INTRODUCTION

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THE MACHINE-PULLED HIGH-IMPEDANCE glass capillary microelectrode (GM) is standard for transmembrane potential recordings. However, the GM is fragile. It is often difficult to maintain stable impalement for extended periods of time, especially in beating ventricular tissues. One alternative method to record action potential (AP) is to use monophasic AP (MAP) recording techniques (2, 4). Although the MAP recording techniques can provide useful information on cellular repolarization properties such as AP duration (APD) and APD restitution characteristics, its recordings are more reliable in paced rhythms (3, 6) than during ventricular fibrillation (VF) (7). For example, single cell APs registered by a GM during VF showed the presence of discrete diastolic intervals (8). However, diastolic intervals were not observed during VF when MAP recordings were used (11). In one study, we simultaneously recorded transmembrane potential during VF using a GM and an MAP electrode (7). The results showed no correlation between the transmembrane potential morphologies even though the two electrodes were very close to each other. Loeb et al. (10) described a pure iridium metal microelectrode (MM) for physiological recordings. This electrode was made of a combination of materials whose properties allowed the construction of an extremely sharp tip and a relatively high impedance, making it possible to record from a very small area (a few cells). However, it was unclear if iridium MMs could be used to record cardiac APs during paced rhythm or during VF. The purpose of the present study was to test the following hypotheses: 1) pure iridium MMs can be used as an alternative to GM to assess cardiac APD during regular pacing and during VF, and 2) the MMs are more durable than the GMs, allowing uninterrupted stable impalement in an isolated swine right ventricle (RV) without the need to change electrodes repeatedly during an experiment.

	METHODS

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This animal protocol was approved by the Institutional Animal Care And Use Committee and followed the guidelines of the American Heart Association. The isolated perfused swine RV preparations have been described in detail elsewhere (8). Briefly, six farm pigs of either sex were anesthetized. The hearts were quickly removed. The RV was perfused through the right coronary artery with 37°C Tyrode solution gassed with 95% O₂ and 5% CO₂ at a flow rate of 25 ml/min. The composition of the solution was as follows (in mmol/l): 125.0 NaCl, 4.5 KCl, 0.5 MgCl₂, 0.54 CaCl₂, 1.2 NaH₂PO₄, 24.0 NaHCO₃, and 5.5 glucose, with a pH of 7.35. The isolated RV was placed with the endocardial side up in a tissue bath. The entire tissue was also superfused with warmed 37°C oxygenated Tyrode solution. A bipolar electrode was attached to the endocardial surface for pacing.

GM. The GMs used in the study were machine-pulled standard capillary electrodes filled with 3 mol/l KCl with a tip resistance of ~20 M (9). These microelectrodes were coupled with a silver-silver chloride wire leading to an amplifier with a high-input impedance and variable-capacity neutralization (IE-251; Warner Instrument, Hamden, CO).

MM. Pure iridium MMs (World Precision Instruments) used in the study were uniformly coated with 3-µm-thick parylene-C insulation. The length of the electrodes was 76 mm, and the shaft diameter was 0.406 mm. The tip diameter was microscopically measured. The exposed tip was sharpened to 1 µm. It was beveled for the specified length in micrometers (±20%) with microscopic inspection. The MMs had a nominal impedance of 3.5-4.0 M. The signals were amplified by an alternate current amplifier with a filter setting of 0.1 Hz (high pass) and 100 Hz (low pass) and a gain set at 10. The iridium MMs could be reused after cleaning. We applied 2-3 V direct current between the electrode and a saline bath, with the electrode connected to the cathode. This cleaning procedure took ~10 min to complete.

Study protocol. The swine RV usually fibrillated during excision. The fibrillation continued after the RV was mounted in a tissue chamber. Simultaneous GM and iridium MM recordings were made at two adjacent endocardial sites less than 2 mm apart. The two signals were acquired by AXON TL-1-40 analog-to-digital acquisition hardware and Axoclamp 2A software (Axon Instruments, Foster City, CA), with a sampling rate of 200 µs.

Data analyses. The APDs to 50 and 90% repolarization were measured. Dynamic APD restitution curves were constructed by plotting the APD at 90% repolarization vs. the preceding diastolic interval. If an AP occurred before 90% repolarization of the previous one, its diastolic interval was considered to be zero. The maximum slope of the dynamic APD restitution curve was calculated by fitting the data to a biexponential equation using ORIGIN (Microcal Software, Northampton, MA). The mean APDs at 50 and 90% repolarization during pacing were also compared using paired t-tests. Pearson correlation coefficient was calculated to determine whether or not there was a linear correlation. A P value  0.05 was considered significant.

	RESULTS

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Stability of recordings. At least 20 (range, 20-38) GMs were used to complete a single study (3-6 h). In contrast, only one MM was necessary to complete the study. The MM recordings remained stable throughout the entire duration of the study. There was no need for replacement.

AP recordings during fixed-rate pacing. Figure 1 shows simultaneous endocardial AP recordings from a GM and an MM during pacing at 1,000-ms cycle length. The basic morphology of the AP recorded with the MM and the GM was comparable. However, the MM recording showed a gradually increasing phase 4 potential similar to that observed during spontaneous phase 4 depolarization. We believe that this was an artifact created by alternate current coupling. Attempts to record with MMs using direct current coupling failed due to large baseline drifts.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Simultaneous recordings of transmembrane potential (TMP) with a glass microelectrode (GM; top) and of action potential with an iridium metal microelectrode (MM; bottom) during pacing at a cycle length of 1,000 ms.

AP recordings during VF. Figure 2 shows simultaneous recordings by a GM and an MM during VF. The GM recording and the MM recording showed similar AP morphologies on a beat-to-beat basis. In both recordings, transient low-amplitude activity occurred, compatible with the visitation of the electrode by the core of a spiral wave (1). Also registered was a large excitable gap. Note that, as a result of alternate current coupling, the excitable gap recorded by the MM showed up-sloping membrane potential changes as mentioned above.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   GM (top) and MM (bottom) recordings during ventricular fibrillation (VF). GM and MM recordings had similar morphologies. Note the ability of the MM to duplicate the low-amplitude activities (asterisk) and the excitable gap (EG) recorded with the GM. The recording from MMs can only be made with alternate current (AC) coupling. The early portion of the plateau shows a truncated artifact produced by AC filtering.

View larger version (54K): [in this window] [in a new window]   Fig. 3.   Simultaneous recordings with a GM (top) and MM (bottom) for 30 s during VF. The MM recordings demonstrated the amplitude despite loss of impalement by GM at the middle of the recording.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Action potential duration at 90% repolarization (APD90) restitution curves during VF. Note the similarity of the curves determined with the GM (left) and the MM (right). The maximum slopes (Slopemax) of the two curves are similar to each other.

MM and MAP. Figure 5 shows simultaneous recordings with the MM and MAP during pacing at a cycle length of 400 ms. The MM signals were stable during pacing, whereas the recording from the MAP electrode showed unstable AP amplitude with morphology distortion.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Simultaneous recordings with an MM (top) and with monophasic action potential (MAP; bottom) during pacing at a cycle length of 400 ms. Note that the MM signals were stable during pacing, whereas the recording from the MAP electrode showed unstable action potential amplitude with morphology distortion.

	DISCUSSION

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Metal electrode as an alternative to the GM. In the present study, we simultaneously recorded with a standard GM and with an MM from the endocardium in isolated swine RVs during regular (pacing) and irregular (VF) activations. We found that the MM is more stable than the GM for long-term recordings in vitro. The APD and slope of the APD restitution curves determined by the MM were not different from that determined by the GM. Furthermore, we demonstrated that the AP registered by the MM during VF closely correlates with the AP recorded by the GM. These findings indicate that the pure iridium MM may be used as a reliable alternative for GM for the study of APD and APD restitution curve characteristics in the isolated swine RV.

MM and cardiac APD. The pure iridium MM was designed for recording neuronal APs (5, 10). In this study, we demonstrated that the same electrode can also be used to register cardiac APs. The APD restitution curve recorded by the MM was not different from that recorded by the GM. Because the MM was more stable and much more durable and easier to manipulate than the GM, this recording technique should be very beneficial to investigators in the study of cardiac AP characteristics and APD restitution dynamics, which are known to be important in ventricular arrhythmogenesis (12).

Limitations of the MMs. Although the tip of the MM is sharpened to only 1 µm, it is unclear whether or not the tip is recording from a single or from several cells. Furthermore, whether or not the MM tip causes cellular damage remains to be defined. Because the (dV/dt)_max of the MM was considerably smaller and more variable than that of the GMs, it appears that the MM most likely registered extracellular signals. It is therefore not useful to study the rate of rise in phase 0 depolarization of the AP.