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Am J Physiol Heart Circ Physiol 295: H1460-H1469, 2008. First published July 25, 2008; doi:10.1152/ajpheart.00414.2008
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New insights into the atrial electrophysiology of rodents using a novel modality: the miniature-bipolar hook electrode

Yoram Etzion,1,2 Michal Mor,1 Aryeh Shalev,2 Shani Dror,1 Ohad Etzion,2 Amir Dagan,1 Ofer Beharier,1,3 Arie Moran,3 and Amos Katz1,4

1Cardiac Arrhythmia Research Laboratory and 2Internal Medicine Division, Soroka University Medical Center; and 3Department of Physiology, Faculty of Health Science and 4Cardiology Department Barzilai Medical Center, Ashkelon Campus, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Submitted 19 April 2008 ; accepted in final form 24 July 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Studies of atrial electrophysiology (EP) in rodents are challenging, and available data are sparse. Herein, we utilized a novel type of bipolar electrode to evaluate the atrial EP of rodents through small lateral thoracotomy. In anesthetized rats and mice, we attached two bipolar electrodes to the right atrium and a third to the right ventricle. This standard setup enabled high-resolution EP studies. Moreover, a permanent implantation procedure enabled EP studies in conscious freely moving rats. Atrial EP was evaluated in anesthetized rats, anesthetized mice (ICR and C57BL6 strains), and conscious rats. Signal resolution enabled atrial effective refractory period (AERP) measurements and first time evaluation of the failed 1:1 atrial capture, which was unexpectedly longer than the AERP recorded at near normal cycle length by 27.2 ± 2.3% in rats (P < 0.0001; n = 35), 31.7 ± 8.3% in ICR mice (P = 0.0001; n = 13), and 57.7 ± 13.7% in C57BL6 mice (P = 0.015; n = 4). While AERP rate adaptation was noted when 10 S1s at near normal basic cycle lengths were followed by S2 at varying basic cycle length and S3 for AERP evaluation, such rate adaptation was absent using conventional S1S2 protocols. Atrial tachypacing in rats shortened the AERP values on a timescale of hours, but a reverse remodeling phase was noted thereafter. Comparison of left vs. right atrial pacing in rats was also feasible with the current technique, resulting in similar AERP values recorded in the low right atrium. In conclusion, our findings indicate that in vivo rate adaptation of the rodent atria is different than expected based on previous ex vivo recordings. In addition, atrial electrical remodeling of rats shows unique remodeling-reverse remodeling characteristics that are described here for the first time. Further understanding of these properties should help to determine the clinical relevance as well as limitations of atrial arrhythmia models in rodents.

atrial effective refractory period; rate adaptation; electrical remodeling

MICE AND RATS ARE USED EXTENSIVELY in cardiac research, and reliable models of cardiac pathologies have been developed and applied in these species (4, 19, 31). Moreover, genetically altered mice have become invaluable tools for studying the molecular basis of cardiac pathologies including ventricular arrhythmias (7, 28). Use of rodents for studying atrial arrhythmias has been largely limited, but in recent years atrial tachyarrhythmias have been repeatedly produced in rats and mice, and rodents are increasingly used for studying various molecular, cellular, and pharmacological aspects of atrial function (1, 5, 8, 24, 36, 37). In addition, mice with genetic alterations are providing important insights in this field as well (17, 23, 27, 32).

Due to the small size of the rodent heart and, more so, the rodent atria, electrode implantation for studies of functional electrophysiology is challenging. Several techniques were developed to increase the applicability of such studies (2, 23). The transesophageal approach, which is least invasive, necessitates the use of high energy stimulus and induces stimulus artifacts that invariably obscure the atrial signal (12, 33). Development of the open-chest epicardial approach (3) and transvenous endocardial approach (11) significantly improved the obtained results. However, even with these modalities in very skilled hands, stimulus artifacts and ventricular signals usually obscure the atrial signals when programmed stimulation protocols are applied to the atria (17). As a result, a detailed description of the basic atrial properties of rats and mice is largely missing, and various studies that apply rapid pacing protocols do so without an ability to monitor the actual maintenance of the 1:1 atrial capture (36, 38). In addition, a detailed description of the atrial electrical remodeling in rodents is currently unavailable.

Here we developed and used a novel type of epicardial electrode, the Miniature-Bipolar Hook Electrode (covered by International Patent Application No. PCT/IL2008/000161 of Mor Research Applications, Israel). In the present study, we show the advantages of this modality for studying the electrical characteristics of the rodent atria. Moreover, we demonstrate the applicability of these electrodes as the first available tool for atrial EP studies in instrumented conscious rats. Using this modality, we could analyze in detail the atrial electrical properties of rats and mice and demonstrate that the in vivo rate adaptation properties of the atria are surprisingly different than those expected based on previous ex vivo recordings in the murine heart. In addition, we show that atrial electrical remodeling of rats shows unique remodeling-reverse remodeling characteristics that are described here for the first time.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
All experiments were approved by the Institutional Ethics Committee, Faculty of Health Sciences, Ben-Gurion University of the Negev (Beer-Sheva, Israel). At the end of all experiments, animals were killed using intravenous KCl injection under deep isoflurane anesthesia.

Design and capabilities of the bipolar mini-hook electrodes. The bipolar mini-hook electrode is designed to be kept in place without suturing and can be easily attached to the rodent epicardial surface using a small lateral thoracotomy. Each electrode is constructed from a pair of Teflon-coated silver wires (A-M Systems, Carlsborg, WA; coated diameter of 140–200 µm) that were twisted on each other to form a double-stranded electrode (Fig. 1, A and B). Distally, the exposed end of each wire is attached to a miniature stainless steel insect pin (100- µm diameter, 12.5-µm tip diameter; FST, Foster City, CA). The electrode head, excluding the tip, is delicately coated with plastic, and the tips are curved. The distance between the tips is 0.5–1 mm, and the curvature of the tip is 0.5–1 mm in diameter. In the proximal part, a pin is used to attach each pole to the electrical cables of the EP apparatus. While the main design of the electrode is for in vivo experiments, it can be used in isolated heart studies as well (as described in Atrial tachypacing).


Figure 1
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  		Fig. 1. Bipolar mini-hook electrode is useful for in vivo studies of atrial electrophysiology (EP) in rodents. A: schematic presentation of the electrode. B: photograph showing the head of a typical electrode. Needle of an insulin syringe (27 G) is shown for comparison. C: schematic presentation indicating the anatomic places in which electrodes were hooked in the present study (arrows). For our standard recordings, 2 electrodes were inserted on the lateral aspect of the right atria (RA): a pacing electrode on the HRA, and a recording electrode on the LRA. Positioning of the electrodes on the lateral aspect of the atria prevented recordings of large signals from the adjacent ventricular mass. A third electrode was used for recordings from the RV. Dashed arrow indicates the approximate position of a left electrode, which was used in some rat experiments to stimulate the left atria (LA; see Fig. 4). D: an example of the recordings obtained in the setting presented in C. LRA, recording obtained from the LRA electrode. RV, recording from the RV electrode. HRA Stim, timing of pacing induced through the HRA electrode (dashed lines are added for clarity). Note the high resolution of the atrial signal in the LRA recording, as well as the small stimulus artifact that is completely separated from the atrial signal. E: similar recordings as in D in an ICR mouse.

 
Anesthesia and ventilation for acute EP studies. Acute in vivo experiments on anesthetized rats were performed on a total of 63 males (Sprague-Dawley rats, 250–350 g). These experiments included animals in which initial validation of the experimental setup was applied and from which baseline EP characteristics of anesthetized rats were analyzed (n = 45), along with detailed AERP rate adaptation experiments (n = 7) and atrial tachycardia remodeling experiments (n = 11). Anesthesia was induced and maintained using isoflurane (1). Animals were ventilated (Inspira; Harvard Apparatus) in a weight-adjusted manner using either an endotracheal tube or tracheotomy (n = 35 and 27, respectively). Positive end expiratory pressure of 3 cm H2O was initially used and was increased to 5 cm H2O upon thoracotomy. For endotracheal intubation, a 14-G catheter was used that was coated with silicone grease excluding the tip to prevent air leakage. Blood gases under open-chest conditions for at least 4 h (n = 7) demonstrated normal values (pH = 7.34 ± 0.01, bicarbonate = 23.8 ± 0.7 mM, and PCO2 = 35 ± 2.5 mmHg). Body temperature was rectally monitored and maintained at 37 ± 0.5°C by a heating pad. For experiments longer than 1 h, 0.9% saline was injected intravenously at a rate of 1 ml·100 g–1·h–1 through a syringe pump. Experiments on ICR mice were preformed on a total of 25 animals (28–35 g). Five of these animals were initially used for calibration of ventilation condition (see below), and 20 additional animals were used for EP recordings. Mice were anesthetized (isoflurane) and ventilated through a tracheotomy similarly to rats. However, while ventilation was initially performed using the Inspira (Harvard Apparatus) weight-adjusted ventilation parameters, our preliminary blood gases measurements revealed substantial CO2 retention under these conditions that was not related to chest opening and could not be resolved by minimizing dead space. To overcome this problem, we increased the respirator minute volume and could calibrate relatively normal PCO2 values for a ~30-g mouse using a tidal volume of 600 µl and a respiratory rate of 140 beats/min. Later on, we switched to a mice dedicated respirator (Minivent, Harvard Apparatus), which resolved this issue completely. Nevertheless, blood gases were always obtained at the end of mice recordings, and while successful EP recordings could be obtained in 20 ICR mice, only recordings from mice demonstrating PCO2 below 55 mmHg (n = 15; mean PCO2 = 39.3 ± 3.3) were included in the final analysis of baseline characteristics (see Table 2). In six of the above ICR mice detailed analysis of AERP rate adaptation characteristics (see Fig. 5D) were preformed after baseline EP recordings. Confirmatory recordings of rate adaptation properties in C57BL6 mice were done in a similar manner to that described above for the ICR strain (n = 6).


Figure 5
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  		Fig. 5. Atrial rate dependence. A: failed 1:1 atrial capture as a function of AERP150 in anesthetized rats. Note that in 34 out of 35 evaluated animals the failed 1:1 atrial capture was larger than AERP150. Dashed horizontal lines indicate rates at which pacing is usually applied in atrial tachycardia studies of rats. Note that at cycles of 50 and 60 ms, respectively, only 1 animal out of 35 (~3%) and 7 animals out of 35 (20%) clearly retained 1:1 capture at the initiation of rapid pacing. B: failed 1:1 atrial capture as a function of AERP100 in anesthetized ICR mice. Failed 1:1 atrial capture was 31.7 ± 8.3% longer than the AERP100 (P = 0.0001; n = 13). C: AERP as a function of basic CL (BCL) in anesthetized rats using single-beat S1S2S3 protocol ({blacksquare}), 10-beat S1S2 drive train ({blacktriangledown}), and 40-beat S1S2 drive train ({circ}; see MATERIALS AND METHODS for specific description of the pacing protocols). Note the AERP shortening with the single-beat protocol and the complete absence of this phenomenon when 10- or 40-beat drive trains were used. **Lowest BCL at which 1:1 atrial capture was obtained for each animal; the AERP with the single-beat drive train was 80.4 ± 1.7% of the AERP obtained using the 10-beat drive train (P = 0.0001; n = 7). D: similar experiment as in C in anesthetized ICR mice. **Lowest BCL at which 1:1 atrial capture was obtained for each animal; the AERP with the single-beat drive train was 74.2 ± 4.2% of the AERP obtained using the 10-beat drive train (P = 0.005; n = 6). E: similar experiment as in C and D but in anesthetized C57BL6 mice. **Lowest BCL at which 1:1 atrial capture was obtained for each animal; the AERP with the single-beat drive train was 62.3 ± 5.6% of the AERP obtained using the 10-beat drive train (P = 0.0001; n = 6).

 
EP setup for acute recordings. Animals were connected to an electrophysiological system (Nihon Kodhen, RMC1100), and surface ECG was monitored using cutaneous clips fixed on each limb. Under aseptic conditions, consecutive intercostal spaces 2–5 were gently exposed with special care to avoid lung injury. Under direct vision, two pairs of miniature bipolar hook electrodes were inserted on the lateral aspect of the high right atrium (HRA) and low right atrium (LRA) for pacing and recording, respectively (Fig. 1C). A third bipolar electrode was inserted on the right ventricle (RV) for recordings. In some experiments (see RESULTS), an additional pacing electrode was implanted on the LA. In mice, electrode implantation was done under binocular vision (Motic, ZMZ-168). With the use of the above setup, the obtained stimulus threshold levels at the HRA were 0.18 ± 0.04 and 0.13 ± 0.02 mA for rats and mice, respectively, and high resolution atrial EP was consistently obtained (Fig. 1, D and E).

Electrophysiological signals were interfaced with a PC using an A/D converter (PCI-6024E; National Instruments, Austin, TX) and a program developed by YE (using LabView 7.1, National Instruments) to control data acquisition, electrical stimulation, and offline analysis. Electrical stimulation (2-ms square pulses) was applied through an isolation unit (Iso-Flex; AMPI). Cardiac recordings were filtered (1 Hz-1,000 KHz) and sampled to the PC at a digital sample rate of 2 KHz. EP protocols included basal recordings of RR interval, PR interval, and QT interval (if easily detected in the ECG). Thereafter, a programmed S1S2 stimulation protocol was used to determine the atrioventricular (AV) node refractory period (AVERP) and the atrial effective refractory period (AERP) in the millisecond range. A decremental atrial pacing protocol starting from 150- or 100-ms cycle length (CL) in rats and mice, respectively, down to 20-ms CL (in 5-ms steps) was used. Each step consisted of a 5-s long train of 2-ms pulses. This protocol was used to determine the AV node Wenckebach conduction block (AV wenk), the longest interval with 2:1 AV conduction (AV2:1), and the longest interval with failed 1:1 atrial capture. Sinus node recovery time and corrected sinus node recovery time were obtained using 30 S burst pacing protocols (3). Basic CL (BCL) in milliseconds is stated when relevant for each parameter (i.e., AERP150 = AERP at a BCL of 150 ms). For atrial rate adaptation studies, a "single-beat S1S2S3 protocol" was used in which a drive train of 10 S1s at a near normal BCL (100 ms in mice and 150 ms in rat) was followed by a single S2 at varying BCL and a single S3 for AERP evaluation. The longest S2S3 interval that failed to capture was defined as the AERP of the S2 beat. In addition, drive trains of ≥10 S1 at different BCL followed by a single S2 for AERP evaluation ("10-, 20-, or 40-beat S1S2 drive train") were also applied. A recovery time of at least 3 S was allowed between each S1S2 or S1S2S3 protocol. Determination of all AVERP and AERP values in the study was confirmed three consecutive times.

Atrial EP in conscious rats. A total of 14 conscious rats was evaluated in this section after the initial determination of the desired experimental technique (as described in Fig. 2A). From these 14 animals, 9 were used for atrial tachypacing studies (5 for "treatment" and 4 for sham pacing experiments, see RESULTS). In the final setup for this technique, two pairs of bipolar mini-hook electrodes, as well as two peripheral stainless steel electrodes, were connected to a 8-pin "female" connector, which was designed so that it could be implanted in the back of the animal. Sterilization was applied using electron beam radiation. Rats were anesthetized (75/5 mg/kg ip ketamine/xylazine), intubated, and placed supine on a warmed heating pad. Under sterile conditions, a subcutaneous canal was created from the left thorax to the upper back of the animal and the connector (protected by a small latex cover) was inserted. Thereafter, hook electrodes were implanted on both atria through minimal bilateral thoracotomies and the two peripheral electrodes were positioned in the back of the animal for ECG measurements as well as unipolar atrial recordings (Fig. 2C). After chest closure, the animal was placed in a prone position and the back connector was exteriorized. Animals were injected with carprofen (5 mg/kg sc) and 0.9% NaCl (1.5 ml) and were transferred to a normal cage for a recovery period of 3–5 days. During the first 3 days, dypirone (0.4 mg/ml) and ciprofloxacin (0.125 mg/ml) were added to the drinking water. After the recovery period, the animals were placed in a dedicated recording chamber where the back connector was linked to an elastic cable. Pacing and recording and were made in the right (RA) and left atria (LA), respectively (Fig. 2). After a 2- to 4-h adjustment period, baseline electrophysiological measurements were performed (see Table 3), and thereafter 0.5 mg/ml of atenolol was added to the drinking water to reduce AV conduction during atrial tachypacing. This manipulation increased the RR interval by 24.5 ± 3.1% (n = 12; P < 0.001) and the AVERP by 14.6 ± 1.5% (P < 0.001) at 24 h but did not significantly affect the AERP (P = 0.57).


Figure 2
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  		Fig. 2. Atrial EP in instrumented rats. A: ECG (top trace) and bipolar LA recording (bottom trace), through the back connector, after a preliminary surgical procedure in which 1 hook electrode was inserted on the LA of a rat. Note the high resolution recording from the atria indicating the focal attachment of the electrode to the atrial surface. B: similar animal as in A. Pacing through the LA electrode resulted in atrial capture that was documented in the ECG. Nevertheless, atrial EP could not be assessed using this preliminary setup. C: recording from a rat in which each atrium was instrumented with a bipolar mini-hook electrode. In this final setup, right electrode was used for pacing, whereas the left electrode was used for recording. Top trace: unipolar recording from LA (against a peripheral electrode on the back). Middle trace: bipolar recording from the LA. Bottom trace: RA pacing. Note that the LA signal could be easily assessed during RA pacing.

 
Atrial tachypacing. Experiments were done in three experimental setups as follows: 1) isolated heart recordings: use of this setup was mainly dedicated for atrial tachypacing experiments including a total of 12 hearts (6 treatment, 3 shams, and 3 "on-off experiments," see RESULTS). Additionally, confirmatory experiments of atrial rate adaptation were done in this setup as well (n = 6, see RESULTS for details). After intraperitoneal injection of heparin (1,000 U), hearts were quickly removed to a cold Tyrode's solution containing the following (in mM): 140 NaCl, 5.4 KCl, 0.5 MgCl2, 2.5 CaCl2, 0.39 NaH2PO4, 11 glucose, and 10 HEPES (titrated to pH 7.4 with HCl). Hearts were hung on a Langendorff system, and a coronary pressure of 100 mmHg was applied. Mini-hook electrodes were attached to the RA (for pacing), LA (for atrial recordings), and RV (for ventricular recording). After 20 min (recovery time), 3 h of rapid atrial pacing at a CL of 5- to 10-ms higher than the failed 1:1 atrial capture was applied to the RA at double diastolic threshold. Sham hearts were paced at a 200 ms CL for 3 h. 2) Anesthetized rats: experiments were preformed on a total of 11 animals (7 treatment and 4 shams, see RESULTS). A conventional electrode setup (Fig. 1) was used in these experiments. Rapid atrial pacing was applied at a cycle 5–10 ms higher than the failed 1:1 atrial capture for 4 h. Sham animals were paced at a CL of 120 ms (similar to the mean RR interval of the experiment group during tachypacing). 3) Conscious rats: experiments were preformed on a total of nine animals (5 treatment and 4 shams, see RESULTS). After 24 h of adjustment in the pacing cage under application of atenolol in the drinking water, rapid atrial pacing protocol was applied at a 70-ms CL for 24 h. At this pacing rate, a 1:1 atrial capture was maintained and the ventricular response rate was 2:1 resulting in a mean RR interval of 140 ms. AERP was measured at 2, 4, 6, 12, and 24 h at double diastolic threshold intensity. Sham animals (n = 4) went through a similar procedure as the rapid atrial pacing animals but were paced instead at 140 ms for 24 h.

Statistical analysis. Values are means ± SE. Paired or unpaired Student's t-test was used as required. Atrial electrical remodeling data were evaluated using one-way ANOVA of repeated measures. If the initial ANOVA test determined significance, post hoc analysis using Scheffé's F test was done to compare the baseline AERP values and the values at each time point thereafter. Statistical significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
The mini-hook electrodes greatly facilitated the ability to record atrial signals in a simple and accurate fashion. In various previous studies (1, 11, 17, 23, 29, 34, 36, 38), atrial pacing protocols were applied in a rather "blind" manner with regard to the atrial response. Some important misinterpretations which may result from such methodology are illustrated in Fig. 3, A and B. As can be seen in Fig. 3C, our present signal resolution also enabled easy detection of the failed 1:1 atrial capture, which had not been analyzed in vivo in rats and mice to the best of our knowledge. As we will show, analysis of this parameter led to some important and unexpected findings.


Figure 3
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  		Fig. 3. Examples of high resolution atrial signals obtained in anesthetized rats and ICR mice. In all recordings, top trace denotes LRA recording, middle trace denotes RV recording, and bottom trace marks the pacing through the HRA electrode. A: recordings in rat during a decremental pacing protocol. Note the 1:1 atrioventricular (AV) conduction at CL of 150 ms and CL of 95 ms but the failure of 1:1 conduction at CL of 90 ms. While the ventricular recordings could suggest AV conduction block at CL of 90 ms, atrial recordings indicate that failure occurred at the atrial level. B: recordings from a mouse during S1S2 protocol. Note successful AV conduction at an S1S2 of 46 ms and block at S1S2 of 45 ms. While ventricular recordings may suggest that AVERP was reached, the atrial recording indicates failure at the atrial level (i.e., AVERP ≤45 ms). C: recordings of failed 1:1 atrial capture in a mouse. Decremental pacing protocol was used to determine the atrial response to increased pacing rates. Note 1:1 capture at CL of 50 ms and failure at CL of 45 ms.

 
Baseline properties. The supraventricular EP data obtained from a group of 45 anesthetized adult rats are summarized in Table 1. Similar recordings obtained in 15 ICR mice are summarized in Table 2. It can be noted that the heart rates obtained in the present study while the animals were under isoflurane anesthesia were near the physiological range usually seen in rats and mice in a conscious state (>350 beats/min for rats; >500 beats/min for mice). For rats, we also had an opportunity to record these parameters for the first time in the conscious state (Table 3). Comparison with the anesthetized animals indeed demonstrated rather similar values.


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  		Table 1. Summary of basic supraventricular EP in anesthetized rats

 

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  		Table 2. Summary of basic supraventricular EP in anesthetized ICR mice

 

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  		Table 3. Summary of basic supraventricular EP in conscious rats

 
To test the ability of our current modality to be used in studies comparing the properties of both atria, an additional pacing electrode was implanted on the left atria of five anesthetized rats. Successful positioning of all four electrodes (3 electrodes on the standard positions on the right side plus additional pacing electrode on the LA; Fig. 1C) was rather easily obtained in all five preparations. Thereafter, double threshold pacing was performed sequentially at the RA electrode (HRA) vs. the LA electrode (positioned at the posterior left atria), and we could not detect a difference in the obtained AERP or failed 1:1 atrial capture recorded by the LRA electrode (Fig. 4; see DISCUSSION).


Figure 4
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  		Fig. 4. LA vs. RA stimulation in anesthetized rats. Left: atrial effective refractory period (AERP)150 measurements. Note the similarity in the obtained AERP150 values (n = 5; P = 0.56) Right: similar experiments but measurement of failed 1:1 atrial capture. Note the similarity in failed 1:1 atrial capture values (n = 5; P = 0.2). NS, not significant.

 
Rate adaptation properties of the rodent atria in vivo. Taking advantage of the high resolution recordings obtained by the current modality (Figs. 1–3), we next aimed at evaluating the in vivo rate adaptation properties of the atria in rats and mice. Based on previous ex vivo results in the murine mice (16), we could assume that the AERP that follows high pacing rates as well as the failed 1:1 atrial capture of the same tissue should both be shorter than the AERP recorded at near normal pacing rates (see discussion). However, our atrial recordings could not support this prediction (Fig. 5; Tables 1 and 2). In anesthetized rats, for instance, the failed 1:1 atrial capture was longer than the AERP150 in 34 of 35 evaluated animals (Fig. 5A) and was overall 27.2 ± 2.3% longer than the AERP150 (P < 0.0001; n = 35). In ICR mice, a similar phenomenon was noted (Fig. 5B) and the mean duration of the failed 1:1 atrial capture was 31.7 ± 8.3% longer than the AERP100 (P = 0.0001; n = 15). Consistently, we also could not demonstrate a rate-dependent shortening of the AERP in anesthetized (Fig. 5C) or conscious (n = 5, not shown) rats as well as in anesthetized ICR mice (Fig. 5D) using drive trains of ≥10 S1 at different BCL followed by a single S2 for AERP evaluation. However, significant rate-dependent shortening of the AERP of the S2 beat was noted when the single-beat S1S2S3 protocol was applied (Fig. 5, C and D). As a result, at the shortest BCL in which 1:1 atrial capture was obtained for each animal, the AERP of the S2 beat obtained with the S1S2S3 protocol was 80.4 ± 1.7% of the AERP obtained using the 10 beats drive train in rats (P = 0.0001; n = 7) and 74.2 ± 4.2% in ICR mice (P = 0.005; n = 6). To further confirm our findings similar rate adaptation experiments were preformed in C75Bl/6 mice. In agreement with the above results, failed 1:1 atrial capture was 57.7 ± 13.7% longer than the AERP100 (P = 0.015; n = 4) and at the shortest BCL in which 1:1 atrial capture was obtained the AERP of the S2 beat obtained with the single-beat S1S2S3 protocol was 62.3 ± 5.6% of the AERP obtained using the 10-beat S1S2 drive train (Fig. 5E; P = 0.0001; n = 6). To get further insight into whether the current new findings are specific to the in vivo setting only, we studied the rate dependence of the AERP shortening in isolated rat heart (not shown). With the use of a 10-beat S1S2 drive train, the mean AERP value at the shortest CL that retained 1:1 atrial capture was 91.8 ± 3.2% of the AERP value at a 150-ms CL (P = 0.53; n = 6) demonstrating a "flat" rate adaptation curve of the AERP in this preparation as well. In addition, when the single-beat S1S2S3 protocol was preformed in these hearts, shortening of the AERP of the S2 beat values was noted as demonstrated in the in vivo setting. As a result, the AERP derived from the single-beat S1S2S3 protocol was 79.0 ± 8.5% of the AERP obtained using the 10-beat S1S2 drive train at the shortest BCL in which 1:1 atrial capture was maintained (P = 0.017; n = 6). These results indicate that our current findings are not confined to the in vivo setting only and may differ from previous ex vivo findings due to a difference in the recording methodology used [AERP vs. monophasic action potential (MAP) recordings, seeDISCUSSION].

Atrial tachycardia remodeling. Limited data exist regarding atrial tachycardia remodeling of rodents. In the present study, we utilized our novel modality to explore this issue in rats. Initially, we applied rapid atrial pacing in isolated perfused hearts at a cycle 5- to 10-ms longer than the failed 1:1 atrial capture for 3 h. Surprisingly, while AERP rapidly decreased after 1 h of rapid atrial pacing to 62.2 ± 6.8% of baseline (P = 0.012; n = 6), it thereafter returned back toward its baseline value and was not significantly different from the baseline values thereafter (Fig. 6A, left). Sham hearts in which the atrium was paced at a 200-ms cycle had stable AERP values (n = 4; P = 0.41). Additional control experiments in which the rapid pacing was stopped at 1 h and was turned on thereafter (n = 3) demonstrated complete recovery of AERP shortening after discontinuation of pacing and reinitiation of similar AERP shortening after a second period of rapid pacing (Fig. 6A, right), indicating that the remodeling-reverse remodeling did not result from deterioration of the preparations. Application of a similar rapid atrial pacing protocol in anesthetized rats resulted in a similar remodeling of the AERP to 82.5 ± 4.8% of baseline at 2 h (P = 0.047; n = 7) and reverse of the remodeling thereafter (Fig. 6B). Sham animals did not demonstrate similar AERP shortening (n = 4; P = 0.69). Next, we applied atrial tachypacing in the conscious rat up to a period of 24 h at a constant CL of 70 ms. Similar shortening of the AERP to 82.3 ± 1.0% of baseline (P = 0.026; n = 5) was noted in the first 4 h, and thereafter a reverse remodeling phase was noted in this experimental setup as well (Fig. 6C). Sham animals, paced at a 140-ms CL for 24 h, did not demonstrate significant AERP changes during this period (n = 4; P = 0.57).


Figure 6
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  		Fig. 6. Atrial tachycardia remodeling in rats. A, left: tachypacing of isolated-perfused hearts at a cycle 5- to 10-ms longer than the failed 1:1 atrial capture for 3 h. Sham animals were paced at CL of 200 ms. Note that AERP decreased after 1h of rapid atrial pacing to 62.2 ± 6.8% of baseline (P = 0.013; n = 6) but thereafter returned toward baseline. A, right: additional isolated heart experiments in which the rapid pacing was stopped at 1 h and was turned on thereafter after recovery of AERP values (n = 3). AERP was reduced to 70.5 ± 1.7% of baseline after the first episode of rapid pacing and recovered to 97.8 ± 5.2% of baseline after 30 min of discontinued pacing. Reinitiation of rapid pacing reduced the AERP again to 74.2 ± 3.4% of baseline. B: application of a rapid atrial pacing protocol similar to that in A in anesthetized rat preparations resulted in AERP shortening to 82.5 ± 4.8% of baseline at 2 h (P = 0.047; n = 7) and a return back toward baseline thereafter (P = 0.69 and P = 0.63 at 3 and 4 h, respectively). Sham animals paced at a 120-ms cycle did not reduce their AERP (n = 4; P = 0.69). C: application of atrial tachypacing in conscious rats up to a period of 24 h at a constant CL of 70 ms. Note shortening of the AERP to 82.3 ± 1.0% of baseline (P = 0.026; n = 5) in the first 4 h and thereafter a reverse remodeling phase that was noted in this experimental setup as well. Sham animals, paced at 140 ms CL for 24 h, did not demonstrate significant AERP changes during that period (n = 4, P = 0.57).

 

   DISCUSSION
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The present study describes two main achievements of high importance: 1) the development of a novel, simple, and reliable methodology for studying the supraventricular electrophysiology of anesthetized as well as conscious rodents; and 2) a detailed description of several atrial characteristics of rodents that could not be assessed properly until now. These achievements are discussed in detail below.

Bipolar mini-hook electrodes. Our new methodology demonstrates the after advantages over currently available techniques: 1) a low stimulus threshold for pacing and highly improved signal resolution of the atrial recordings, 2) an ability to easily implant various electrodes on the same atria and on both atria in parallel, and 3) the applicability for long-term recordings in conscious freely moving rodents. While the use of rodent models for studying supraventricular function has largely increased in the last decade (1, 5, 8, 11, 17, 23, 29, 34–36, 38), the ability to study atrial electrophysiological properties in the in vivo setting was complicated and limited with the currently available techniques. As a result, various studies (8, 13, 14, 35, 38) were either done fully ex vivo or applied the desired treatment in vivo and extracted the heart thereafter to study the electrophysiology in a proper manner. Although in some instances current techniques can record parameters such as AERP (3, 39), the use of these modalities is currently restricted to a few highly skilled researchers and even then can fail to obtain the desired results (17).

According to our experience, three main factors, all related to the small dimension of the atria, converge and make the rodent atrial recordings so complicated: 1) to obtain proper signals during pacing there is a need to physically attach four different electrical poles on the same atrial tissue (2 for bipolar pacing and 2 for bipolar recording). This mission is rather challenging. 2) Pacing poles should be at least 1–2 mm from the recording poles to obtain reduced stimulus artifacts. 3) Recording poles should be fairly distant from the ventricular tissue, otherwise signals are recorded from the adjacent large ventricular mass. Proper achievement of all these conditions using a blindly inserted electrode is a highly difficult task. The present technique overcomes all these problems, since it allows placement of electrodes under direct vision [as in the open-chest approach of Berul et al. (3)] but with a small lateral incision. Therefore, success can be obtained in the large majority of rats and mice tested. Moreover, the above-noted properties have made the mini-hook electrodes suitable for long-term atrial pacing/recordings of instrumented animals, which opens up a variety of new research options.

Rodent atrial EP characteristics. The ability to easily obtain high-quality atrial EP recordings from several rats and mice has opened a window of opportunity for us to analyze some important characteristics as discussed below.

Comparing the properties of both atria. In humans and large animals, shorter refractoriness of the LA compared with RA (25, 30) is potentially involved in the preferential role of the LA in atrial fibrillation (AF) initiation and maintenance (21, 26). This phenomenon is at least partially related to a larger potassium current related to Ether A Go Go (HERG) potassium channel (IKr) and consequent shorter action potential duration (APD) in the LA (18). Whether a similar phenomenon can be observed in the rodent atria in vivo is currently unknown. However, Lomax et al. (20) found shorter AERP and APD of the LA compared with the RA in C57BL6 mice ex vivo that was related to a larger outward current density in LA cardiomyocytes. Our present results, in contrast, indicate that pacing of the RA and LA induces the same recorded refractoriness in the LRA electrode in anesthetized rats in vivo. These results stress the usefulness of the mini-hook electrodes as a first modality for studying the properties of both rodent atria in vivo and may indicate the presence of rather uniform refractoriness of the rat atrial tissue in that context. While the difference between our current results and Lomax et. al. (20) may be species dependent (rat vs. mice), it may also be related to difference in factors underlying AERP duration in vivo and ex vivo, as it can be noted that the baseline values of AERP that were measured in C75BL6 and ICR mice in the present study (~40 ms; Fig. 5; Table 2) are much longer than the values observed by their recordings in the isolated mice atria (<20 ms). In addition, it should be noted that while the bipolar pacing electrodes were placed on both atria in the present study, the AERP recordings were all done by the same electrode located in the LRA (Fig. 1C). Therefore, a shorter AERP of the LA may have been missed in our present study due to the failure of the LA signal to invade the LRA recording site at S1S2 intervals shorter than the RA AERP. Further studies, preferentially in C57BL6 mice and using an additional recording electrode in the LA, seem feasible and can be of high value for proper understanding of this issue in the future.

In vivo rate adaptation. As in large animals, the refractoriness of the murine myocardium is normally determined mainly by the APD (15). However, rate dependence of the murine APD was only recently evaluated for the first time using ex vivo MAP recordings (16). The results of this study indicated that atrial and ventricular APD CL dependence and restitution properties are surprisingly similar to those of larger mammals and humans. Moreover, at increasing pacing rates, marked shortening of the ventricular steady-state APD was noted. These results imply that the ERP that follows high pacing rates should be shorter than the ERP recorded at slower near normal pacing rates. In contrast, the results of the present study indicate that while CL-dependent shortening of the AERP of the S2 beat can be observed when a single-beat S1S2S3 protocol is applied, such rate adaptation is completely absent when conventional drive trains of ≥10 S1 at different BCL are applied followed by an S2 for AERP evaluation. Since this surprising result is important, we tested and confirmed the absence of conventional AERP rate adaptation not only in rats (anesthetized and conscious) but also in two different strains of mice commonly used in laboratory studies (Fig. 5). Moreover, similar findings could also be observed in the isolated rat heart preparation; therefore, we conclude that this phenomenon is not restricted to the in vivo setting only. In view of that fact that APD has been shown to become shorter with a decrease in the BCL using MAP recordings in the murine heart (16), as well as using the single-beat S1S2S3 protocol in the present study, the flat rate adaptation curves after S1S2 drive trains suggest the presence of a rate-dependent "process" that increases refractoriness at high-firing frequencies regardless of the APD. While an ionic mechanism of such a process needs to be determined, this important finding of the present study can again stress the usefulness of the current methodology for better understanding the EP properties of the rodent atria. Further experiments combining MAP recording with our current methodology may facilitate the understanding of this issue in future studies. Detailed comparison between atrial and ventricular rate adaptation properties will also be of high importance in this context.

In agreement with the above finding, our results also unexpectedly indicated that the failed 1:1 atrial capture of rodents is significantly longer than the AERP recorded at near normal BCLs (150 ms in rats; 100 ms in mice). This finding by itself has important implications for atrial tachypacing studied, since it demonstrates that empirical pacing at a 50- or even 60-ms CL (as done in various studies) will induce a 1:1 atrial response in only a minority of animals (Fig. 5A). For example, Yamashita et al. (38) concluded that the antiarrhythmic drug cibenzoline attenuates the upregulation of Kv1.5 channel gene expression after 4 h of atrial pacing at 60 ms cycle (38). Based on our current results, it may be suggested that effects of the drug on the APD could have switched animals with borderline values of failed 1:1 atrial capture (Fig. 5A) from 1:1 atrial response to a 2:1 response leading to a much lower actual pacing rate in the cibenzoline-treated animals. Such a possibility can be easily confirmed or excluded in future studies using our currently described methodology.

Atrial tachycardia remodeling. Shortening of the AERP is the hallmark of atrial tachycardia remodeling (reviewed in Refs. 6, 22). Even short-term AF (several minutes) was shown to initiate AERP shortening (9, 10). While this issue has been studied in detail in large mammals, rodent experiments are rather sparse. Nevertheless, indications for acute shortening of the AERP by rapid pacing now exist (1, 38), and it was even suggested that long hours of rapid pacing may trigger AF in the rodent atrium ex vivo (24). Since methodological problems have largely limited proper examination of this issue in rodents up to now, we utilized our current technique for more proper evaluation. Our result clearly demonstrates that maximal rate pacing ex vivo and in vivo initiates AERP shortening followed by recovery of the AERP on a timescale of hours. While the above "reverse remodeling" phenomenon may have been claimed to result as a technical artifact (i.e., deterioration or an effect of anesthesia), sham experiments as well as the on-off experiments in the isolated rat heart (Fig. 6A) indicate that our preparations did not loose their ability to respond to rapid pacing with time. Furthermore, using our novel instrumented rat model, we could show that a similar phenomenon can be observed in the conscious rat as well. Interestingly, our current observations (not shown) also indicate that on the timescale of 24–72 h, tachypacing of the rat atria does not by itself initiate atrial arrhythmias as was previously suggested by ex vivo recordings (24). The above-described "reverse remodeling" phenomenon highlights the fact that while rodent models may be very useful to study various aspects of atrial function, they may also have significant limitations in terms of clinical relevance, which must be fully characterized to be able to use these models in a proper manner. Transient increase of Kv1.5 mRNA was described after 2 h of atrial tachypacing in rats (36). Whether this mechanism contributes to the transient AERP shortening described in the present study is yet to be determined.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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 GRANTS
DISCLOSURES
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This work was partially supported by the Yaacov Matzner and Amiram Eldor Physician-Scientist Fellowship from the Bat Sheva de Rothschild Foundation and the Israeli Health Ministry (Y. Etzion), Chief Scientist Grant of the Israeli Health Ministry (Y. Etzion), and German Israeli Foundation Grant 850/2004 (AK, AM, and Y. Etzion).


   DISCLOSURES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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REFERENCES
 
Y. Etzion, A. Moran, and A. Katz have rights in the mini-hook electrode patent.


   ACKNOWLEDGMENTS
 
We thank Dr. Stanley Nattel for fruitful discussion and important scientific insight and Dr. Morton Mower for generous support. Y. Etzion thanks the Dr. Gabi and Eng. Max Lichtenberg Career Development Chair in Medicine, Ben-Gurion University of the Negev, for generous support of research activities.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Etzion, Cardiac Arrhythmia Research Laboratory and Internal Medicine Division, Soroka Univ. Medical Center, P.O. Box 151, Beer-Sheva 84101, Israel (e-mail: tzion{at}bgu.ac.il)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Y. Etzion and M. Mor contributed equally to this work.


   REFERENCES
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 RESULTS
 DISCUSSION
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 REFERENCES
 

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