Machine-pulled high-impedance glass capillary microelectrode is standard for transmembrane potential (TMP) recordings. However, it is fragile and difficult to impale, especially in beating myocardial tissues. We hypothesize that a high-impedance pure iridium metal electrode can be used as an alternative to the glass microelectrode for TMP recording. The TMPs were simultaneously recorded from isolated perfused swine right ventricles with a metal microelectrode and a standard glass microelectrode during pacing and during ventricular fibrillation. The basic morphology of TMP recorded with these electrodes was comparable. The action potential duration (APD) at 90% repolarization was 241 ± 29 ms for the metal microelectrode and 236 ± 31 ms for the glass microelectrode with a good correlation (r = 0.99, P < 0.0001). The maximum slope value of the APD restitution curves during pacing was also significantly correlated. One metal microelectrode and >20 glass microelectrodes were needed per study. We conclude that, in isolated perfused swine right ventricles, the TMP recorded by the metal microelectrode is comparable with that recorded by the glass microelectrode. Because the metal microelectrode is more durable than the glass microelectrode, it can serve as an alternative for APD recording and for restitution analyses.
- transmembrane action potential
- ventricular fibrillation
- action potential duration restitution curve
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.
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% CO2at 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.
The GMs used in the study were machine-pulled standard capillary electrodes filled with 3 mol/l KCl with a tip resistance of ∼20 MΩ (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).
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 MΩ. 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.
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.
We first recorded APs during VF for 5 min. After being cardioverted to quiescence, the RVs were then paced at regular intervals. By employing different pacing protocols, we obtained a broad spectrum of AP characteristics for comparison between the two recording methods. First, simultaneous recordings were made during fixed-rate pacing at a cycle length of 1,000 ms. The APD restitution curves were then constructed by using a dynamic pacing protocol. This protocol initially paced the RVs at a 400-ms cycle length at two times the diastolic threshold current for eight beats, followed immediately by eight beats of pacing at 350-, 300-, 280-, 260-, 240-, 230-, 220-, 210-, 200-, 190-, 180-, 170-, and 150-ms cycle lengths, respectively. Simultaneous MM and MAP recordings were made at two adjacent endocardial sites <5 mm apart.
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.
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 increasingphase 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.
The APD at 50% repolarization recorded by the MMs (206±34 ms) and the GMs (209±41 ms) were not significantly different. There was an excellent correlation (r = 0.99, P < 0.0001). Also the APD at 90% repolarization recorded by the MMs (241±29 ms) was not significantly different from that recorded by the GM (236±31 ms). There was an excellent correlation between the two electrodes (r = 0.93, P < 0.0001) as well. In contrast, large differences in the maximum derivative of voltage in relation to time [(dV/dt)max] were noted between that recorded by the GM (87.5 ± 4.3 V/s) and that recorded by the MM (0.58 ± 0.14 V/s). There were no statistically significant correlations between them (P = not significant). This lack of correlation was due to a considerably smaller AP amplitude recorded by the MMs (range 0.8–3.8 mV, mean 2.6 ± 0.8 mV).
The dynamic APD restitution curves recorded by the GMs and by the MMs in one RV showed that the two curves closely matched each other (maximum slope value 1.83 vs. 1.97). There was a significant correlation in the maximum slope of the APD restitution curves between that recorded by MMs and by GMs in all RVs (1.65 ± 0.54 vs. 1.80 ± 0.98, r = 0.91, P < 0.05).
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.
Figure 3 demonstrates the stable recordings of an MM over 30 s during VF, whereas GM shows loss of impalement in the middle of recording. In all RVs, the MMs demonstrated stable and undistorted recording during the extended period.
Figure 4 shows APD restitution curves constructed by a GM and an MM during VF in one RV, showing that the two curves closely matched 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.
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.
A significant difference between the MMs and the MAP electrodes is that the sharper tip (1 μm) of the MM records from fewer cells than the larger-tipped MAP electrode that simultaneously records from a large number of cells. Therefore, the pure iridium MM may be in fact a “mini MAP” electrode. Size-based differences at the contact site appear to have important influences on the morphology of the signals during irregular rhythms, such as VF. The MM, but not the MAP electrode, recorded AP that correlated well with the single-cell AP during VF.
It is possible that the pure iridium MMs can also be used to record from the left ventricle if the contraction is reduced by an excitation-contraction coupler. However, in our experience, it could not be used to record from a beating heart in situ because the greater motion of a whole heart damaged the tip of the electrode.
Another limitation is that the environment of our in vitro preparation is sufficiently noisy that recording from MM can only be made with filters and with alternate current coupling. Therefore, the MM is not suitable for studying spontaneous phase 4 depolarization or other pacemaker activity.
In conclusion, MMs may be used as a substitute for GMs for recording APD and APD restitution curves in vitro. The MM is preferable over the GM because it is stable in beating ventricular tissue and simpler to use. However, the MMs are not useful in determining AP amplitude and (dV/dt)max or in the study of pacemaker activity.
We thank Avile McCullen and Meiling Yuan for technical assistance and Elaine Lebowitz for secretarial assistance.
This study was done during the tenure of a Fellowship Grant from the College of Medicine, Yonsei University (M.-H. Lee) and was supported by a Grant from Myung-Sun Kim Memorial Foundation (M.-H. Lee), a Cedars-Sinai Electrocardiographic Heartbeat Organization Foundation and Sweepstakes Award (H. S. Karagueuzian), a Pauline and Harold Price Endowment (P.-S. Chen), an National Heart, Lung, and Blood Institute Specialized Center of Research Grant in Sudden Death (P50-HL-52319), an American Heart Association National Center Grant-in-Aid (9750623N, 9950464N), University of California-Tobacco Related Disease Research Program Grant 6RT-0041, and the Ralph M. Parsons Foundation (Los Angeles, CA).
Address for reprint requests and other correspondence: P.-S. Chen, Rm. 5342, CSMC, 8700 Beverly Blvd., Los Angeles, CA 90048-1865 (E-mail:).
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- Copyright © 2000 the American Physiological Society