets of animals (12 rats/group) at a cycle length of 150 ms (pulse width 2 ms, 2⫻ threshold current) to determine the RA and atrioventricular (AV) conducting system effective refrac-
Heart Rhythm, Vol 9, No 9, September 2012 tory period (ERP). Atrial and AV conducting system ERPs were defined as the longest S1-S2 coupling interval that failed to generate a propagated beat. To assess atrial tachyarrhythmia inducibility, 25-Hz burst pacing (pulse width 2 ms, 4⫻ threshold current) was applied for 3 seconds, with six 3-second burst cycles separated by 1-second intervals. AF was defined as a rapid (⬎800 bpm) irregular atrial rhythm, and AF inducibility was defined as AF lasting for at least 5 seconds immediately following the 6-burst cycle protocol. If AF was induced after fewer than 6 burst pacing cycles, burst pacing was suspended so as not to interfere with the evolution of AF. Wenckebach cycle length was defined by failure of 1:1 AV conduction as determined by RA pacing with decremental steps of 5 ms. Sinus node recovery time was determined by 30-second RA pacing with a cycle length of 150 ms.
OSA simulation OSA was mimicked by turning off the ventilator and closing the airway for 40 seconds. Fifteen seconds after ventilator arrest, the same AF induction protocol was applied during apnea (Figure 1A). A nonobstructive control intervention was also studied, in which the same protocol was repeated in each rat during ventilatory arrest with an open airway (Figure 1B).
Hemodynamic study A 2F pressure transducer catheter (Scisense P catheterRAT) was introduced into the LV of subsets of animals (6 rats/group) through the left internal carotid artery. Systolic, diastolic, and LV end-diastolic pressures were determined during apnea. LV catheter position was adjusted to avoid catheter-induced arrhythmia during apneic episodes. In
Figure 1 Schematic of experimental protocol. A: Obstructive apnea was induced by stopping the ventilator at end-expiration and closing the airway for 40 seconds. Forced inspiration against a closed airway generated large negative intrathoracic pressures. B: To obtain a nonobstructive control period, the same protocol was repeated during ventilatory arrest with an open airway. During both apnea paradigms, 25 Hz-burst pacing (2-ms pulse width, 3-second duration) was begun 15 seconds after apnea onset as 6 burst cycles separated by 1-second intervals.
Iwasaki et al Table 1A
AF Mechanism in Obesity and Obstructive Apnea
Results
Electrophysiologic variables Lean (n ⫽ 12) Obese (n ⫽ 12)
Sinus cycle length (ms) 181.3 ⫾ 6.4 RA ERP (ms) 40.0 ⫾ 1.7 AV ERP (ms) 80.3 ⫾ 3.9 Wenckebach cycle length (ms) 95.9 ⫾ 2.3 Sinus node recovery time (ms) 228.9 ⫾ 9.1
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178.5 39.5 84.2 101.1 219.7
⫾ ⫾ ⫾ ⫾ ⫾
7.2 2.0 2.2 3.5 7.8
AV ⫽ atrioventricular; ERP ⫽ effective refractory period; RA ⫽ right atrial.
some animals, the role of LA dilation was tested by preventing LA dilation during obstructive apnea with a balloon catheter (Maveric 2.0 mm ⫻ 2.0 cm, Boston Scientific, MA, USA) inserted in the inferior vena cava (IVC) from the right femoral vein through a 0.014-inch guidewire. The IVC balloon was inflated to 12 atm during obstructive apnea, after AF inducibility during obstructive apnea had been confirmed at baseline.
Echocardiography Transthoracic echocardiographic studies were performed at baseline and during obstructive apnea with a phased-array 10S-probe (4.5–11.5 MHz) in a Vivid 7 Dimension system (GE-Healthcare Ultrasound, Horten, Norway). M-mode echocardiography was used to measure left atrial (LA) dimension (LAD); LV dimension at end-diastole (LVDd) and end-systole (LVDs); and LV anterior wall (LV-AWT), LV posterior wall (LV-PWT), and right ventricular anterior wall (RV-AWT) thickness. LV fractional shortening (LVFS) was calculated as (LVDd – LVDs)/LVDd ⫻ 100%. LV mass was calculated following Reffelmann and Kloner19 and corrected by LVDd. Apical 4-chamber view was recorded to measure LA area and RA diameter. To study LV diastolic properties, pulsed-wave Doppler was used to evaluate transmitral flow and pulmonary venous flow (PVF). Systolic (S) and (D) diastolic PVF were measured and the S/D ratio calculated. LV isovolumic relaxation time (IVRT) was measured with continuous-wave Doppler and corrected isovolumic relaxation time (IVRTc) by the simultaneously recorded R-R interval. LV diastolic filling patterns were classified according to published criteria.20 The average of 3 consecutive cardiac cycles was used for each measurement. Detailed echocardiographic characterization was obtained in subsets of 6 rats/group, with studies performed in additional animals to relate dynamic changes in LA diameter with OSA to AF inducibility.
Statistical analysis Data are expressed as mean ⫾ SEM. Unpaired t tests were used to compare single nonrepeated means from different groups The Fisher exact test with Bonferroni correction was used to analyze frequencies. For time-dependent comparisons, two-way repeated measures ANOVA with time as a repeated factor were used. Two-tailed P ⬍.05 was considered significant.
Electrophysiologic parameters There were no significant differences between obese and lean rats in baseline electrophysiologic (Table 1A) or hemodynamic (Table 1B) parameters.
Negative intrathoracic pressure generation during obstructive apnea Time from ventilatory arrest to spontaneous breathing onset was not different between obese and lean rats (obese 7.1 ⫾ 0.8 second vs lean 8.2 ⫾ 1.0 second, P ⫽ NS). Negative intrathoracic pressure gradually increased during obstructive apnea (Figure 1A). During nonobstructive control interventions, the same rats showed only very small intrathoracic pressure changes (Figure 1B). Maximum negative intrathoracic pressure increased similarly during obstructive apnea in both groups (obese ⫺39.6 ⫾ 2.5 mm Hg vs ⫺18.2 ⫾ 1.0 mm Hg, P ⬍.001; lean ⫺44.1 ⫾ 3.4 mm Hg vs ⫺15.0 ⫾ 0.7 mm Hg, P ⬍.001). Spontaneous premature atrial contractions (PACs) were observed during obstructive apnea in both groups (Online Figure 1); however, there were no differences in the numbers of PACs between groups (obese 3.3 ⫾ 0.7 beats/40 seconds [n ⫽ 9] vs lean 2.6 ⫾ 0.7 beats/40 seconds [n ⫽ 9], P ⫽ NS). PACs were rare in nonobstructive control periods (obese 0.2 ⫾ 0.2 beats/40 seconds vs lean 0.1 ⫾ 0.1 beats/40 seconds).
AF vulnerability Figure 2 shows recordings during typical burst pacing cycles during obstructive apnea and the nonobstructive control period in one obese rat. Under obstructive apnea, a longlasting AF episode immediately followed the burst cycle (left panel). In contrast, no arrhythmia was induced by burst pacing in the same rat during open airway apnea (right panel). Overall, no AF was induced by burst pacing under baseline conditions or nonobstructive control periods in either the obese or the lean group. Obstructive apnea significantly increased AF inducibility in both lean and obese rats (Online Figure 2), but the AF-promoting effects of obstructive apnea were greater in obese animals. Under obstructive apnea, AF was induced in 24 of 28 obese rats (86%) vs only 5 of 18 lean rats (28%) (P ⬍.001). Once AF was induced, there were no significant differences in AF duration between obese (91 ⫾ 35 seconds) and lean (100 ⫾ 35 seconds) rats.
Cardiac remodeling Echocardiography (Table 2) showed significant increases in LV-PWT, LV mass, and LV mass/LVDd, indicating LV Table 1B
Basic hemodynamic parameters
SBP (mm Hg) DBP (mm Hg) LVEDP (mm Hg)
Lean (n ⫽ 6)
Obese (n ⫽ 6)
121.3 ⫾ 2.1 84.2 ⫾ 3.5 7.3 ⫾ 0.6
118.0 ⫾ 2.0 87.1 ⫾ 3.3 6.0 ⫾ 0.4
DBP ⫽ diastolic arterial pressure; LVEDP ⫽ left ventricular end-diastolic pressure; SBP ⫽ systolic arterial pressure.
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Figure 2 Examples of burst pacing-induced atrial fibrillation (AF) during obstructive apnea (left) and failure to induce AF during a nonobstructive control period (right) in an obese rat.
hypertrophy, in obese rats. LV diastolic filling patterns indicated impaired diastolic function in obese rats, with decreased systolic PVF and S/D ratio for PVF, as well as prolonged IVRT and IVRTc. No appreciable chamber size differences were observed between obese and lean rats.
Table 2
LAD (mm) LA area (mm2) RA (mm) LV-AWT (mm) LV-PWT (mm) LVDd (mm) LVDs (mm) LV FS (%) LV mass (mg) LV mass/LVDd (mg/mm) RV-AWT (mm) PVF left upper S (cm/s) PVF left upper D (cm/s) S/D ratio PVF left lower S (cm/s) PVF left lower D (cm/s) S/D IVRT (ms) IVRTc
5.4 30.6 4.69 1.8 1.70 7.6 3.5 54.9 915 120 0.49 33.1 41.8 0.80 53.1 31.7 1.69 16.6 1.28
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.2 1.3 0.2 0.04 0.06 0.3 0.4 3.4 50 3 0.04 2.4 1.9 0.06 2.0 1.5 0.08 1.5 0.11
Arterial blood samples were obtained from the left internal carotid artery at baseline and after 30 seconds of obstructive apnea. Severe hypoxia and mild hypercapnia occurred with obstructive apnea in both groups of rats (Table 3), with no significant differences in pH, pO2, pCO2, and SaO2 between groups.
Hemodynamic changes
Echocardiographic parameters Lean (n ⫽ 6)
Arterial blood gases during obstructive apnea
Obese (n ⫽ 6) 5.1 27.8 4.94 1.94 1.91 7.8 4.1 47.8 1058 136 0.72 20.3 43.7 0.47 39.0 28.00 1.40 21.9 1.61
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.2 1.8 0.2 0.06 0.06* 0.3 0.2 1.5 22* 4† 0.05* 1.2‡ 1.7 0.04‡ 2.6‡ 1.69 0.09* 1.9* 0.11*
AWT ⫽ anterior wall thickness; D ⫽ diastolic; FS ⫽ fractional shortening; IVRT ⫽ isovolumic relaxation time; IVRTc ⫽ corrected isovolumic relaxation time; LA ⫽ left atrium; LAD ⫽ left atrial dimension; LV ⫽ left ventricle; LVDd ⫽ left ventricular dimension at end-diastole; LVDs ⫽ left ventricular dimension at end-systole; PVF ⫽ pulmonary venous flow; PWT ⫽ posterior wall thickness; RA ⫽ right atrium; RV ⫽ right ventricular; S ⫽ systolic; S/D ratio ⫽ systolic/diastolic PVF ratio. *P ⬍.05, †P ⬍.01, ‡P ⬍.001 vs lean.
There were no significant differences in systolic, diastolic, and LV end-diastolic (LVEDP) pressures at baseline between obese and lean rats (Table 1B). During obstructive apnea, LVEDP increased on a beat-to-beat basis during each expiratory phase and also showed a progressive increase during obstructive apnea cycles (Figure 3A). The maximum value of LVEDP during obstructive apnea was significantly greater in obese than in lean rats (Figure 3B).
LA enlargement during obstructive apnea We suspected that the greater increases in LVEDP during obstructive apnea in obese rats might be associated with acute LA dilation, which could contribute to AF; therefore, we performed echocardiography to study dynamic changes during obstructive apnea conditions. Figure 4A shows representative M-mode images across the LA. LA diameter was significantly increased vs baseline after 30 seconds of obstructive apnea in both obese and lean rats, but LA enlargement was more prominent in obese rats (Figure 4B). The RA was also significantly enlarged after 30 seconds of obstructive apnea, with no significant difference between lean and obese rats (obese 5.4 ⫾ 0.2 mm vs 4.9 ⫾ 0.2 mm at baseline [n ⫽ 6], P ⬍.01; lean 5.3 ⫾ 0.2 mm vs 4.7 ⫾ 0.2 mm [n ⫽ 6], P ⬍.05).
Iwasaki et al Table 3
AF Mechanism in Obesity and Obstructive Apnea
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Arterial blood gases and electrolytes during obstructive apnea Lean (n ⫽ 7) Baseline
pH pCO2 (mm Hg) pO2 (mm Hg) SaO2 (%) Na⫹ (mEq/L) K⫹ (mEq/L) Base excess (mmol/L)
7.42 37.7 80.9 94.7 136.7 4.1 0.7
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
Obese (n ⫽ 6) Apnea 30 seconds
0.01 2.8 3.7 0.7 0.6 0.1 0.5
7.36 49.0 24.3 36.0 137.7 4.7 2.3
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.01† 2.2† 1.8‡ 2.3‡ 0.7 0.1* 0.5*
Baseline 7.43 38.2 81.7 93.7 134.2 4.4 1.5
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
Apnea 30 seconds 0.02 4.3 8.9 1.4 0.8 0.2# 0.9
7.32 54.5 26.3 37.2 135.2 4.9 1.3
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.04† 6.7 2.3‡ 15‡ 0.5# 0.2 1
SaO2 ⫽ oxygen saturation. *P ⬍.05, †P ⬍.01, ‡P ⬍.001 vs baseline. # P ⬍.05 vs lean.
Response of AF vulnerability to various interventions To investigate the potential mechanisms underlying increased AF susceptibility with OSA and obesity in our model, intervention studies were performed. Twenty-four AF-susceptible obese rats with reproducibly inducible AF during obstructive apnea were randomly assigned to 1 of 4 intervention groups: saline injection (control intervention), pharmacologic autonomic blockade, rocuronium (to induce ventilatory muscle paralysis), and IVC balloon occlusion (to reduce LA volume). Results are shown in Figure 5. After saline injection (1 mL), AF remained inducible in all 5 rats tested, demonstrating the stability of the model over the evaluation study period. Pharmacologic autonomic block with intravenous propranolol (1 mg/kg) and atropine (1
mg/kg) prevented AF induction in 3 of 7 rats (43%), indicating that although autonomic changes may contribute to AF inducibility, AF still can be induced during OSA despite the absence of autonomic tone in many rats. To clarify whether forced respiration against a closed airway contributes to increased AF inducibility during obstructive apnea, rocuronium (1 mg/kg) was administered intravenously immediately before the obstructive apnea cycle. Pretreatment with rocuronium prevented spontaneous respiration (Online Figure 3), and AF inducibility occurred during obstructive apnea in only 2 of 6 rats after drug administration. Based on the greater LA dilation during obstructive apnea in obese vs lean rats as well as the recognized effect of LA dilation to promote AF,21 we considered the possibility that LA dilation is a key factor in AF promotion. Therefore, we pre-
Figure 3 Left ventricular pressure changes during obstructive apnea. A: Obstructive apnea progressively increased left ventricular end-diastolic pressure (LVEDP), with a gradual increase (dashed arrow) superimposed on periodic changes associated with respiration (open arrows). B: Maximum LVEDP during obstructive apnea was higher in obese vs lean rats.
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Figure 4 Acute left atrial (LA) distension during obstructive sleep apnea (OSA). A: M-mode images showing changes in LA dimensions with OSA in a lean rat (LR), an obese rat (OR), and the obese rat after inferior vena cava (IVC) balloon occlusion. B: LA diameter changes (mean ⫾ SEM).
vented LA dilation via IVC blood flow occlusion with IVC balloon catheter inflation. IVC balloon occlusion completely prevented LA distension during obstructive apnea in AF-susceptible obese rats (Figure 4). Prevention of LA dilation was associated with substantial suppression of AF inducibility, with AF induction totally prevented by balloon inflation in 5 of 6 rats (83.3%) that had been inducible during obstructive apnea at baseline.
Discussion In this study, we examined the effects on AF vulnerability of obesity and acute obstructive apnea episodes, alone and
Figure 5 Atrial fibrillation (AF) inducibility after various interventions in obese rats with AF inducibility at baseline. After AF inducibility was confirmed, interventions were performed in distinct sets of rats to assess the role of autonomic function, forced inspiration, and left atrial dilation. SR ⫽ sinus rhythm.
in combination. In our model, obesity greatly enhanced the capacity of obstructive apnea to promote AF. Forced respiration–induced acute LA stretch superimposed on LV diastolic dysfunction proved to be central to AF promotion.
Comparison with previous studies of AF mechanisms in obstructive apnea and obesity Few experimental analyses of AF mechanisms related to OSA have been reported. Ghias et al16 studied obstructive apnea in dogs, with apneic periods up to 2.5 minutes. Autonomic neural activity was enhanced, and ganglion plexus ablation prevented AF induction. Linz et al9 applied negative intrathoracic pressure during 2-minute tracheal occlusions in pigs and noted enhanced AF inducibility that was prevented by atropine. Similar to the results of those studies, we observed a reduction in AF inducibility after autonomic blockade. However, unlike the results of the prior studies, protection was incomplete. Our data suggest a role for acute LA dilation in AF inducibility. To our knowledge, this is the first such demonstration in an animal model of OSA. We were unable to find any previous experimental studies of AF promotion associated with obesity. In our Zucker obese rats, basic electrophysiologic properties were unchanged, and AF inducibility was not enhanced in the absence of obstructive apnea. However, in the presence of obstructive apnea, obesity clearly enhanced AF vulnerability. The increase in AF vulnerability that we observed was related to increased LA dilation in response to obstructive apnea and was suppressed by precluding negative intrathoracic pressure rises with rocuronium or abrogating obstructive apnea-induced LA dilation by IVC occlusion. Obese rats showed significantly greater LA dilation under obstructive apnea conditions than did lean rats (Figure 4), apparently because of LV diastolic dysfunction (Table 2) that
Iwasaki et al
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augmented LVEDP rises (Figure 3) during obstructive apnea. Thus, obesity and obstructive apnea interacted to promote AF.
Potential role of acute LA enlargement Negative intrathoracic pressure draws blood into the thorax (increasing venous return), enhancing atrial filling and volume. The AF promoting properties of LA dilation are well recognized.22 A variety of mechanisms have been implicated in these effects, including altered atrial refractoriness, conduction, and (with long-term stretch) fibrosis.22 Acute atrial distension increases refractoriness heterogeneity,23 an important determinant of atrial vulnerability to AF induction.24 A variety of ion channels modulated by stretch are known to affect electrophysiologic properties and arrhythmia susceptibility.25 Modest increases in atrial pressure can cause appreciable AF promotion in man.26 Of note, although obesity greatly enhanced the AF-promoting effect of obstructive apnea and augmented the associated LA enlargement, even without concomitant obesity (i.e., in lean rats) obstructive apnea caused significant LA dilation (Figure 4B) and increased AF inducibility.
Cardiac remodeling in obesity In this study, obesity caused diastolic dysfunction that conspired with increased venous return during obstructive apnea to enhance LA dilation and increase AF inducibility. We compared Zucker obese rats, which have a leptin-signaling deficiency, with Zucker lean control rats, which lack the leptin defect.17,27 Our findings of LV dysfunction associated with cardiac hypertrophy in Zucker obese rats are consistent with recent reports.28 It has been suggested that lipotoxicity due to lipid deposits29 and/or oxidative stress28 might contribute to adverse remodeling in this model.
Role of hypoxia Hypoxia has been considered an important factor in AF promotion by OSA.9 In the present study, hypoxia itself was insufficient to promote AF because there were no significant differences in blood gas abnormalities during obstructive apnea between lean and obese rats (Table 3), yet AF inducibility was clearly much greater during obstructive apnea in obese vs lean rats. However, this observation does not exclude a contribution of hypoxia to apnea-related AF promotion; it merely indicates that hypoxia alone was insufficient to substantially increase AF inducibility in our rats. Indeed, 2 of 6 rocuronium-treated rats still had inducible AF during obstructive apnea. Hypoxia is known to impair LV contractile and diastolic function,30 which are important factors in AF susceptibility. Repeated exposure to intermittent hypoxia causes structural remodeling in Zucker obese rats.31
Novelty and potential clinical relevance To our knowledge, our study is the first experimental work to address mechanisms of AF promotion associated with obesity and the first to examine the potential interactions
1415 between obesity and OSA in an animal model. Obesity and OSA are increasingly recognized to be important but poorly understood contributors to population risk of AF.32 Our findings may help in appreciating why the risk of paroxysmal atrial tachyarrhythmia is increased 18-fold immediately after a disturbed breathing episode.15 By providing mechanistic insights, our work may contribute to improved understanding and ultimately to new therapeutic approaches to AF associated with these conditions.33 Our work also could lead to mechanistic hypotheses for testing in future clinical studies.
Study limitations In this study, we examined only acute effects of obstructive apnea related to the short-term increase in atrial arrhythmia risk immediately following an OSA episode.15 A recent study reported atrial enlargement and conduction abnormalities in AF patients with repetitive nocturnal OSA.34 Longterm repeated obstructive apnea episodes might cause structural changes leading to conduction disturbances and electrophysiologic abnormalities that further contribute to the AF substrate. Further work is needed to examine the long-term effects of repeated nocturnal OSA episodes on cardiac structure and AF susceptibility. Our work was performed in a rat model, and, as with all studies of animal models of human disease, extrapolation to clinical AF should be cautious, ultimately requiring confirmation by clinical investigation. Although considered an excellent model for clinical obesity and the metabolic syndrome,27 the Zucker obese rat is only one experimental obesity paradigm among many, and further work in other animal models and human subjects is needed. Although our observations point to LA dilation as an important contributor to AF promotion in the presence of combined obesity and OSA, in no way do they exclude contributions from other factors. Indeed, our finding that autonomic blockade prevented AF induction in 3 rats that had been AF-susceptible prior to autonomic blockade suggests a role for autonomic regulation, albeit less significant than the contribution of LA dilation in the present model. The detailed electrophysiologic mechanisms of AF in our model are still uncertain. ERP shortening associated with acute LA distension might play an important role in AF promotion; however, accurate measurement of ERP during obstructive apnea was difficult because of unstable catheter contact due to important chest wall motion and because of the dynamic and short-lasting nature of electrophysiologic changes during apneic episodes. Conduction abnormalities during acute LA dilation also might play an important role. However, accurate measurement of conduction would require optical or dense multielectrode atrial mapping methods that are challenging to apply in the closed-chest rats needed to study obstructive apnea. Obesity increased AF inducibility but not AF duration. Although AF was much more likely to be induced during obstructive apnea in obese than lean rats, once it was induced its duration did not differ between groups. Obstruc-
1416 tive apnea episodes were relatively short (40 seconds total), with rapid resolution of LA dilation upon breathing restoration. Therefore, any effects of obstructive apnea/LA dilation on AF maintenance would have been quite self-limited, making it difficult to detect underlying differences in the AF-maintaining substrate between groups.
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15. 16. 17. 18.
Acknowledgements We thank Nathalie L’Heureux, Chantal St-Cyr, and Audrey Bernard for technical help, Ange Maguy for help in establishing experimental methods, and France Thériault for secretarial assistance.
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1416.e1
Online Figure 1 PACs during OSA. Very few PACs occurred with open-airway apnea.
Online Figure 2 AF-induction rates. No rats could be induced into AF under baseline and open-airway conditions. During obstructive apnea, AF was inducible in 24/28 obese rats versus 4/18 lean rats (P⬍0.001).
Online Figure 3 Examples of AF-induction during obstructive apnea before rocuronium (left) and failure to induce AF after rocuronium (right) in an obese rat. Rocuronium suppressed forced respiration against the closed airway during obstructive apnea, preventing intra-thoracic pressure changes and AF-induction.