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Cardiovascular Research 2003 59(4):863-873; doi:10.1016/S0008-6363(03)00540-6
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Copyright © 2003, European Society of Cardiology

Cholinergic atrial fibrillation: IK,ACh gradients determine unequal left/right atrial frequencies and rotor dynamics

Farzad Sarmast, Arun Kolli, Alexey Zaitsev, Keely Parisian, Amit S Dhamoon, Prabal K Guha, Mark Warren, Justus M.B Anumonwo, Steven M Taffet, Omer Berenfeld* and José Jalife

Institute for Cardiovascular Research and Department of Pharmacology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA

berenfeo{at}upstate.edu

* Corresponding author. Tel.: +1-315-464-8006; fax: +1-315-464-8014.

Received 3 April 2003; revised 8 July 2003; accepted 10 July 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: We tested the hypothesis that left atrial (LA) myocytes are more sensitive to acetylcholine (ACh) than right atrial (RA) myocytes, which results in a greater dose-dependent increase in LA than RA rotor frequency, increased LA-to-RA frequency gradient and increased incidence of wavelet formation during atrial fibrillation (AF). Methods and results: AF was induced in seven Langendorff-perfused sheep hearts in the presence of ACh (0.1–4.0 µM) and studied using optical mapping and bipolar recordings. Dominant frequencies (DFs) were determined in optical and electrical signals and phase movies were used to identify rotors and quantify their dynamics. DFs in both atria increased monotonically with ACh concentration until saturation, but the LA frequency predominated at all concentrations. Rotors were also seen more often in the LA, and although their life span decreased, their frequency and number of rotations increased. Patch-clamp studies demonstrated that ACh-activated potassium current (IK,ACh) density was greater in LA than RA sheep myocytes. Additionally, ribonuclease protection assay demonstrated that Kir3.4 and Kir3.1 mRNAs were more abundant in LA than in RA. Conclusions: A greater abundance of Kir3.x channels and higher IK,ACh density in LA than RA myocytes result in greater ACh-induced speeding-up of rotors in the LA than in the RA, which explains the ACh dose-dependent changes in overall AF frequency and wavelet formation.

KEYWORDS Acetylcholine; Arrhythmia (mechanisms); Atrial function; K-channel; Mapping


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Atrial fibrillation (AF) is maintained by reentry but its precise pathophysiological bases remain unclear [1,2]. In the 1920s, Lewis [3] postulated that AF was due to activation by a single rapidly firing reentrant circuit. More recently, Schuessler et al. [4] demonstrated in the isolated canine right atrium that acetylcholine (ACh) could convert multiple reentrant circuits into a single, relatively stable circuit that resulted in fibrillatory conduction. Work from our laboratory in the isolated heart has demonstrated that AF in the presence of ACh is maintained by fast reentrant sources (rotors) in the left atrium (LA), with fibrillatory conduction toward the slower right atrium (RA) [5–7].

We surmise that, during acute AF maintained by ACh, high frequency reentrant sources stabilize in the LA primarily because LA myocytes are more sensitive than RA myocytes to the repolarizing effects of ACh. If this is correct, then LA myocytes should have a greater ability to adapt to higher excitation frequencies than RA myocytes. We have therefore compared ACh concentration-dependent changes in AF dynamics and activation frequency in the LA and RA of the isolated sheep heart. We have also investigated the density of the ACh-activated potassium current (IK,ACh) in LA and RA myocytes, as well as chamber-specific differences in the levels of mRNA encoding Kir3.x (GIRKx) proteins that form the channels responsible for IK,ACh [8]. Our results strongly suggest that LA-to-RA gradients in excitation frequency and fibrillatory conduction in this model are the result of significant chamber-specific differences in the functional expression of IK,ACh channels.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
This investigation conformed to the current Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 85-23, revised 1996).

2.1 Langendorff-perfused heart preparations
Young sheep (20–30 kg) were anesthetized with sodium pentobarbital (35 mg/kg). The heart was removed and connected to a Langendorff apparatus for continuous perfusion at 200 ml/min with warm (36–38°C), buffered HEPES–Tyrode’s solution whose composition is described elsewhere in detail [5–7].

2.2 AF induction and maintenance
AF was induced in the presence of 0.1 µM ACh by rapid pacing. ACh concentration was increased stepwise up to 4.0 µM. More than 10 min were allowed to achieve stable perfusion at any given ACh concentration and three movies were made at 1-min intervals.

2.3 Optical and electrical recordings
Two synchronized CCD cameras were used to record di-4-ANEPPS fluorescence (~3x3 cm2 each field) from both LA and RA free walls as described in detail elsewhere [5–7]. Five-second movies (128x128 pixels, 300 frames/s) were made during AF at different ACh concentrations. Concurrently, epicardial bipolar electrograms were recorded from the left and the right atrial appendages (RAA and LAA) using a Biopac data acquisition system (Biopac, Santa Barbara, CA, USA).

2.4 Dominant-frequency maps
Frequency analysis of optical and electrical recordings was performed as previously described [6,9]. Briefly, the power spectral density was estimated for each pixel and the frequency with the maximum power was assigned to be the dominant frequency (DF). DFs are presented on maps that describe their spatial distribution in the optical field of view. We used DFs from the highest frequency domains in the LA and RA (LAHi, RAHi) to create ACh dose–response curves for both atria.

2.5 Phase maps
To highlight the formation of wavelets and rotors [10–12], the phase of the action potential in each pixel, {theta}(t), was computed using the Hilbert transform. A phase singularity (PS) was defined as a point where all phases converged. Individual PSs were tracked over 500 consecutive frames to determine their trajectories and lifespan. The average number of PSs per cm2 per second (PS density) was determined for each AF movie. Rotor was defined as a wave of excitation rotating around a PS for ≥l cycle. The five longest living rotors were identified during AF at each concentration of ACh in each experiment. Rotor excitation frequency was calculated as the inverse of rotation cycle length (1/CL).

2.6 RPA analysis of channel expression
We utilized a ribonuclease protection assay (RPA) to determine the relative concentration of Kir 2.x and 3.x gene products. Since there are no published sequences of Kir2.x or Kir3.x channels derived from sheep, probes to assess the expression of these genes were cloned using PCR with sheep DNA as a template. The RPA probes (Kir2.x, Kir3.x channels and the housekeeping protein cyclophilin) were transcribed to form [33P]-radiolabeled cRNA, which was hybridized to total cellular RNA isolated from the atria of five sheep using a Riboquant Ribonuclease Protection Assay kit (BD Pharmingen, San Diego, CA, USA). RNA was quantified by PhosphorImager (Molecular Dynamics) and the signal for each channel RNA was normalized to the cyclophilin signal in that lane.

2.7 Isolation of sheep atrial myocytes
The Langendorff-perfusion technique was used for the isolation of sheep atrial myocytes as described in detail elsewhere [13,14]. Whole-cell patch clamp recordings were carried out also as previously described [12,13,15]. For the acetylcholine-sensitive current (IK,ACh), voltage-clamp protocols (1.6 mV/s ramps) were applied from –100 to 0 mV (–80 mV holding potential (HP)). Currents were measured in control and during ACh superfusion. IK,ACh was analyzed as the difference current; i.e. current at ACh 0.05, 0.1 or 0.5 µM minus control current. For IK1, ramps were applied from –100 to +10 mV (HP=–80 mV) in control and in the presence of Ba2+ (1 mM). Current was measured as the Ba2+-sensitive component. All currents were normalized by cell capacitance.

2.8 Statistical analysis
Values are shown as mean±S.E.M. Two-variable comparisons were analyzed using two-way ANOVA. Paired and unpaired comparisons were conducted using the Student’s t-test. P<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 ACh dose–response relationships
Simultaneous optical recordings from LA and RA free walls during AF were made in six experiments to study the effects of ACh (0.1–4.0 µM) on AF frequency and organization. DF analysis produced maps containing multiple frequency domains within each atrium. Fig. 1 illustrates typical findings at 0.2 and 4.0 µM ACh in one experiment. Both the highest and lowest frequency domain values of the LA are larger than those in the RA at both concentrations. At 0.2 µM, LA frequencies ranged between 9.0 and 11.7 Hz; RA frequencies ranged between 8.4 and 9.4 Hz. At 4.0 µM, AF frequencies increased in both atria. However, a large domain within the LA activated at a maximum frequency of 33 Hz (red). This frequency was appreciably higher than the frequency in the RA (24 Hz). At both concentrations, the frequency dispersion in the LA (0.2 µM, 2.7 Hz; 4.0 µM, 11.0 Hz) was greater than the RA (0.2 µM, 1.0 Hz; 4.0 µM, 6.0 Hz. P<0.01).


Figure 1
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  		Fig. 1 Examples of dominant frequency maps on the LA (left) and RA (right) during AF at two different ACh concentrations. (A) At 0.2 µM, the domain frequencies as well as the frequency dispersion are greater in the LA. (B) At 4.0 µM, the LA-to-RA difference in frequency and dispersion is larger suggesting that ACh has a more pronounced effect on the LA. Arrows indicate location of single pixel recordings.

 
Fig. 2A shows dose–response plots for the highest frequency domains for each atrium (LAHi and RAHi) constructed from maps such as those in Fig. 1. Both LAHi and RAHi curves show a positive relationship with ACh (P<0.05). However, the LA demonstrated higher excitation frequencies throughout the entire range of ACh concentrations (P<0.05). In Fig. 2B, dose–response curves plotted from electrographic recordings validated the optical data: the LAA frequency was higher than the RAA frequency at all ACh concentrations (P<0.05). In addition, in both panels, the LA and RA curves plateau at similar concentrations and approach a slope of zero at 4.0 µM. Also, in both panels, before reaching the plateau, the LA curves demonstrate steeper slopes than the RA curves. The differences between optical and electrographic data were not significant.


Figure 2
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  		Fig. 2 DF dose–response curves for ACh 0.1–4.0 µM. (A) LA (squares) and RA (circles) plots of highest frequency domain values from optical data (n=6). (B) Plots from LAA (squares) and RAA (circles) electrograms (n=7). In both cases, DFs are higher in LA than RA at all ACh concentrations.

 
3.2 Wavelet formation and rotor lifespan
The effects of ACh on the dynamics of rotors and the incidence of wavelet formation were analyzed using phase mapping [10]. Fig. 3 presents snapshots from individual episodes at 0.1 and 4.0 µM ACh. Phase singularities are labeled with black circles; they represent sites of wavebreak and wavelet formation and are also the instantaneous centers around which rotational activity occurs [10–12]. As shown in Fig. 3A, at 0.1 µM ACh, the LA demonstrated two PSs organizing a pattern of figure-of-eight reentry, whereas the RA had only one PS. In Fig. 3B, at 4.0 µM, the number of PSs increased in both atria. However, at any given time there were more PSs in LA than in RA. The bar graphs in Fig. 4 summarize the results in all six experiments. In Fig. 4A, both atria demonstrated a direct ACh dose–response relationship for PS (i.e. wavelet) density. However, LA densities were always larger than RA densities. At 0.1 µM, 2.9±0.6 PS/cm2/s were counted in LA versus 1.8±0.4 PS/cm2/s in RA (P<0.01); at 4.0 µM, PS density increased to 13.5±1.8 in LA and 8.0±0.9 in RA (P<0.01). These data demonstrate that the incidence of wavelets is greater in the LA than in the RA regardless of the ACh concentration.


Figure 3
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  		Fig. 3 Phase analysis of simultaneous LA (left) and RA (right) optical movies during AF. (A) Single frame example of phase at 0.1 µM ACh. (B) Same experiment at 4.0 µM ACh. Black circles indicate location of phase singularities (PSs). PS density is larger in LA than in RA at both ACh concentrations.

 

Figure 4
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  		Fig. 4 Properties of PS in LA and RA during AF at 0.1 and 4.0 µM ACh. (A) Density of PSs increases with ACh. At both concentrations, PS density is significantly larger in LA than RA. (B) Rotor lifespan vs. ACh concentration. Increasing ACh decreases lifespan in both atria. (C) Number of rotations significantly increases with ACh in both atria.

 
We also analyzed dose-dependent effects of ACh on rotor lifespan (see Fig. 4B). Rotors were defined as those PSs that lasted more than one rotation. The average lifespan decreased in both atria when ACh changed from 0.1 to 4.0 µM. Specifically, in the LA, rotor lifetime decreased from 171±28 to 79±3 ms (P<0.05), while in the RA it shortened from 182±6 to 98±12 ms (P<0.05). Lifespan in both atria was similar at each concentration. In Fig. 4C, further analysis demonstrated that on average, rotors in both atria lasted more cycles at the higher ACh concentration (RA: 1.18±0.3 vs. 2.07±0.13 cycles, respectively; LA: 1.62±0.12 vs. 2.27±0.07 cycles, respectively; P<0.05). There was no significant difference between the atria at any of the two concentrations.

3.3 DF mapping and rotation period
Power spectral analysis (see Figs. 1 and 2Go) allows quantification of LA–RA differences in activation frequency during AF but does not convey direct physiological meaning [6,9]. On the other hand, phase mapping provides a more direct demonstration of the underlying mechanisms of the arrhythmia. We therefore used the phase-analysis data to quantify mean rotor frequency as the inverse of cycle length (1/CL). Fig. 5A shows results from six hearts; a direct ACh dose-dependence of rotor frequency was demonstrated in both atria (P=2x10–11), with frequency being higher in LA than RA at all concentrations (P=2x10–4). The LA–RA rotor frequency gradient also increased with ACh concentration (1.0 Hz at 0.1 µM vs. 6.2 Hz at 4.0 µM; P<0.05). Furthermore, as shown in panels B and C, the excitation frequencies of the longest-lasting rotors in each atrium correlated well with the high frequency domains of their respective DF maps described above (unity slope and R2>0.96 for both atria). Similarly, the average rotor frequency in LA and RA also demonstrated strong correlation to the LAA and RAA electrogram frequencies (R2=0.99 in the RA and R2=0.93 in the LA; data not shown).


Figure 5
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  		Fig. 5 Relationship between ACh concentration and rotor frequency (1/CL). (A) ACh dose–response curves of LA (squares) and RA (circles) for the five longest living rotors for each of the six experiments. Rotor frequency is greater in LA than RA at all concentrations. (B) LA rotor frequency vs. LA frequency measured by spectral analysis. (C) RA rotor frequency vs. RA frequency measured by spectral analysis.

 
3.4 RPA analysis of Kir channel distribution
As an initial approach to determining the molecular mechanism underlying the different sensitivities of LA and RA to the effects of ACh during AF, we measured the relative abundance of mRNA encoding Kir3.1 and Kir3.4 proteins. RPA hybridization probes for Kir3.1 and Kir3.4 were developed by PCR using sheep DNA as a template (Fig. 6A) [15,16]. As shown in Fig. 6B, using these RPA probes, we found that Kir3.1 was ~50% more abundant in the LA than in the RA (P=0.03) and Kir3.4 was ~35% more abundant in the LA than the RA (P=0.03). Note that the Kir3.x channel complex responsible for IK,ACh is a heterotetramer of Kir3.1 and Kir3.4, although recently it has been shown in the bovine atrium that a significant proportion of channels exist as Kir3.4 homotetramers [8]. Finally, we also measured the relative abundance of mRNA encoding constituting channel proteins for IK1. Fig. 6 shows that the Kir2.3 channels, which have been suggested to be solely responsible for IK1 in the sheep atria (Dhamoon, submitted) are about 14% more abundant in the LA compared to the RA but this difference is not statistically significant (P=0.35). Thus, our measurements of the density of channels that may be implicated in the LA and RA differences in AF frequency [6] suggest that IK,ACh is more important than IK1 in establishing that difference.


Figure 6
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  		Fig. 6 RPA analysis of Kir3.1, Kir3.4 and Kir2.3 in the LA and RA. (A) Data from one sheep heart. Abundance of the isoform bands (arrows) was compared after scaling to the cyclophilin band in each lane. (B) mRNA concentrations from the LA and RA of five sheep (see Methods) are 1.28±0.18 vs. 0.84±0.1, 1.94±0.21 vs. 1.44±0.11, and 0.65±0.16 vs. 0.57±0.11 RNA/cyclophilin for Kir3.1, Kir3.4 and Kir2.3, respectively. Asterisks indicate P<0.05.

 
3.5 Acetylcholine-sensitive currents in atrial myocytes
The data presented thus far suggest that the significantly different responses of the LA and RA to ACh during AF are somehow related to the dissimilar mRNA levels in the two atria of Kir3.1 and Kir3.4 channels responsible for IK,ACh. To provide more definite proof for such a correlation, we examined the effects of ACh (0.05, 0.1, and 0.5 µM) on IK,ACh current density of LA and RA myocytes [13]. We did not find any significant differences in IK,ACh density at 0.05 µM (data not shown). However, IK,ACh density was significantly higher in the LA than in the RA at 0.1 and 0.5 µM.

Fig. 7A shows ramp-generated currents in an LA myocyte in control and in the presence of 0.1 and 0.5 µM ACh. The superimposed washout trace demonstrates the reversibility of the effects. The bottom of panel A is the ramp protocol used to generate the current traces. In Fig. 7B, the top graph shows IK,ACh current density–voltage relations at 0.1 µM ACh. The inward (at –100 mV) and peak outward (at –40 mV) current densities were significantly higher in LA than RA, as shown by the bottom bar graph (P<0.05). Fig. 7C (top) shows mean IK,ACh current density–voltage relationships at 0.5 µM ACh. Clearly, the LA has a higher IK,ACh density than the RA, as also demonstrated by the bottom graph showing significant differences in both inward and outward currents (P<0.05). Also, note that at the higher ACh concentration, there was an apparent reduction in current rectification. Therefore, peak outward current was measured at 0 mV (see top panel of Fig. 7C). Fig. 7D shows that IK1 density is similar in LA and RA cells.


Figure 7
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  		Fig. 7 IK,ACh and IK1 densities in sheep atrial cells. (A) Top, v-clamp ramp generated currents in an LA cell in control and in the presence of 0.1 µM and 0.5 µM ACh. Note the reversibility of ACh effect. Bottom, the voltage-clamp ramp protocol. (B) ACh=0.1 µM. Top, current density in left (LA, n=6) and right (RA, n=6) atrial cells. Bottom, peak inward (at –100 mV) and peak outward (at –40 mV) current densities from top graph. (C) ACh=0.5 µM. Top, current densities in left (LA, n=7) and right (RA, n=6) atrial cells. Bottom, peak inward (at –100 mV) and peak outward (at 0 mV) current densities from top graph. LA IK,ACh density is higher than RA at both concentrations (P<0.05). Note the apparent reduction in current rectification at the higher ACh concentration. (D) Top, IK1 density in LA (n=5) and RA (n=10) cells. Bottom, voltage-clamp ramp protocol.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The most important new results of this study are: (1) during AF, ACh changes activation frequency in a dose-dependent manner in both atria, with the frequency being higher in LA than RA at all concentrations. (2) The LA–RA frequency gradient also increases with the ACh concentration. (3) Rotor frequency in each atrium correlates well with the highest frequency domains of the corresponding DF map, and there is a greater sensitivity of LA rotors than RA rotors to the effects of ACh. (4) mRNA levels of Kir3.1 and Kir3.4 channels are higher in LA than RA. (5) IK,ACh density is significantly higher in LA myocytes than RA myocytes. Altogether, the results provide strong support to the idea that LA-to-RA gradients of excitation frequency and fibrillatory conduction in acute, cholinergically mediated AF are the result of significant differences in the functional expression of IK,ACh channels in RA and LA myocytes.

4.1 Cholinergic input and AF maintenance
Both vagal stimulation and administration of ACh may result in AF [17,18]. In animal models, vagal stimulation results in sustained AF as long as the vagus nerve is continuously stimulated [18]. Catheter ablation of the cardiac parasympathetic nerves abolishes vagally-mediated AF [19]. This has been attributed to increased spatial dispersion of refractoriness due to heterogeneous vagal innervation. Li et al. [20] demonstrated significant intrinsic differences in the APD of LA myocytes with respect to RA myocytes of the dog heart. In addition, they showed that LA myocytes had a larger IKr density and greater ERG protein expression compared to the RA. At 6 Hz, APD in the LA and RA were 100 and 110 ms, respectively. It is possible that similar differences contributed somehow to LA-to-RA frequency gradients during acute AF in our experiments through the resultant LA-to-RA differences in ERP. Yet, intrinsic APD differences alone are insufficient to explain the mechanism of AF maintenance or the exceedingly high frequency that can be achieved in some parts of the LA. The highest concentration of ACh yielded a frequency domain of 33 Hz in the LA free wall (see Fig. 1). While such a high frequency would not be expected at more physiologic levels of cholinergic input, the data do bring attention to the fact that somewhere in the LA the APD was <30 ms, which cannot be explained on the basis of activation of a time-dependent potassium current such as IKr whose time constant is about 135 ms at +10 mV [20]. Thus, under acute condition, continuous vagal stimulation, ACh perfusion or other pro-fibrillatory ministrations that are capable of abbreviating atrial APD to extreme values are necessary for the arrhythmia to be established and maintained.

Traditionally, the ability of cholinergic input to promote AF maintenance in the normal heart has been attributed to the heterogeneous distribution of vagal innervation and muscarinic ACh receptors throughout the atria, which increases spatial dispersion of refractory periods [21]. Recently published data from our laboratory in the Langendorff-perfused sheep heart [6] showed that increasing the ACh concentration from 0.2 to 0.5 µM, increased the frequency of the dominant source, as well as the LA-to-RA frequency gradient, suggesting that the LA and RA are indeed different in their response to ACh in this species. Here we have extended such observations by determining the effects of a wide range of ACh concentrations on rotor frequency and AF dynamics and supported these data by measurements of mRNA levels of specific membrane channels and patch clamp results. This enables us to postulate a hypothesis of a heterogeneous response to cholinergic input in a much more specific and precise way; that is, IK,ACh activation is larger in the LA than in the RA, which sets the stage for the development of AF by internal or external triggers through two distinct mutually complementary mechanisms: firstly, it greatly abbreviates APD, and thus leads to an increase in the frequency of reentrant sources in both atria as rigorously demonstrated here for the first time; secondly, it increases resting membrane conductance and thus reduces the space constant and conduction velocity in the atrial tissue [22]. This allows stabilization of a dominant rotor in the region of greatest APD abbreviation (e.g. the LA); outside this region, slower rotors could appear. However, unless somehow protected, such rotors would be entrained and eventually annihilated by the wavefronts emanating from the fastest source. In addition, in the face of high frequency excitation emanating from the rotor, the lower excitability of the tissue, together with the highly complex atrial structure, contributes to intermittent block. As such, ACh enhances sink-to-source mismatch at branching sites and other areas of changing cell-to-cell coupling and/or geometry and facilitates the development of spatially distributed delays and intermittent block, the hallmark of fibrillatory conduction [7]. The experiments presented here demonstrate the manner in which, over a wide range of concentrations, ACh is capable of increasing rotor frequency in the LA to a significantly greater extent than in the RA, thus increasing interatrial frequency gradients and AF complexity.

4.2 The role of the rotor in AF
We have used varying concentrations of ACh to demonstrate that the global AF frequency strongly correlated with the inverse of rotor cycle length. However, with the increase in rotor frequency, rotor lifespan decreased. On the other hand, rotor frequency and PS density were directly correlated in that the faster the activity, the more wavebreaks were observed. The simultaneous occurrence of all three phenomena can be explained by the observation that a matching dose–response increase in frequency dispersion occurred as well. The data support the previously proposed hypothesis that the increase in dispersion at higher ACh concentrations is a manifestation of increasing electrophysiologic heterogeneities in the tissue, which provide more numerous opportunities for intermittent conduction block to occur [23,24]. Therefore, while at low ACh concentrations the rotors remain relatively stable, at non-physiologically high concentrations, these faster spinning rotors encounter a substrate of augmented heterogeneity where there is a greater likelihood for wavelet production to occur. This logical sequence of events accounts for the decreased rotor lifespan as well as increased PS generation. The fact that the wavelet offspring propagate at frequencies similar to the rotation frequency of any simultaneously existing rotor, is evidence that the wavelets emanate from such rotor sources. In brief, while it has long been known that elevated ACh concentrations are a recipe for more complex fibrillatory patterns, our results provide definite evidence that this is the direct result of an increased rotor frequency.

4.3 IK,ACh and acute AF: LA vs. RA frequency differences
In the acute setting of experimental AF, and in the presence of ACh, left-to-right differences in AF frequency in the isolated sheep heart may result from chamber-specific differences in parasympathetic signaling at the level of muscarinic receptors, inhibitory G-protein and/or density of Kir3.x channels. Chamber-specific differences in muscarinic (M2) receptor density have been demonstrated in rabbit hearts [25] but, to our knowledge, no such studies have been made in the sheep heart. Stimulation of muscarinic receptors in the heart activates the G-protein coupled Kir3.x channel complex [8]. Upon exposure to ACh, the heterotrimeric G-protein complex dissociates into its {alpha} and β{gamma} subunits, which results in the interaction of β{gamma} with the Kir3.x subunits causing an increase in the open-state probability of the channel, with consequent activation of IK,ACh. Kovoor et al. [26] demonstrated that AF could not be induced in Kir3.4 (GIRK4) knockout mice. More recently, numerical simulations by Kneller et al. [24] suggested that IK,ACh, is a determinant of frequency and stability during AF. However, prior to our study, it was unknown whether IK,ACh is differentially expressed in the two atria.

Our results show that IK,ACh density is higher in myocytes obtained from the LA than the RA, and this correlates well with larger Kir3.1 and Kir3.4 mRNA levels in LA than RA. We were unable to determine the level of Kir3.x protein due to the unavailability of highly specific Kir3.x antibodies. While previous studies have shown that the Kir3.x channel complex is composed of Kir3.1 and Kir3.4, it has also been demonstrated that in atrial myocytes, a large fraction of Kir3.4 subunits exist as homomultimers [8]. Hence, though precise contribution of each of the Kir3.x channel could not be determined here, their left-to-right difference in the mRNA levels, together with the patch-clamp results, support the hypothesis that in the sheep heart, LA myocytes can adapt to higher excitation frequencies than RA myocytes as a result of a differential distribution of IK,ACh density. Clearly, such differences provide a robust mechanistic explanation for the observed ACh concentration-dependent changes in rotor frequency and organization during AF in this species.

4.4 Clinical implications
Recent work in man [27] and animals [5] suggests that the posterior left atrium and pulmonary veins play important roles in the initiation and maintenance of AF. Whether autonomic alterations are involved in the mechanisms of AF triggering and/or maintenance in these structures is not known. On the other hand, while a correlation between enhanced vagal tone and paroxysmal AF has been observed in humans [28], it is unclear whether in the clinical setting most AF episodes in patients with structurally normal hearts are related to alterations in the autonomic tone.

Several studies have shown that parasympathetic signaling is altered in human atrial myocytes from chronic AF patients. Bosch et al. [29] showed a reduced action potential duration and a decreased rate response of atrial repolarization, which correlated with a decrease in ICa,L and increases in IK1 and IK,ACh at hyperpolarizing potentials. Subsequently, however, Brundel et al. [30] demonstrated that mRNA and protein levels of Kir3.1–3.4 were significantly decreased in chronic AF patients. Dobrev et al. [31] also found that chronic AF induces a transcriptionally-mediated increase in IK1. However, unlike Bosch et al. [29], these authors found that IK,ACh was downregulated to levels that correlated well with the above mentioned Kir3.x protein changes. More recently Dobrev et al. [32] confirmed their own previous results and suggested that the discrepancy with the data of Bosch et al. may have been related to differences in cell populations in the two studies, as well as technical differences in the manner in which IK,ACh was measured. Thus, it seems safe to conclude that IK,ACh reduction in patients with chronic AF occurs as an adaptive response to the continuously high excitation frequency. Interestingly, however, according to one report [33] cholinesterase activity is reduced in chronic AF, which suggests that the reduced IK,ACh may be partially compensated by a reduced level of degradation of the released ACh. Therefore, while important information about the underlying mechanisms of rate adaptation and electrical remodeling has been obtained in the last several years, the issue of the molecular mechanism of AF maintenance and perpetuation is by no means settled.

The studies presented here are far from answering the above question. Nevertheless, the human studies outlined above, together with the demonstration in two different experimental models that fibrillatory activity, whether atrial or ventricular [12] is related to differential expression of ion channels, provide some clues for future research on the mechanisms of chronic AF, particularly in regards to the roles of changes in the expression of specific Kir2.x, and Kir3.4 and possibly Ca,L channels. As discussed above, chronic AF in man is associated with reductions in IK,ACh, and ICa,L but normal or increased IK1. However, the consequences of such a reduced response to parasympathetic activity on wave propagation dynamics in the spontaneously fibrillating atria have never been studied. We speculate that the decrease in the expression of specific Kir3.x channels will reduce IK,ACh and the ability of the fibrillating atria to respond to ACh. However, downregulation of Ca,L channels in the presence of normal or enhanced chamber-specific expression of Kir2.x channels in the sheep heart, should lead to sufficient reduction of local refractoriness to allow stabilization of rotors and maintenance of the arrhythmia in the long term.

Time for primary review 25 days.


   Acknowledgements
 
Supported in part by grants PO1-HL39707, RO1-HL60843 from the NIH and Scientist Development grants from the AHA (A.Z. and O.B.). We thank You Li, Jiang Jiang and Jenny Dang for technical assistance.


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

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